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This is to certify that the

dissertation entitled

RAPID FORMATION OF MULTIPLE SYNAPSES IN THE
SUPRAOPTIC NUCLEUS OF ADULT RAT HYPOTHALAMIC
SLICES

presented by
BARBARA K. MODNEY

has been accepted towards fulfillment
of the requirements for

PhD degree in Psychology/Neuroscience

F

Major professor

Date November 6, 1990

 

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

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RAPID FORMATION OF MULTIPLE SYNAPSES
IN THE SUPRAOPTIC NUCLEUS OF
ADULT RAT HYPOTHALAMIC SLICES
By

Barbara K. Modney

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Psychology and Neuroscience Program

1 990

L

WT ’

ABSTRACT
RAPID FORMATION OF MULTIPLE SYNAPSES
IN THE SUPRAOPTIC NUCLEUS OF
ADULT RAT HYPOTHALAMIC SLICES

By

Barbara K. Modney

The adult rat supraoptic nucleus (SON) is a known model system for
investigations of neuronal plasticity. Previous research has demonstrated that new
multiple somatic synapses form in SON during chronic dehydration i_n mo. In the
present experiments hypothalamic slices containing the SON were maintained in
yitro in either high or low osmolality media after which multiple synapses were
measured at the electron microscopic level.

In the first experiment slices were maintained in a static bath chamber with
constant infusion of dilute medium to compensate for evaporation. The average
firing rate of neurons from slices incubated in high osmolality medium was higher
than low osmolality slices and a significant increase in multiple synapses was found
in the high osmolality slices.

In Experiment 11, slices were incubated in low or high osmolality medium in
a perifusion chamber which allows for continuous perifusion of fresh medium over
the slice. In this experiment there was no detectable osmotic effect on multiple
synapses. It could be that multiple synapses were forming in both groups since a
significant increase was found in low osmolality slices from this chamber relative to

slices from Experiment I.

In Experiment III, the static bath chamber was used again and a time course
experiment was designed to determine if the sequence of anatomical events that
occurs during multiple synapse formation could be discerned. Slices incubated for
5.5, 2.5 or 1.0 hours in low or high osmolality media. In contrast to the results
from Experiment I there was no detectable osmotic effect on any morphometric
measure. There was a significant increase in multiple synapses in the 2.5 and 5.5
hour groups relative to the 1.0 hour group.

These results suggest that multiple synapses may form in the SON i_n vitro
(as they do i_n vilo) in response to deafferentation. This formation is rapid since
significant increases were found within 2.5 hours. The SON provides a unique
neural system with which to compare and contrast natural and reactive
synaptogenesis. The hypothalamic slice preparation may provide a useful tool to

investigate a rapid response to deafferentation.

This dissertation is dedicated to my mother
whose strength and capacity for love
was truly remarkable
and to Dad and Gary for their unlimited

unconditional love and understanding.

iv

ACKNOWLEDGEMENTS

I have many people to thank for various things they have done for me since
I came to Michigan State. Were I to honestly and faithfully acknowledge you all I
would have to write a book.

To Glenn Hatton, who not only provided generous financial support
throughout my training, but more importantly provided a role model for what a
multidisciplinary neuroscientist should be - Thank You is a gross understatement.

To my dissertation committee members - Drs. Cobbett, Nunez and Tweedle,
your willingness to meet with me for many data discussions during these
experiments was truly appreciated - your advise and encouragement will never be
forgotten.

To A. Salm, K. Smithson, C. Wagner and R. Cole - the distinctions between
friends, colleagues and peers simply don’t exist, you’ve been all these and much
more. I will consider myself to be truly blessed if I continue to meet people like
you who enrich so many aspects of my life.

To the many able technicians who have run the lab and have helped me
enormously: K. Grant, L. Koran, P. Rusch, I. Smithson & K. Smithson - Thank
you for everything, especially the things that I never realized you did and probably

took for granted.

The following people at PRB have been helpful in many ways: G. Beagley,
L. Bergland, J. Harper, F. Marzban, C. Sisk, M. Weiss, H. Wieferich, and Q. Yang.
Last but not least, to Ginny, Erik, D’Lynn, Margie, Steve, Pat, Dick, Phyllis,
Emy, Jeremy, Jordon, John, Debbie, Katy, Gregor, Betsy, Al, Jen and Annie and
Bob, Maribeth, Megan, Mom & Dad—in law and of course Dad and Gary - thanks

for constant support, you are the best part of my life.

vi

TABLE OF CONTENTS

PAGE

List of Tables .......................................... ix
List of Figures .......................................... xi
Abbreviations ......................................... xiv
INTRODUCTION ........................................ 1
The Hypothalamo-neurohypophysial System .................... 1

The Reorganized HNS .................................. 5
STATEMENT OF THE PROBLEM ............................ 12
GENERAL METHODS .................................... 16
Slice Preparation ..................................... 16
Tissue Processing ..................................... 17
Morphometrics ....................................... 18
Statistical Analysis .................................... 19

EXPERINIENT I-OSMOTIC STIMULATION OF HYPOTHALAMIC SLICES . 21

Somatic Region ...................................... 21
Results .......................................... 21
Dendritic Region ..................................... 32
Results .......................................... 35
Conclusion ......................................... 39

vii

EXPERIMENT II - SLICES IN THE PERIFUSION CHAMBER .......... 40

Results ........................................... 41
Conclusion ......................................... 42
EXPERIMENT III - TIME COURSE FOR SYNAPSE FORMATION ....... 45
Results ........................................... 45
DISCUSSION .......................................... 62
Methodological Considerations ............................. 64
Morphometric Measures ............................... 64
Experiment I vs Experiment III .......................... 65
Electron Microscopic Evaluation of Brain Slices ................. 67
Reactive and Rapid Synapse Formation ....................... 71
Multiple Synapse Formation .............................. 75
Multiple Synapses in Developing Systems ................... 76
Multiple Synapses in Adult Animals ....................... 78

Direct Appositions and Multiple Synapses ..................... 80
SUMMARY ........................................... 83
LIST OF REFERENCES ................................... 84
APPENDIX A - TISSUE EMBEDDING PROTOCOL ................. 96
APPENDIX B - SUPPLIES AND EQUIPMENT .................... 98
APPENDIX C - DATA TABLES - EXPERIMENT I ................ 101
APPENDIX D - DATA TABLE - EXPERIMENT II ................ 110
APPENDIX E - DATA TABLES - EXPERIMENT HI ............... 111

viii

LIST OF TABLES

PAGE

Table 1 - Multiple Synapse Measures From Slices Incubated in Low or
High Osmolality Medium ........................... 27

Table 2 - Correlation Coefficients Between Morphometric Measures and
Incubation Medium Osmotic Pressure ................... 47
Table 3 - ANOVA Tables Experiment HI ....................... 49
Table 4 - Group Means and Standard Errors For Experiment III ......... 52
Table 5 - Electron Microscopic Investigations of Brain Slices .......... 68
Table 6 - Cellular Elements Contacting MNC Somatic Membrane ....... 101
Table 7 - Multiple Synapse Measures ........................ 102

Table 8 - Mean Length (mm) of Apposition Between Axon Terminals
and MNC Somatic Membrane ....................... 103
Table 9 - Somatic Size and Shape Measures .................... 104
Table 10 - Average Firing Rates ............................ 105
Table 11 — Dendritic Synapses .............................. 106
Table 12 - Dendritic Multiple Synapses ........................ 107

Table 13 - Percentage of Dendritic Membrane Contacted by Cellular
Elements .................................... 108

Table 14 - Average Length of Dendritic Synapses and Percentage of
Dendritic Axonal Contact Made by Multiple Synapses ....... 109
Table 15 - Percentage of Synapses That were Multiple Synapses ........ 110
Table 16 - Structures Contacting MNC Somata ................... 111

Table 17 - Percentage of Synapses that were Multiple and Percentage of
Total Axonal Contact Made by Multiple Synapses .......... 114

Table 18 - Average Length (um) of Single and Multiple Synapses ....... 115

Table 19 - Number of Single and Multiple Synapses per 100 um of
Somatic Membrane .............................. 116

Table 20 - Number of Direct Soma-Somatic Appositions Per 100 pm of

Somatic Membrane and Their Average Length ............ 117
Table 21 - Somatic Size and Shape Measures .................... 118
Table 22 - Measured Osmolality of Chamber Medium ............... 119

Figure 1 -

Figure 2 -

Figure3-

Figure 4 -

LIST OF FIGURES

Schematic diagram of the methodology used for the present
experiments. Briefly horizontally cut hypothalamic slices
were incubated in low or high osmolality medium and
extracellular single unit activity was monitored. Following
fixation the SON was prepared for electron microscopy and

morphometric measures were obtained from micrographs .....

Single unit activity of neurons from slices incubated in low
or high osmolality medium in Experiment I. A — Sample
traces from neurons incubated in low osmolality (first trace)
or high osmolality medium. B - A significant increase in
average firing rate was obtained in slices incubated in high

osmolality medium. p < 0.05 ......................

Photomicrograph of a semi-thin (1 pm) section from a
hypothalamic slice. The SON is roughly delimited by the
dotted white lines. White arrows point to the dorsal cut
surface of the slice where some tissue damage is present.
Black arrows point to extensive vacuolization in the area
lateral to SON. The nucleus is largely devoid of such
damage since is never placed directly on the chamber net.

OC - Optic chiasm. Bar = 100 um ..................

Electron micrographs showing SON morphology from
conventionally prepared tissue (A; transcardial perfusion) and
a hypothalamic slice (B). Portions of SON somata (S) as
well as several dendrites (D) are indicated in both panels.
As is common in control animals astrocytic processes
(arrowheads) separate adjacent postsynaptic elements. Single
synapses are indicated by arrows. Comparisons between
these two micrographs show that the morphology of slice
tissue was often as good as conventionally prepared tissue.
Panel A is from a normal male rat transcardially perfused
with the same fixative used in the present experiments with
the exception that dimethylsulfoxide was not included. This
tissue was embedded in Epon-Araldite resin and sections
were mounted on Butvar coated slot grids. Tissue from
panel B was prepared according to the protocol listed in the

Methods section. Bar = 2 pm .....................

xi

PAGE

...14

...22

...24

...25

Figure 5 — Multiple somatic synapses from hypothalamic slices incubated
for at least 4 hours. In A a single synapse is formed by an
axonal terminal (*) on one of the SON somata (S). A
second terminal (**) contacts both SON somata. A small
portion of the two somatic membranes is in direct apposition
near the multiple synapse (arrowhead). Astrocytic processes
(arrows) are interposed between the adjacent soma and the
single terminal (*). Bar = 1 urn. In B an axonal terminal
(**) forms a synapse with both an SON soma (S) and an
adjacent dendrite (D). A thin astrocytic process separates a
large portion of the terminal membrane (arrows) from the
cell body. Direct apposition between the cell body and the
dendrite is marked by arrowheads. Bar = 0.5 pm ............ 28

Figure 6 - Photomicrographs showing direct soma-somatic and soma-
dendritic membrane appositions. In A a large portion of
somatic membrane of two adjacent somata (S) is in direct
apposition (delimited by arrows). A thin astrocytic process
(arrowheads) separates a portion of the two cell membranes.
Bar = 1 pm. In B a portion of the extensive apposition
shown in panel A is shown at a higher magnification.
The apposition is indicated by arrows while the glial
process is indicated by the arrowhead. Bar = 0.2 pm. In C
the direct apposition between a dendrite (D) and an MNC
soma (S) is delimited by arrows. These elements are also
contacted by an axonal terminal (*) that forms a multiple
synapse. Bar = 0.5 pm ............................ 30

Figure 7 — Photomicrographs of the SON dendritic region from an
animal that was conventionally perfused for electron
microscopy (A; see Figure 4 for tissue information) and
from a hypothalamic slice (B). In both panels a few of the
dendrites are labeled (D). Astrocytic processes separate
many of the dendrites (arrowheads) and dendrites in direct
apposition are also present (arrows). Bars = 2 pm ........... 33

Figure 8 - Cellular organelles associated with synapse formation. In A
an axonal terminal (*) is apposed to an SON soma (S) and a
dendrite (D). The long thin arrow points to a coated vesicle
that is fused at the dendritic membrane immediately adjacent
to the axonal terminal. Portions of the dendritic and somatic
membranes are in direct apposition (large arrow). Bar = 0.5
pm. In B an axonal terminal (*) is apposed to two dendrites
(D). Long arrows point to polyribosomal clusters subjacent
to the terminal apposition. Bar = 0.5 pm. In C a large
cluster of polyribosomes is present immediately subjacent to
the axonal terminal (*). The terminal is also apposed to
an SON soma (S) that is largely out of the photomicrograph.
Bar = 0.5 pm ................................... 36

xii

Figure 9 - Dendritic bundles from a slice that incubated 2.5 hours in
high osmolality medium. A bundle of 3 dendrites (labeled
with 1’s) and a bundle of 2 dendrites (labeled with 2’s)
are indicated. Thin astrocytic processes (arrowheads) separate
adjacent dendrites from the bundles. Bar = 1 pm ............ 38

Figure 10 -Photomicrographs of hypothalamic slices incubated in the
perifusion chamber. In A the general characteristics of the
somatic region of SON are shown. Portions of two somata
(S, one nucleus is labeled) are shown with several dendrites
(D) in the center of the field. One axonal terminal forms a
single somatic synapse (*) and two terminals form dendro—
dendritic (**) multiple synapses. Bar = 2.0 pm. In B
dendrites (D) from the dendritic region are shown. Two
dendritic bundles of 2 dendrites (labeled 1 and 2) as well as
a bundle of 4 (labeled with 3’s) are in the field. Axonal
terminals forming single (*) and multiple synapses (**) are
indicated. Bar = 1 pm ............................. 43

Figure 11 -Comparisons of the percentage of synapses that were multiple
and the percentage of total axonal contact made by multiple
synapses. Significant increases over time were found on both
measures ...................................... 57

Figure 12 -Multiple synapse measures for the three incubation times.
The percentage of somatic membrane contacted by multiple
synapses did not reach the p < 0.05 level (p < 0.07). In
contrast, the number of multiple synapses per 100 um of
somatic membrane did increase significantly. *p < 0.05
compared to 5.5 hour, #p < 0.05 compared to 2.5 hour ........ 58

Figure 13 -Comparisons of cell area and perimeter over the three
incubation times. Both measures decreased over time. * p <
0.05 compared to 5.5 hour, # p < 0.05 compared to 2.5 hour ..... 60

Figure 14 -The percentage of multiple synapses associated with dendritic
polyribosomes (pr) and coated vesicles (cv). No obvious
or consistent differences were seen over the incubation
times, perhaps due in part to extreme variability between
animals ....................................... 61

xiii

ABBREVIATIONS

ANOVA — Analysis of Variance

cv — Coated Vesicle

HNS - Hypothalamo-neurohypophysial system
Hz - Hertz

MNC - Magnocellular neuroendocrine cell
mOsm - Milliosmoles

NL - Neural lobe

Osm - Osmolality

OX - Oxytocin

pr - Polyribosomes

psd - Postsynaptic density

SE - Standard Error

SON - Supraoptic nucleus

VP - Vasopressin

xiv

INTRODUCTION

While fulfilling its traditional role as a model system for neurosecretion, the
hypothalamo-neurohypophysial system (HNS) has emerged as a unique system with
which to investigate adult neuronal plasticity. Associated with changes in the
internal as well as external environment of the animal are alterations in many of the
morphological characteristics of the HNS. These morphological changes, including
the formation of new synapses and changes in neuronal-glial interrelationships, are
often clearly related to increased demand for the HNS’s secretory products. Few
mammalian systems have such a clear and malleable structure-function relationship.
By studying this system insight is gained into the ways in which the adult nervous
system can respond to required changes in neuronal functioning.

H 1 Ne h h i m

Consideration of the HNS as a model for the study of neural plasticity has
evolved from its more prominent status as a model for the study of neurosecretion
(see Bissett & Chowdrey, 1988; Forsling, 1986; Hatton, 1990 for reviews). One of
the most convenient characteristics of this system is the anatomical configuration of
the magnocellular neuroendocrine cells (MNCs) and their massive efferent projection
to the neural lobe (NL) of the pituitary gland. MNCs are scattered in several nuclei
in the anterior hypothalamus (Peterson, 1966). The emphasis here is on the

supraoptic nucleus (SON) of the rat, since most of the experimental work discussed

2
below involves the SON (see Hatton, 1990; Theodosis & Poulain, 1987 for
reviews; and see Gregory, Tweedle & Hatton, 1980; Theodosis & Poulain, 1989;
and Tweedle & Hatton, 1976; 1977 for relevant work in other magnocellular
neuroendocrine cell groups).

The bilateral supraoptic nuclei are located at the ventral surface of the brain
just lateral to the optic chiasm and tracts. The nucleus proper can conveniently be
divided into a dorsal somatic region and a ventral dendritic region. These regions
are not mutually exclusive (i.e. there are dendrites in the somatic region), but rather
describe the relative predominance of somata vs. dendrites in the two regions.
Neurons are densely packed within the nucleus and are almost exclusively
neurosecretory, although the rare non-neurosecretory neuron has been described
(Itoh, Iijima & Kowada, 1986). At the ultrastructural level, the dense packing of
MNCs is readily apparent and adjacent somata are typically separated by thin
astrocytic processes. MNCs are characterized by an abundance of cellular organelles
involved in peptide processing (e.g. Golgi apparatus, rough endoplasmic reticulum,
ribosomes etc.) as well as the presence of dense core vesicles. MN Cs have simple
dendritic trees consisting of 1-3 dendrites which project ventrally initially and then
turn and course in a rostral-caudal orientation parallel to the ventral surface of the
brain (e.g. Armstrong, Scholer, & McNeil], I982; Dyball & Kemplay, 1982). Many
dendrites in this region contain dense core vesicles similar to those seen in SON
somata and terminals in the neural lobe (Armstrong et al., 1982; Yulis, Peruzzo &
Rodriguez, 1984). Adjacent dendrites are usually separated by thin astrocytic
processes in a manner similar to that described between somata in the somatic

region (Perlmutter, Tweedle & Hatton, 1984b; Yulis et al., 1984). The most ventral

3

portion of the SON, adjacent to the pia-arachnoid is the ventral glial limitans which
consists of an extensive blanket of interwoven and interconnected astrocytic
processes (Armstrong et al., 1982; Yulis et al., 1984). Within this region lie most
of the astrocytic cell bodies that send processes dorsally through the nucleus and
separate the adjacent dendrites and somata (Salm & Hatton, 1980). The dense
packing of MNCs in the SON, the relative segregation of somata and dendrites as
well as the homogeneity of cell type within the nucleus (i.e. few intemeurons) make
a morphometric analysis of these neurons under various experimental conditions
relatively straightforward.

MNCs contain several neumpeptides (see Bondy, Whitnal, Brady & Gainer,
1989 for review), the best known and characterized being oxytocin (OX) and
vasopressin (VP). These two hormones are manufactured by separate cell
populations in the SON that are rougmy segregated. That is, oxytocinergic neurons
are generally located in the more antero—dorsal regions of the nucleus, while
vasopressinergic neurons are located in the more posterior-ventral regions of the
nucleus. There is considerable intermingling of the two cell populations so that
anatomical identification of the hormone content of a particular cell requires
immunocytochemical staining (e. g. Hou-Yu, Lamme, Zimmerman & Silverman,
1986). OX vs. VP containing cells can often be identified on the basis of their
electrophysiological characteristics (see Poulain & Wakerley, 1982).
Vasopressinergic neurons often display a phasic firing pattern consisting of
alternating periods of action potential bursts and silence. In lactating animals with
suckling pups a synchronous high frequency burst of action potentials of most

oxytocinergic neurons reliably precedes milk ejection.

4

A major axonal projection from SON neurons is to the NL, although
collaterals that terminate in the area lateral to SON (Mason, Ho & Hatton, 1984)
and oxytocinergic and vasopressinergic synapses within the nucleus proper have also
been described (Choudhury & Ray, 1990; Ray & Choudhury, 1990; Theodosis,
1985). In the NL neurosecretory axons and terminals are intimately associated with
pituicytes (astrocytic cells of the NL, Salm, Hatton & Nilaver, 1982; Suess &
Pliska, 1981). Pituicytes often surround and/or compleme engulf axonal terminals
(Tweedle & Hatton, 1980b). Thin pituicyte processes and axonal terminals contact
the basal lamina which surrounds the capillaries. Action potentials generated by
MNCs invade the temtinals and cause calcium dependent hormone release.
Hormone that gains access to the fenestrated capillaries enters the pituitary-portal
system for distribution to peripheral tissues. Peripheral effects of both hormones
have been known for many years (See Amico & Robinson, 1985 and Gash & Boer,
1987 for comprehensive reviews). Oxytocin promotes milk ejection during lactation
and uterine contraction during parturition, while VP or anti-diuretic hormone, is best
known for its ability to act on the collecting tubules of the kidney to promote water
reabsorption.

Even though the conventional characterization of the HNS focuses on hormone
release from the NL and hormone action at peripheral tissues, evidence for central
projections of MNCs is strong (Buijs, Swaab, Dogterom & van Leeuwen, 1978).
Release of these hormones from the axon collaterals that terminate lateral to SON
and within the nucleus proper has been demonstrated (Mason, Hatton, Ho, Chapman
& Robinson, 1986). Recent evidence suggests that hormone detected within the

nucleus may be the result of dendritic release (Pow & Morris, 1989; Tweedle,

5
Smithson & Hatton, 1988). The functional significance of dendritic hormone release
is unclear. Several studies suggest that OX excites MNCs (Freund-Mercier &
Richard, 1984; Inenaga & Yamashita, 1986; Yamashita, Okuya, Inenaga, Kasai,
Uesugi, Kannan & Kaneko, 1987) and VP may have a similar effect (Inenaga &
Yamashita, 1986). It may be that dendritic release of hormone serves to increase
MNC activity. A second possibility is that dendritic release of OX participates in
the anatomical reorganization of the SON since chronic infusion of OX
intracerebroventricularly results in characteristic changes in SON morphology
(Montagnese, Poulain & Theodosis, 1990; Theodosis, Montagnese, Rodriguez,
Vincent & Poulain, 1986b).
r i HN

The most common experimental manipulations used to study anatomical
changes in the HNS are dehydration, parturition and lactation. Dehydration,
produced by water deprivation or substitution of 2% saline for drinking water, alters
virtually every measurable characteristic of both oxytocinergic and vasopressinergic
neurons (see Amico & Robinson, 1985; Gash & Boer, 1987 for reviews).
Messenger ribonucleic acid (mRNA) levels for OX and VP, as well as several other
peptides are elevated by drinking 2% saline. Protein synthesis, post-translational
processing of the prohormones, axonal transport of hormones to the NL are all
increased during dehydration. Concurrent with this increased synthesis and transport
of OX and VP are significant alterations in the electrical activity of MN Cs.
Oxytocinergic neurons increase their firing rate, while vasopressinergic neurons
either evolve a phasic firing pattern or alter existing burst characteristics (e.g. burst

duration, firing rate within burst). This generalized increase in the activity of

6
MNCs during osmotic challenge is probably due, at least in part, to the intrinsic
osmosensitivity of MNCs. Ultimately the chronic increased activity of MNCs leads
to increased hormone release from the NL and progressive depletion of
neurohypophysial hormone. Given that osmotic stimulation is such of robust and
powerful stimulus for this system, it is not surprising that the anatomical
characteristics of these neurons are also dramatically altered.

Parturition and lactation are most often associated with the activation of
oxytocinergic neurons (see Forsling, 1988), although the VP content of the NL is
depleted during parturition (Fuchs & Saito, 1977). Also, mapping of metabolic
activity of MNCs using the 2-deoxyglucose method has suggested that
vasopressinergic neurons are also activated following a bout of suckling (Allen,
Stern & Adler, 1984). During parturition oxytocinergic neurons exhibit a
generalized increase in firing rate and bursts of action potentials precede expulsion
of each fetus. Progressive decreases in neurohypophysial OX and increases in
plasma OX occur as parturition progresses. During lactation afferent information
produced by suckling pups is relayed through an elusive neural pathway to MNCs
in the hypothalamus. Milk ejection follows the synchronized high frequency firing
of most of the oxytocinergic neurons in the hypothalamus. This high frequency
firing is required for sufficient OX release from the NL to cause milk ejection. As
with osmotic stimuli, the activation of oxytocinergic neurons during parturition and
lactation is associated with morphological changes in the HNS.

Changes in the activated HNS observed at both the light and electron
microscopic level are as ubiquitous as the other indicators of activation. Light

microscopic studies have concentrated on changes in cell size, proliferation of Nissl

7

substance and nucleolar size (Armstrong, Gregory & Hatton, 1977; Bandaranayake,
1974; Ellman & Gan, 1971; Enestrom, 1967; Hatton & Walters, 1973; Kalirno,
1965; Morris & Dyball, 1974). Most early electron microsc0pic studies of the
stimulated system concentrated on proliferation and/or depletion of intracellular
structures (e. g. rough endoplasmic reticulum, Golgi apparatus, dense core vesicles
etc.). More recent studies have focussed on changes in the synaptic and neuronal-
glial relationships in the SON and NL following activation.

Two particular anatomical changes which occur in five are detected at the
electron microscopic level and serve as a starting point for the experiments
discussed below. First, the synaptic characteristics of SON of experimentally treated
animals are different from control animals. The most obvious and best documented
synaptic change m yi_v_o is the appearance of novel multiple synapses. A multiple
synapse is defined as a single axonal terminal which simultaneously contacts at least
two adjacent post-synaptic structures, i.e. two adjacent somata, two dendrites, or
adjacent soma and dendrite. While this type of synapse is relatively rare in normal
animals, their frequency is increased in the dendritic region during parturition and
lactation (Perlmutter et al., 1984b) and in the somatic region during lactation and
dehydration (Chapman, Theodosis, Montagnese, Poulain & Morris, 1986; Hatton &
Tweedle, 1982; Montagnese, Poulain, Vincent & Theodosis, 1987; Modney &
Hatton, 1989; Theodosis, Chapman, Montagnese, Poulain & Morris, 1986a;
Theodosis & Poulain, 1984; Theodosis, Poulain & Vincent, 1981; Tweedle &
Hatton, 1984a). That the multiple synapses formed under these conditions are truly
related to the physiological demand for hormone release is supported by the fact

that they disappear when hormone demand returns to basal levels (e. g. pup weaning

8

and rehydration). Although this type of synapse was first described using the
conventional stimuli of dehydration, parturition and lactation they also form in SON
under a number of different experimental conditions including: 1)
innacerebrovenuicular administration of OX for 8 days (Theodosis, et al., 1986b;
Montagnese, et al., 1990), 2) transcardial perfusion with hypertonic solution
immediately prior to transcardial fixation (Tweedle, Smithson & Hatton, 1989), and
3) following removal of an afferent projection from the subfomical organ to the
SON (Weiss, Tweedle, Marzban, Modney & Hatton, 1988).

While the formation of multiple synapses is well-established, very little is
known about their precise function. Indeed that they are functional synapses is
based only on morphological data. That they often form along with alterations in
the electrical activity of these neurons suggests that they participate in the control of
the SON during periods of high hormone demand. There is immunocytochemical
evidence that some somatic multiple synapses contain GABA (Theodosis, Paut &
Tappaz, 1986, see also van den Pol, 1985 Figure 5 and Buijs, van Vulper &
Geffard, 1987) and evidence suggests that some may contain dopamine (Buijs,
Geffard, Pool & Hoomeman, 1984). There is also evidence that some dendritic
multiple synapses are noradrenergic (Tweedle & Hatton, 1984b). Even though the
morphological characteristics of the terminals that form somatic multiple synapses
are similar following chronic dehydration and lactation it is not known if the same
afferent system forms these synapses under the two conditions.

That synaptogenesis occurs in the SON of adult animals is well established.
The mechanisms(s) by which these new synapses form has not been determined.

There is a suggestion that multiple synapses are formed by the conversion of pre-

9
existing single synapses (Hatton & Tweedle, 1982; Perlmutter et al., 1984b, 1985;
Tweedle & Hatton, 1984a). According to this hypothesis a single terminal may
initially contact one SON soma but be separated from another adjacent soma by a
thin astrocytic process. Retraction of this process from between the second soma
and the terminal would then allow formation of a multiple synapse. This
mechanism provides an attractive explanation for the very rapid (20 minutes)
formation of multiple synapses that occurs during intravascular perfusion of
hypertonic solutions (T weedle et al., 1989).

The extent to which multiple synapse formation in the SON conforms to the
events that occur during synaptogenesis in developing animals or during reactive
synaptogenesis has not yet been investigated. For example, in developing animals
synaptogenesis is associated with aggregates of polyribosomes in the postsynaptic
element (typically dendritic spines) near the new synapse (Palacios-Prii, Palacios &
Mendoza, 1981; Steward & Falk, 1986). Similar aggregates are also seen in
dendritic spines of dentate granule cells during the reinnervation that follows
removal of the ipsilateral entorhinal cortex (Steward, 1983). A second organelle
that has been implicated in synapse formation in developing systems (e. g. Altman,
1971) and during reactive synapse formation (McWilliams & Lynch, 1981) is the
coated vesicle. Both of these organelles have been hypothesized to participate in
the formation and/or maturation of postsynaptic specializations. Coated vesicles
have been shown to contain acetylcholine receptors and could be involved in
depositing receptors at the postsynaptic site (Bursztajn, & Fischbach, 1984). Coated

vesicles in both pre- and postsynaptic elements may also play a role in cell-cell

10
recognition during the early stages of synapse formation in developing neural
systems (see Vaughn, 1989 for review).

Another anatomical change in the SON described at the electron microscopic
level occurs both with dehydration and with parturition and lactation. As mentioned
earlier, in the normal animal, thin astrocytic processes typically separate adjacent
somata and dendrites. When the HNS is activated adjacent somata/dendrites are not
always separated by glial processes, instead extensive portions of somatic/dendritic
membrane are seen in direct apposition (Chapman et. al, 1986; Hatton & Tweedle,
1982; Montagnese et. a1 1987; Theodosis et. al, 1981; 1986a; Theodosis & Poulain,
1984; Tweedle & Hatton, 1976; 1977). In the dendritic region of SON the
increased direct membrane apposition between adjacent dendrites results in the
appearance of dendritic bundles (Perlmutter et al., 1984b; Perlmutter, Tweedle &
Hatton, 1985), i.e. two or more dendrites having portions of their membrane in
direct apposition.

As with multiple synapse formation, glial process retraction has been
suggested as the mechanism through which direct neuronal apposition can occur.
One study has shown a marked redistribution of immunoreactive staining for glial
fibrillary acidic protein in the SON, but not control areas of the hypothalamus,
during lactation (Salm, Smithson & Hatton, 1985). A similar study has yet to be
done in dehydrated rats. Many functional implications for the absence of glial
processes from between adjacent somata and dendrites have been suggested (see
Hatton, 1990). Given the ability of astrocytes to buffer extracellular K‘ and to
actively uptake various neurotransmitters, several neuronal consequences can be

envisioned. For example the accumulation of extracellular K+ could slightly

11
depolarize the neurons. Similarly the accumulation of neurotransmitters in the
extracellular space could theoretically extend the duration and/or magnitude of their
effect.

Accompanying the reorganization of the SON are anatomical changes that
occur at the level of the terminals of these neurons. In the NL the frequency of
axonal terminals completely surrounded by pituicyte cytoplasm declines in post-
partum, lactating and dehydrated animals (Tweedle & Hatton, 1980a; 1982). In
addition, the percentage of basal lamina contacted by pituicyte processes decreases
(relatively more contact of the basal lamina by axonal terminals) when the HNS is
activated in 21°19 (Tweedle & Hatton, 1987). Similar changes have also been
induced in an in yigo NL preparation (Luckrnan & Bicknell, in press; Perlmutter,
Hatton & Tweedle, 1984a; Smithson, Suarez & Hatton, in press). The release of
pituicyte engulfed terminals and retraction of processes from the basal lamina allows
greater temrinal contact at the basal lamina. It is thought that these alterations
ultimately permit more hormone direct access to the circulation. Again the active
role of the glial elements is thought to participate in the reorganization of the NL

during periods of high hormone demand.

STATEMENT: OE THE PROBLEM
The experimental work discussed above has used exclusively in 2119 models.

While these experiments have provided and will continue to provide vital
information about SON’s plasticity, they do not have the advantages that an in 111:9
system could provide. Hypothalamic slices and explant preparations maintained in
m have been invaluable in elucidating many neurophysiological characteristics of
MNCs (see Armstrong, Gallagher & Sladek, 1985). These preparations allow
investigators to asses the actions of neurotransmitters, the intrinsic membrane
properties of MNCs and hormone release in response to experimental manipulations.

In tango preparations have also been useful in studying the plastic capabilities
of the SON and NL. Hypothalamic slices have been used to investigate the extent
and modifiability of dye-coupling (an indirect measure of electrotonic coupling)
among SON neurons (see Hatton, 1990 for review). For example, the incidence of
coupling among SON neurons is considerably higher in slices prepared from
lactating animals relative to slices prepared from virgin female animals (Yang &
Hatton, 1987). An i_n v_igo neural lobe preparation has been used successfully to
investigate dynamic neural-glial changes in response to manipulations of the
incubation media (Luckrnan & Bicknell, 1990; Perlmutter et al., 1984a; Smithson et
al., in press).

The success of these i_n ximo studies provided the impetus to determine if an
in yin hypothalamic slice preparation could be used to investigate anatomical
restructuring in the SON. The potential advantages of such an i_n vi_go system
include: a) the ability to record the electrical activity of neurons and then examine

the tissue anatomically, b) greater knowledge of and control over the external

12

13
environment of the tissue relative to in mg manipulations, c) greater precision in
examining the time course of anatomical changes that lead to a reorganized SON.
A horizontal hypothalamic slice preparation that has been used extensively for
electrophysiological and dye coupling studies (Hatton & Yang, 1989; 1990; Weiss,
Yang & Hatton, 1989; Yang & Hatton 1987; 1989) was used to answer the
questions and issues concerning plasticity in the SON listed below.

Does an i_n yitm SON preparation respond to manipulations (e.g. increases in
osmolality) of incubation media with anatomical changes that resemble those seen i_n
yi_Q? Assuming that changes do occur i_n vim what is the time course and
sequence of events that lead to such changes? If synapse formation does occur Ln
firm will cytoplasmic organelles associated with this process (e.g. ribosomes) be

associated with the increase in synapses?

14

Figure 1 - Schematic diagram of the methodology used for the present experiments.
Briefly horizontally cut hypothalamic slices were incubated in low or high
osmolality medium and extracellular single unit activity was monitored. Following
fixation the SON was prepared for electron microscopy and morphometric measures
were obtained from photomicrographs.

 

  

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Figure 1

li ' n

A schematic illustration of the methods used in these experiments is shown in
Figure 1. Adult male rats (40-60 days old) maintained on ad lib food and water
were used. Rats were killed by rapid decapitation without anesthesia within the first
five hours after lights on. Following decapitation the brain of each rat was quickly
removed and placed in artificial cerebral spinal fluid of low osmolality (290
mOsm/kg) or high osmolality (340 mOsm/kg) containing (mM, low/high osmolality)
NaHCO3 (23.0/27.0), KCl (4.6/5.4), NaHzPO4 (1.2/1.4), MgSO, (1.2/l.4) D—glucose
(9.4/11.0, NaCl 117.0/138.0, 3-[N-morpholinerropanesulfonic acid (4.6/5.4), CaCl2
(2.2/2.6). For all experiments media were supplied coded so that data were
collected without awareness of treatment group membership. The fresh brain was
blocked by making two cuts with a razor blade just lateral to the lateral olfactory
tract to remove the extreme portions of the cortex, and a single cut just rostral to
the pons. The remaining tissue block was mounted dorsal surface down on a
Vibratome chuck. A horizontal slice(s) (500—600 pm thick) which included the
SON was cut on a Vibratome filled with oxygenated medium maintained at room
temperature. These slices were then placed ventral surface down in either of two
recording chambers (see specific experiments). Temperature of the bathing medium
for both chambers was maintained between 34-36 degrees C.

Neuronal activity was monitored using conventional extracellular recording
techniques. Extracellular electrodes were pulled with a Kopf Vertical Pippette Puller
(Model 700C) and the tip was manually broken back to 2-3ttm. Electrodes were

filled with 1M sodium acetate. Spontaneously active cells were recorded on either a

16

17

Vetter Model B tape recorder or an Akai Stereo Cassette deck (Model CS-34D).
Average firing rates were determined for cells with activity that could be clearly
distinguished from noise and that were recorded for at least 2 1/2 minutes. Average
firing rates were obtained by playing back the tape and triggering an oscilloscope
sweep on the peak of each action potential. The number of sweeps per 10 second
interval was recorded by an electronic counter (Hewlett Packard Counter, Model
5512A). The total number of action potentials was divided by total time (seconds)
to obtain the average firing rate.
Ti Pr sin

Following incubation (see specific experiments for total incubation time) slices
were immersed overnight in fixative containing 2.5% glutaraldehyde, 1%
paraformaldehyde, 0.5% dimethylsulfoxide in 0.10 M cacodylate buffer, pH 7.4.
The following day, coronal sections (300-400 um) were cut from the horizontal
slices. The SON was excised from three adjacent tissue sections and following
three buffer (0.15 M cacodylate) rinses the tissue was osmicated for one hour in a
1:1 mixture of 2% osmium and 3% potassium ferricyanide in 0.2M cacodylate
buffer, pH 7.4. The tissue was e_n_ Lloc stained in 4% uranyl acetate overnight,
dehydrated in a series of graded alcohols, and embedded in Spurr’s resin (see
Appendix A for embedding protocol). Semi-thin sections (0.5-l um), cut on a
Reichert Ultracut E microtome, were heat mounted on glass slides. Sections with
adequate tissue preservation were further analyzed at the electron microscopic level.
Note that these slices did not necessarily correspond to slices used in the

electrophysiological analysis above. Thin sections (60-90 nm) were cut and

18
collected on 200 mesh thin-bar copper grids, and were stained with lead citrate. A
single thin section from each slice was used for data collection.
Morphometric:

Semi-thin sections from slices used in electron microscopic data collection
were analyzed using an Olympus 02 Image Analysis system. Cells were viewed
through the microscope and on a video screen. The perimeter of each cell was
manually delimited with an optical cursor and the follow parameters were generated
by the computer for each cell: 1) Cell area (m2), 2) Cell perimeter (um) and 3)
Cell Shape (41tarca)/P€rimeter). In each slice at least 18 cells which contained a
nucleolus were measured.

Length measurements were made from electron micrographs with either a
Houston Instruments digitizing table interfaced with a Zenith-200 computer and in-
house software (Experiment I - Somatic Region) or a Summagraphics Digitizing
table and SigmaScan software. Twenty electron micrographs of the somatic region
(12,000X final magnification) per slice were taken. On each micrograph the lengths
of the following cellular elements contacting neuronal somatic membrane were
measured a) astrocytic processes, b) axonal terminals that contacted only one SON
soma (i.e. single synapses), c) axonal terminals that contacted both a soma and an
adjacent soma or dendrite (i.e. multiple synapses), (1) adjacent soma-somatic/dendritic
membrane, e) unidentified elements. (It should be noted that a synapse was inferred
each time an axonal terminal apposed a postsynaptic element, regardless of whether
or not clearly defined synaptic specialization were present.) The total length of
somatic membrane was obtained by summing the length of all the elements listed

above. Data from individual micrographs were summed for each animal and the

19

percentage of somatic membrane contacted by each cellular element was calculated.
Additionally, the percentage of synapses that were multiple synapses and the
percentage of total axonal contact made by multiple synapses was obtained (total
length of axonal contact made by multiple synapses/total length of all axonal
contacts * 100). The number of multiple synapses per 100 pm of somatic
membrane and the average length (pm) of single and multiple synapses was
determined.
smegma/mam

Data are presented as the Mean i Standard Error unless otherwise noted. The
hypothesis that neurons from slices incubated in high osmolality medium had a
higher firing frequency (Hertz, Hz.) than cells from low osmolality slices was tested
using a one-tailed Mann-Whitney U test. A one-tailed test was deemed appropriate
since numerous i_n yi_vo AND in LIES: studies have demonstrated that these neurons
respond to increased osmolality

Morphometric data from Experiments I and II were analyzed using a two-
tailed t-test for differences between means. Two-tailed tests were used since no
previous morphometric measures of hypothalamic slices were available and increases
or decreases in many of the measures were conceivable. For example, many of the
measurements made are dependent to some extent on cell size which could have
increased or decreased in the high osmolality group. Similarly the synapses
measures could have decreased based on previous studies of axotomized neurons.
Morphometric data from Experiment III which had 6 groups were analyzed using a
Two-Factor Analysis of Variance (ANOVA) with medium osmolality (low and high)

and incubation time (5.5 hours, 2.5 hours, 1 hour) as factors. All comparisons

20
between treatment group means were made following a significant F ratio using
Tukey’s test modified for unequal sample size (Winer, 1962). Correlation
coefficients (Pearson’s r) between several variables were also calculated. In all

cases probability levels of p < 0.05 were considered significant.

R NTI- MTI TIN FHYPTHALAMI LIE
Mansion

As discussed earlier osmotic challenge is a potent stimulus for activation of
the HNS and is associated with numerous changes in the somatic region of SON.
Several electrophysiological experiments have demonstrated that MNCs in yitro are
capable of responding to osmotic challenges (Bourque, 1989; Bourque & Renaud,
1984; Hatton, Armstrong & Gregory, 1978; Mason, 1980), and anatomical changes
similar to those seen i_n m after dehydration occur in the in filo NL incubated in
high osmolality medium (Perlmutter et al., 1984a). A primary goal of this
experiment was to determine if slices incubated in high osmolality medium exhibited
anatomical changes similar to those seen during osmotic challenge i_n mg.

A static bath chamber in which the slice is placed on a nylon net in a
medium filled well with constant infusion of dilute media to compensate for
evaporation (see Hatton, Doran, Salm & Tweedle, 1980) was used for this
experiment. Slices were incubated for between 4-6 hours prior to fixation and
tissue processing.

Results - A total of 32 animals were used for this experiment. All slices
included for data analysis were alive as determined by the presence of single unit
activity. This activity was either spontaneous or elicited by electrode advancement.
Slices in which no single unit activity was detected (n=2) were discarded from the
study. A total of 21 spontaneously active cells from 14 slices had action potentials
that were clearly distinguishable from noise (see Figure 2). All of these cells fired
in what could be considered a continuous manner and included 12 cells from slices

that incubated in low osmolality media and 9 cells from high osmolality slices. As

21

FIRING RATE (HZ)

00

ON

A

N

 

2 sec

 

 

\V

 

 

 

 

 

 

LOW HIGH
OSMOLALIT Y OS MOLALITY

Figure 2 - Single unit activity of neurons from slices incubated in
low or high osmolality medium in Experiment I. A - Sample traces
from neurons incubated in low osmolality (first trace) or high
osmolality medium. B - A significant increase in average firing
rate was obtained in slices incubated in high osmolality medium.

* p < 0.05

23
shown in Figure 2 a significant elevation in average firing rate of neurons from
slices incubated in the high .osmolality medium compared to low osmolality was
obtained (p<0.04).

Examination of the semi-thin sections at the light microscopic level was used
to determine if tissue quality was adequate for electron microsc0pic evaluation. As
shown in Figure 3, the SON is free from most tissue damage due to Vibratome cuts
or placement on the chamber net. Slices in which the SON was obviously damaged
by the cutting procedure were excluded, as were slices that had extensive
vacuolation within the nucleus. On this basis eighteen slices were deemed adequate
for electron microscopic analysis. One slice was eliminated based on inadequate
membrane preservation which was not detected until electron microscopic evaluation.
Nine slices from the 290 group and eight slices from the 340 group were used for
morphometric analysis.

At the ultrastructural level the tissue from slices was quite well preserved. A
comparison between the characteristic morphology of MNC somata from a
conventionally perfused animal and a hypothalamic slice is illustrated in Figure 4.
Accurate identification of the cellular elements apposed to SON somatic membrane
was readily accomplished in the slice tissue. Multiple synapses (see Figure 5) were
more frequent in the high osmolality slices relative to the low osmolality slices. As
shown in Table 1, significant increases in the percentage of somatic membrane
contacted by multiple synapses (p < 0.02), the number of multiple synapses per 100
pm of somatic membrane (p < 0.05), the percentage of synapses that were multiple
synapses (p < 0.05), and the percentage of total axonal contact made by multiple

synapses (p < 0.02).

 

 

Figure 3 - Photomicrograph of a semi-thin (1 pm) section from a hypothalamic
slice. The SON is roughly delimited by the dotted white lines. White arrows point
to the dorsal cut surface of the slice where some tissue damage is present. Black
arrows point to extensive vacuolization in the area lateral to SON. The nucleus is
largely devoid of such damage since is never placed directly on the chamber net
OC - Optic chiasm. Bar = 100 um

25

Figure 4 - Electron micrographs showing SON morphology from conventionally
prepared tissue (A; transcardial perfusion) and a hypothalamic slice (B). Portions of
SON somata (S) as well as several dendrites (D) are indicated in both panels. As
is common in control animals astrocytic processes (arrowheads) separate adjacent
postsynaptic elements. Single synapses are indicated by arrows. Comparisons
between these two micrographs show that the morphology of slice tissue was often
as good as conventionally prepared tissue. Panel A is from a normal male rat
transcardially perfused with the same fixative used in the present experiments with
the exception that dimethylsulfoxide was not included. This tissue was embedded in
Epon-Araldite resin and sections were mounted on Butvar coated slot grids. Tissue
from panel B was prepared according to the protocol listed in the Methods section.
Bar = 2 pm

 

27
TABLE I - MULTIPLE SYNAPSE MEASURES FROM SLICES INCUBATED IN

LOW OR HIGH OSMOLALITY MEDIUM

Group % Somatic #/ 100 % %

Osmolality Membrane pm of Synapses Axonal
Somatic Contact

Membrane

Low (n=9) 0.810.12 0.7812016 8.0i1.1 7.4:t0.8

High (n=8) 1.36i0.14’ 1.25i0.14' 11.6:t1.0' 11.0.t1.1’

* p < 0.02

# p < 0.05

There were no significant differences in any of the following ultrastructural
measures (low vs high osmolality): 1) the percentage of somatic membrane
contacted by glial processes (82 i 1.7 vs 77 i 2.2), 2) the percentage of somatic
membrane in direct apposition with adjacent somata and or dendrites (see Figure 6,
2.5 :1: 0.7 vs 3.2 :1: 1.0), 3) the percentage of somatic membrane contacted by axonal
terminals (10.9 i 1.1 vs 12.4 i 1.0), 4) the percentage of somatic membrane
contacted by unidentified elements (4.5 :t 0.8 vs 6.5 :l: 1.1), 5) the mean length
(pm) of axonal terminals (1.16 :1: 0.04 vs 1.15 :1: 0.05). No significant differences
between the groups in cell area were detected (um’, low vs high osmolality, 431 i
13 vs 430 i 23), cell perimeter (um, 80 :i: 1.1 vs 78 i 1.9) or cell shape (0.84 i

0.01 vs 0.87 :l: 0.01).

28

Figure 5 - Multiple somatic synapses from hypothalamic slices incubated for at least
4 hours. In A a single synapse is formed by an axonal terminal (*) on one of the
SON somata (S). A second terminal (**) contacts both SON somata. A small
portion of the two somatic membranes is in direct apposition near the multiple
synapse (arrowhead). Astrocytic processes (arrows) are interposed between the
adjacent soma and the single terminal (*). Bar = l um. In B an axonal terminal
(**) forms a synapse with both an SON soma (S) and an adjacent dendrite (D). A
thin astrocytic process separates a large portion of the terminal membrane (arrows)
from the cell body. Direct apposition between the cell body and the dendrite is
marked by arrowheads. Bar = 0.5 pm.

 

30

Figure 6 - Photomicrographs showing direct soma-somatic and soma-dendritic
membrane appositions. In A a large portion of somatic membrane of two adjacent
somata (S) is in direct apposition (delimited by arrows). A thin astrocytic process
(arrowheads) separates a portion of the two cell membranes. Bar = 1 um. In B a
portion of the extensive apposition shown in panel A is shown at a higher
magnification. The apposition is indicated by arrows while the glial process is
indicated by the arrowhead. Bar = 0.2 pm. In C the direct apposition between a
dendrite (D) and an MNC soma (S) is delimited by arrows. These elements are
also contacted by an axonal terminal (*) that forms a multiple synapse. Bar = 0.5

pm.

 

32

Given the anatomical changes detected in the somatic region it was of interest
to see if dendritic changes were also detectable in osmotically stimulated slices.
Dehydration i_n 21.19 by substitution of saline for drinking water or water deprivation
results in the formation of dendritic bundles, but does not increase dendritic multiple
synapses (Perlmutter et al., 1985) Transcardial perfusion with hypertonic solution
results in the formation of multiple synapses and dendritic bundles (Tweedle et al.,
1989) suggesting that multiple synapses could be forming in the dendritic zone of
SON of the slices used for the somatic analysis discussed above. Five slices from
the low osmolality group and five slices from the high osmolality group were used
for morphometric analysis. '

Measurements of dendritic bundling and dendritic multiple synapses were
similar to the measurements made in the somatic region. Twenty photomicrographs
were taken at a magnification of 5,800X and enlarged 3.5X for a final magnification
of 20,300X (these were printed on 11" X 14" photographic paper). The percentage
of dendritic membrane contacted by adjacent dendrites, single synapses and multiple
synapses was determined using a semi-circular test grid and intersection counting
(see Mayhew, 1979). Quantification of dendritic multiple synapses included the
percentage of terminals that contacted more than one dendrite as well as the
percentage of total axonal contact which was made by such terminals. The mean
length (um) of multiple and single synapses was obtained by tracing the lengths
with the Summagraphics Tablet. The percentage of multiple synapses with

polyribosomes and/or coated vesicles located near the synapse was also determined.

33

Figure 7 - Photomicrographs of the SON dendritic region from an animal that was
conventionally perfused for electron microscopy (A; see Figure 4 for tissue
information) and from a hypothalamic slice (B). In both panels a few of the
dendrites are labeled (D). In A a portion of an astrocyte characteristic of this
region is shown (nucleus labeled ast). Astrocytic processes separate many of the
dendrites (arrowheads) and dendrites in direct apposition are also present (arrows).
Bars = 2 pm.

 

35

Results - Similar to the somatic region the ultrastructural integrity of the SON
dendritic region was quite good. Comparisons between perfused tissue and slice
tissue (Figure 7) did not reveal any obvious differences .in dendritic, synaptic or
astrocytic morphology.

In contrast to the somatic region no significant differences were detected
between the two groups (low vs high osmolality) in three multiple synapse
measures: 1) percentage of dendritic membrane contacted by multiple synapses
(2.39 :l: 0.14 vs. 2.98 :1: 0.44), 2) percentage of terminals that formed multiple
synapses (18.73 :1: 1.57 vs 22.52 i 2.00), 3) percentage of total axonal contact made
by multiple synapses (29.54 i: 2.04 vs 32.82 :1: 7.10). There was a significant
increase in the percentage of multiple synapses that were associated with dendritic
polyribosomes (Figure 8B & 8C) in the high osmolality group (18.09 :I: 3.10)
compared to the low osmolality group (10.21 i 1.60, p < 0.05) while no significant
difference was found in the percentage of multiple synapses associated with coated
vesicles (Figure 8A, low osmolality 13.41 i 3.38 vs high osmolality 17.14 :1: 3.44).
There was no significant difference in the dendritic bundling measure (Figure 9),
i.e., the percentage of dendritic membrane apposed to adjacent dendrites (low
osmolality 9.72 :i: 0.85 vs high osmolality 9.59 :1: 0.58). No significant differences
were found in the remaining morphometric measures (low vs high osmolality): 1)
mean length (pm) of multiple synapses (0.74 :l: 0.01 vs 0.72 :l: 0.02), 2) mean
length (urn) of single synapses (0.87 :1: 0.05 vs 0.83 :l: 0.02), 3) percentage of
dendritic membrane contacted by single synapses (5.74 :1: 0.36 vs 6.01 i 0.35).

36

Figure 8 - Cellular organelles associated with synapse formation. In A an axonal
terminal (*) is apposed to an SON soma (S) and a dendrite (D). The long thin
arrow points to a coated vesicle that is fused at the dendritic membrane immediately
adjacent to the axonal terminal. Portions of the dendritic and somatic membranes
are in direct apposition (large arrow). Bar = 0.5 pm. In B an axonal terminal (*)
is apposed to two dendrites (D). Long arrows point to polyribosomal clusters
subjacent to the terminal apposition. Bar = 0.5 um In C a large cluster of
polyribosomes is present immediately subjacent to the axonal terminal (*). The
terminal is also apposed to an SON soma (S) that is largely out of the
photomicrograph. Bar = 0.5 pm

37

 

38

 

Figure 9 - Dendritic bundles from a slice that incubated 2.5 hours in high
osmolality medium. A bundle of 3 dendrites (labeled with 1’s) and a bundle of 2
dendrites (labeled with 2’s) are indicated. Direct membrane appositions are
indicated by the arrows. Thin astrocytic processes (arrowheads) separate adjacent
dendrites from the bundles. Bar = 1 pm

39

Conclusion

In this experiment two changes that are seen i_n vi_vo during osmotic challenge,
an increase in average firing rate of MNCs and the formation of somatic multiple
synapses, were also seen in the slices incubated in high osmolality medium. These
results, together with previous i_n yitro electrophysiological studies, demonstrate that
osmotic challenge can be an effective i_n \_Ii_tLQ stimulus and suggest that the in _v_itLQ
slice preparation can be used to investigate further the mechanisms by which new

multiple synapses are formed in the SON during osmotic challenge.

L”.u~;ll- 0! IL I!!,'i_la,._\ j 3-’

An important advantage of using an i_n Litre preparation is the ability to control
precisely the duration of the experimental treatment, in this case exposure to the
high osmolality medium. A time course experiment utilizing the slice preparation
was designed to determine if the sequence of anatomical events in somatic multiple
synapse formation could be elucidated. A constant perifusion used for
electrophysiological studies (e.g. Yang & Hatton, 1989) was used for this
experiment. The slice is placed ventral surface down on a small piece of filter
paper that is embedded in bone wax. Small strands of cotton gauze are laid on top
of the slice to facilitate fluid movement over the slice and fresh oxygenated medium
is constantly perifused over and around the slice for the entire incubation period.
One advantage of this chamber is that solutions can be changed rapidly. In this
experiment slices equilibrated in the low osmolality medium for 1.5 hours in an
attempt to establish a common baseline for all the slices. Approximately half of the
slices had their medium switched to high osmolality for four hours while the
remaining slices were constantly perifused with low osmolality medium.

In order to determine whether data obtained in this chamber would replicate
the results from Experiment I counts of multiple synapses were made while the
tissue was viewed on the electron microscope. The percentage of terminals that
formed multiple somatic synapses was calculated by examining temtinals apposed to
SON somata. As in Experiment I, when a terminal also apposed an adjacent soma
or dendrite a multiple synapse was recorded. For each slice 100 terminals were

classified as single or multiple synapses.

41
32511115

A total of 16 animals were used for this study. All slices incubated for 5.5
hours. Two slices were eliminated since no electrophysiological responses were
obtained. The remaining 14 slices had action potentials which were either
spontaneous or elicited in response to electrode advancement. Relative to slices
incubated in the static bath chamber, slices incubated in the perifusion chamber
seemed to yield less stable recordings, although this is difficult to assess
quantitatively since twice as many slices were prepared for the static bath in
Experiment I. Only four cells were spontaneously active for at least 2 1/2 minutes,
two in the low osmolality medium (12.5 and 7.7 Hz.) and two in the high
osmolality medium (2.7 and 3.7 Hz).

Examination of semi-thin sections showed that eight slices (n=3 low osmolality,
n=5 high osmolality) had adequate tissue morphology for electron microscopic
evaluation. In general, and based in part on slices not included in the present
experiments, excellent tissue quality was more difficult to obtain in the perifusion
chamber relative to the static bath. This was most obvious at the ventral surface of
SON which was often extensively vacuolated. It is likely that some of this damage
was due to placement of gauze on the dorsal surface of the slice (near but not on
SON) which added additional pressure to the ventral surface of the tissue.
Nonetheless, as shown in Figure 10, adequate tissue preservation for morphometric
analysis was possible with the perifusion chamber.

In contrast to the results from Experiment I, there was not a detectable increase
in the percentage of synapses that were multiple synapses between the two groups

(low osmolality, 14.3 :i: 1.2 vs. high osmolality 13.6 i 1.2, p > 0.05). An average

42

of 7.9 :i: 1.1 of the synapses from slices incubated in low osmolality medium were
multiple synapses in Experiment I. This is significantly lower than the percentage
obtained in this experiment (p < 0.01). No difference between the chambers was
detected in the slices incubated in high osmolality medium (p > 0.05).
Qonolosion

Two methodological differences existed between Experiments I and II. First
and obviously was the different chamber and second was the pre-incubation of all
slices in low osmolality medium for 1.5 hours prior to incubation in high osmolality
medium. The significant increase in multiple synapses in slices from the perifusion
chamber relative to those from the static bath suggests that synapses formed in both
groups in the perifusion chamber regardless of osmolality. Since the results from
the static bath chamber more closely followed the Ln mo situation, i.e. an osmotic

induction of multiple synapses, the static bath chamber was used for the time course

experiment.

43

Figure 10 - Photomicrographs of hypothalamic slices incubated in the perifusion
chamber. In A the general characteristics of the somatic region of SON are shown.
Portions of two somata (S, one nucleus is labeled) are shown with several dendrites
(D) in the center of the field. Axonal terminals form single somatic synapses (*)
and two terminals form dendro-dendritic (**) multiple synapses. Bar = 2.0 urn. In
B dendrites (D) from the dendritic region are shown. Two dendritic bundles of 2
dendrites (labeled 1 and 2) as well as a bundle of 4 (labeled with 3’s) are in the
field. Axonal terminals forming single (*) and multiple synapses (**) are indicated.
Bar = 1 pm

 

Figure 10

’R-ltltl I REF 12' FOORMATI

The methodological procedure for this experiment was essentially identical to
that used for Experiment I, with the exception that a new chamber with two inner
wells was built. This allowed slices from two animals to incubate simultaneously in
two separate media. The animals used on a given day were usually siblings. Each
day both low and high osmolality medium were provided coded. Slices incubated
continuously for 5.5 hours, 2.5 hours or 1 hour in either the low or high osmolality
medium. Single unit recordings were obtained from the 5.5 hour and 2.5 hour
slices only. For all but two slices medium samples were taken from the incubation
chamber at the end of the experiment. Samples were frozen until the osmotic
pressure of the solution was determined at a later date.

In addition to the morphometric measures discussed in the General Methods
section the following measures were also made: 1) the average length (um) and
number of direct soma-somatic/dendritic appositions per 100 pm of somatic
membrane, 2) the percentage of soma-dendritic multiple synapses associated with
dendritic polyribosomes and, 3) the percentage of multiple synapses with coated
vesicles in the post-synaptic element.

Results

A total of 80 animals used in this experiment were equally divided into low
and high osmolality groups (32 slices incubated for 5.5 hours, 28 slices for 2.5
hours and 10 slices for 1 hour). Four slices were eliminated from the 5.5 hour
group due to technical difficulties during the incubation time.

All slices in the 5.5 hour and 2.5 hour groups had single unit activity, either

spontaneous or elicited by electrode advancement. Of the 17 cells recorded, six

45

46
were recorded from high osmolality slices, three in each time period. The
remaining 11 cells were recorded from slices in the low osmolality group (five from
the 5.5 hour group and six from the 2.5 hour group). There was no detectable
increase in the average firing rates (Hz) of cells from slices incubated in the high
osmolality medium (6.6 :t 0.6) compared to cells from the low osmolality medium
(7.6 :l: 1.1).

A total of 38 slices were used for electron microscopy which included 14
slices from the 5.5 hour group (low osmolality, n = 8; high osmolality n = 6), 12
slices in the 2.5 hour and 12 slices in the 1 hour incubation groups were equally
distributed between low and high osmolality groups (i.e. 6 each). Two slices, one
from the 2.5 hour high osmolality group and one from the 1.0 hour low osmolality
group were not included in any of the analyses since they were statistical outliers
(Dixon & Massey, 1969) on the multiple synapse measures, although their values on
the other morphometric measures were not extreme. The high osmolality, 2.5 hour
slice had no multiple synapses; a situation not encountered in any of the slices
included in these experiments and also never seen in perfused control animals
analyzed with an identical sampling and morphometric method (data from Modney
& Hatton, 1989). The 1.0 hour slice had an enormous number of multiple synapses
(28%), a value not seen even in chronically dehydrated animals in 31129 (high value
fiom Modney & Hatton, 1989 was 22%).

Analysis of the samples taken from the incubation chambers revealed that the
osmotic pressure of the chamber media was not necessarily 290 mOsm/kg or 340
mOsm/kg. In the 5.5 hour group, the average osmotic pressure was 272.8 :1: 12.9 in
the low osmolality slices and 308.3 :1: 18.8 in the high osmolality slices, values that

47

 

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48
were not significantly different. In the 2.5 and 1.0 hour groups the average osmotic
pressure of low osmolality slices was 299.1 :1: 4.1 and 292.4 i 5.3 respectively and
337.7 :1: 5.7 (2.5 hour) and 334.3 :1: 8.0 (1 hour) for the high osmolality slices. In
the 2.5 hour and the 1 hour groups the high osmolality group media osmotic
pressures were significantly higher than the 1 hour group.

Given the variability in the osmotic pressure of the incubation medium
samples, particularly in the 5.5 hour groups, it was important to know if the
osmotic pressure at the end of the incubation time was significantly correlated with
the major dependent variables (i.e. multiple synapse measures, percentage of somatic
membrane contacted by adjacent somatic membrane). This was especially true since
the results of Experiment I suggested that an increase in multiple synapses occurs
with increased osmolality. The four multiple synapse measures and the percentage
of somatic membrane contacted by adjacent somata or dendrites were not
significantly correlated with the osmotic pressure of the incubation media (see Table
2). This was true if all 36 slices were used to calculate the correlation coefficient
or if the correlations were calculated with only the slices from a single time period.

The ANOVAs did not reveal a main effect of osmolality on any of the
dependent variables, nor were there any significant interactions between incubation
time and osmolality. A complete summary of the AN OVAs can be found in Table
3 and group means for each measure are listed in Table 4.

As shown in Figures 11 and 12 three of the four multiple synapse measures
increased over time, the exception being the percentage of somatic membrane
contacted by multiple synapses (p < 0.07). Increases in these measures were
detectable by 2.5 hours with no further increases exhibited at the 5.5 hour time

period. Significant decreases in cell area and cell perimeter (Figure 13) occurred

49
TABLE - 3 ANOVA TABLES EXPERIMENT IH

PER NTA E F MATI MEMBRANE NTA D
B! GLIAL PREESSES

SOURCE df MEAN SQUARE F-test p
Osmolality l 0.55 0.73 0.788
Time 2 28.43 3.77 0.034
Interaction 2 17.34 2.30 0.1 17
Error 30 7.53

ER NT A F MATT MEMB NE NTA D B

AD A ENT DENDR MN MA
SOURCE df MEAN SQUARE F-test p
Osmolality 1 0.10 0.13 0.909
Time 2 5.45 0.70 0.503
Interaction 2 12.81 1.65 0.209
Error 30 7.76
ER NTA E F MATI MEMBRANE NTA D
B! SINGLE SYNAPSES

SOURCE df MEAN SQUARE F-test p
Osmolality l 0.66 0.17 0.683
Time 2 2.04 0.53 0.597
Interaction 2 1.20 0.31 0.635
Error 30 8.44

PER NTA E F MATI MEMBRANE NTA D
BY MULTIPLE SYNAPSES

SOURCE df MEAN SQUARE F-test p
Osmolality 1 32915-3 8.2E-3 0.928
Time 2 1.12 2.85 0.074
Interaction 2 1.16 1.47 0.244
Error 30 0.39

PER NTA E F MATI MEMBRANE NTA D BY
UNIDENTIFIED ELEMENTS

SOURCE df MEAN SQUARE F-test p
Osmolality l 0.32 0.95 0.761
Time 2 3.12 9.11 8.0E-4
Interaction 2 0.27 0.79 0.42
Error 30 0.34

50
TABLE 3 (Cont’d.)

kl 1: ' 0 , IN L YNA-’ _ _ ' ’ 1U it 0 0 ‘ MEMB ' ‘
SOURCE df MEAN SQUARE F-test p
Osmolality l 0.31 0.12 0.725
Time 2 2.07 0.84 0.439
Interaction 2 0.48 0.20 0.822
Error 30 2.45
WWW
SOURCE df MEAN SQUARE F-test p
Osmolality 1 3. lE-4 1.3E-3 0.971
Time 2 1.14 4.98 0.013
Interaction 2 0.29 1.26 0.298
Error 30 0.23

ER F MA- MATI ENDRITI MEMBRANE APP ITI N
PER 11!) gm OF SOMATIC MEMBRANE

SOURCE df MEAN SQUARE F-test p
Osmolality 1 0.60 0.41 0.528
Time 2 2.71 1.83 0.178
Interaction 2 2.63 1.77 0.187
Error 30 1.48

E NTA E F AX NA NTA MADE B LE YNAP E
SOURCE df MEAN SQUARE F-test p
Osmolality 1 0.62 0.02 0.884
Time 2 103.89 3.61 0.039
Interaction 2 46.60 1.62 0.214
Error 30 28.74

ER ENTA E F YNAP E T WERE TIP

SOURCE df MEAN SQUARE F-test p
Osmolality l 0.15 6.7E-3 0.935
Time 2 118.40 5.25 0.011
Interaction 2 33.64 1.49 0.241

Error 30 22.56

51
TABLE 3 (Cont’d.)

AN L N IN N E
SOURCE df MEAN SQUARE F-test p
Osmolality 1 9.0E-4 0.07 0.791
Time 2 0.01 1.09 0.349
Interaction 2 3.6E-3 0.28 0.745
Error 30 0.01
MEAN LENGTH (um) QE MUEIIIBLE SYNAPSES

SOURCE df MEAN SQUARE F-test p
Osmolality 1 0.01 0.19 0.664
Time 2 6.3E-4 0.01 0.988
Interaction 2 0.14 2.58 0.093
Error 30 0.05

flAN LENGTH (3W
SOURCE df MEAN SQUARE F-test p
Osmolality l 0.12 0.67 0.420
Time 2 0.18 1.02 0.374
Interaction 2 0.29 1.61 0.216
Error 30 0.18

Wm)
SOURCE df MEAN SQUARE F-test p
Osmolality 1 4.86 0.48 0.495
Time 2 49.33 4.85 0.015
Interaction 2 0. 15 0.02 0.985
Error 30 10.17
CELLAREAJum’)
SOURCE df MEAN SQUARE F-test p
Osmolality 1 25.58 0.05 0.830
Time 2 9031.71 16.51 1.0E-4
Interaction 2 325.61 0.59 0.558
Error 30 547.02
CELL SMPE

SOURCE df MEAN SQUARE F-test p
Osmolality 1 1.4E-3 1.13 0.296
Time 2 5.6E-6 4.54 0.019
Interaction 2 2.0E-2 1.63 0.212
Error 30 1.2E-3

52

TABLE 4 — GROUP MEANS AND STANDARD ERRORS
FOR EXPERIMENT III

 

 

 

 

ER ENTA F ME B NTA D
B LIAL PR E

INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
Low 86.37 1 0.22 82.99 1 1.50 84.02 1 0.60 84.31 1 0.55
HIGH 85.50 1 1.30 85.41 1 0.94 81.72 1 1.10 84.14 1 0.77
TOTAL 85.89 1 0.87 84.09 1 0.96 83.04 1 0.75 84.23 1 0.50

PER NTA E F MATT MEMBRANE NTA D BY
AD NT DEND MN

INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
Low 3.73 1 0.50 6.52 1 1.65 4.75 1 0.67 5.04 1 0.63
HIGH 4.58 1 1.15 3.99 1 0.47 6.09 1 1.60 4.94 1 0.70
TOTAL 4.19 1 0.65 5.37 1 0.97 5.32 1 0.77 4.99 1 0.46

PERCENTAGE OF soMATIo MEMBRANE
CONTACED BY eroLE sYNAPsEs

INCUBATION TTME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
Low 9.07 1 0.99 8.25 1 0.75 8.69 1 0.71 8.65 1 0.41
HIGH 8.76 1 1.10 8.45 1 0.71 9.62 1 0.66 8.97 1 0.48
TOTAL 8.90 1 0.71 8.34 1 0.50 9.09 1 0.45 8.80 1 0.31

ER NTA F MATT MEMBRA

NTA D B LE YNAP E

INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
Low 0.74 1 0.25 1.74 1 0.34 1.35 1 0.20 1.31 1 0.17
HIGH 1.10 1 0.94 1.23 1 0.12 1.55 1 0.25 1.30 1 0.11
TOTAL 0.94 1 0.20 1.51 1 0.20 1.44 1 0.15 1.31 1 0.11

53
TABLE 4 (Cont’d.)

 

ER NTA E F MA MBRA NTA D BY
UNIDEN IIEED ELEMEN IS
INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 0.09 :l: 0.09 0.51 :l: 0.25 1.18 i 0.27 0.68 :1: 0.17
HIGH 0.06 :t 0.06 0.91 :1: 0.34 0.99 :i: 0.25 0.64 :t 0.16
TOTAL 0.08 :1: 0.05 0.69 :l: 0.20* 1.10 :t 0.18* 0.66 i 0.12

* p < 0.05 compared to 1 hour

 

E F LE YN P E PER F MATI MEMBRA
INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 7.38 i 0.80 6.87 i 0.47 7.55 :1: 0.46 7.29 :1: 0.31
HIGH 7.11 i 0.93 7.18 :l: 0.47 8.07 i 0.63 7.47 i 0.41
TOTAL 7.23 :t 0.60 7.01 :I: 0.32 7.78 :t 0.37 7.38 i 0.25

NUMBER OF MULTIPLE SYNAPSES PER 113) gm F MATI MEMBRANE
INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL

LOW 0.72 :t 0.21 1.51 :l: 0.21 1.26 :l: 0.18 1.20 :l: 0.13
HIGH 0.89 :l: 0.24 1.15 i 0.06 1.47 i 0.18 1.17 :l: 0.12
TOTAL 0.81 i 0.16 1.34 :i: 0.12* 1.35 :l: 0.12* 1.18 :1: 0.12

p < 0.05 compared to 1.0 hour

urn: '0 DIRE 011‘ 0.11.1.1 MENDR 1". HON-
EERImngESQMAflLI/W

INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 2.55 :1: 0.37 3.93 :1: 0.74 2.76 :l: 0.37 3.07 :l: 0.31
HIGH 2.25 :1: 0.33 2.76 :l: 0.25 3.45 :l: 0.66 2.80 :1: 0.29

TOTAL 2.40 :l: 0.24 3.40 :l: 0.44 3.06 :1: 0.31 2.95 i 0.21

54
TABLE 4 (Cont’d.)

 

ER NTA F AX NAL ONTA MADE B TIPLE YNAP E
INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 7.35 :l: 1.92 17.29 :t 2.74 13.85 :1: 2.01 13.23 :1: 1.53
HIGH 11.10 i 2.68 12.79 i 0.89 13.79 :1: 1.85 12.55 :1: 1.14
TOTAL 9.40 :1: 1.75 15.25 :t 1.65* 13.82 :1: 1.36 12.91 :1: 0.96

*p < 0.05 compared to 1 hour

 

ER ENTA E YN T RE M TIPLE
INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 8.62 :1: 1.87 17.98 :1: 2.04 14.43 :1: 1.82 14.02 i 1.35
HIGH 11.15 :t 2.60 13.91 :1: 0.87 15.57 i 1.60 13.52 :1: 1.14
TOTAL 10.00 i 1.62 16.13 :1: 1.30”“ 14.92 :1: 1.22* 13.79 :1: 0.88

*p < 0.05 compared to 1 hour

MEAN LENGTH (Em) GF SINGLE SYNAPSES
INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL

LOW 1.24 :1: 0.08 1.19 i 0.04 1.15 i 0.03 1.19 :1: 0.03
HIGH 1.24 :1: 0.03 1.17 :1: 0.05 1.20 :1: 0.04 1.21 :1: 0.02
TOTAL 1.24 i 0.04 1.18 i 0.03 1.17 i 0.03 1.20 :1: 0.02

MN LENGTH (Em) GF MULTIPLE SYNAPSES
INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR TOTAL
LOW 0.95 :1: 0.14 1.12 :1: 0.04 1.16 :l: 0.12 1.09 :1: 0.06
HIGH 1.23 :1: 0.05 1.07 :1: 0.06 1.04 :1: 0.07 1.11 :1: 0.04

TOTAL 1.10 :1: 0.04 1.09 :1: 0.05 1.11 i 0.07 1.10 :1: 0.04

55
TABLE 4 (Cont’d.)

MEAN LENGTH (gm) GF SGMA-SGMATIG/DENDRIIIG

MEMBRANE APPGSIIIGNS
INCUBATION TIME

 

OSMO 1.0 HR 2.5 HR 5.5 HR
LOW 1.48 :1: 0.12 1.55 :1: 0.13 1.75 :1: 0.18
HIGH 1.97 :1: 0.24 1.44 i 0.14 1.70 :l: 0.15
TOTAL 1.75 :1: 0.16 1.50 :1: 0.09 1.72 :l: 0.12
Wm.)
INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR
LOW 68.0 :1: 1.6 65.8 :1: 1.3 63.9 :1: 0.9
HIGH 68.8 :1: 1.0 66.3 :t 1.1 64.8 :1: 1.8
TOTAL 68.4 :1: 0.09 66.1 :1: 0.8 64.3 :1: 1.0*

*p < 0.05 compared to 1 hour
ELL AREA r_n_2)

INCUBATION TIME

OSMO 1.0 HR 2.5 HR 5.5 HR
LOW 316.0 :1: 11.3 294.1 :1: 13.5 271.9 :1: 7.0
HIGH 327.4 :1: 10.5 296.8 :1: 9.8 262.8 :1: 4.1
TOTAL 322.2 :1: 7.5 295.6 i 8.1* 268.0 :1: 4.4"‘#

*p < 0.05 compared to 1 hour
#p < 0.05 compared to 2.5 hour

LL HAPE

INCUBATION TIME
OSMO 1.0 HR 2.5 HR 5.5 HR

LOW 0.849 :t 0.013 0.850 i 0.011 0.834 i 0.011
HIGH 0.860 :1: 0.009 0.840 :1: 0.003 0.794 :1: 0.026

TOTAL 0.855 1 0.008 0.846 :l: 0.006 0.817 :1: 0.013

TOTAL

1.61 :1: 0.09
1.72 :1: 0.11

1.66 i 0.07

TOTAL

65.6 :1: 0.77
66.7 :1: 0.86

66.1 :1: 0.57

TOTAL

290.5 :1: 7.0
295.3 :1: 8.2

292.9 :1: 5.3

TOTAL

0.843 :1: .006
0.831 :1: .012

0.837 d: .006

56

with increased incubation time. Cell perimeter did not decrease significantly until
5.5 hours while cell area was decreased by 2.5 hour and a further significant
decrease occurred by 5.5 hours. A significant decrease in the cell shape factor was
found between the 1 hour and the 5.5 hour groups suggesting that cells were not
shrinking in a uniform manner. The percentage of somatic membrane contacted by
glial processes decreased between the 1 hour and the 5.5 hour groups and an
increase in the percentage of membrane contacted by unidentified elements occurred
by 2.5 hours.

Correlation coefficients were calculated to determine the relationships among
several dependent variables. Given that cell size can alter the interpretation of
morphometric data based on the somatic membrane (e. g. # of multiple synapses per
100 um of somatic membrane) and that cell size decreased over time in this
experiment, correlation coefficients were determined between cell perimeter and the
multiple synapse measures based on somatic membrane. Cell perimeter was used as
the most meaningful variable in this instance since synapses are measured along the
perimeter of the cell. Contrary to the expected negative correlation between cell
size and multiple synapses, there was no significant correlation between the
percentage of somatic membrane contacted by multiple synapses and cell perimeter
(r = - 0.08, p > 0.05) or the number of multiple synapses per 100 pm of somatic
membrane and cell perimeter (r = - 0.18, p > 0.05). The second relationship of
interest, between direct soma-somatic/dendritic apposition and multiple synapses, did
not correlate significantly (r = 0.15, p > 0.05).

Data collected on the percentage of multiple synapses with dendritic

polyribosomes and coated vesicles were not appropriate for statistical analysis due to

PERCENT

57

1:] IHOUR
2.5 HOUR

 

 

 

151

*#
m, T
51

 

 

 

 

 

 

 

 

SYN APSES AXON AL CONTACT

Figure 11 - Comparisons of the percentage of synapses that were
multiple and the percentage of total axonal contact made by
multiple synapses. Significant increases over time were found
on both measures.

* p < 0.05 compared to 5.5 hour
# p < 0.05 compared to 2.5 hour

#OF MULTIPLE SYNAPSES PER 100nm

OF SOMATIC MEMBRANE

58

|___] IHOUR
2.5 HOUR

  

   

 

 

 

 

 

 
 

 

 

2.01 2.0 -
5.5 HOUR
m
1.5 . z 1.5 4
<1:
or
m
2
u:
2
1.0- U 1.0 -
1~
<1:
2
O
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8°
0.5 - 0.5 -
INCUBATION TIME INCUBATION TIME

Figure 12 - Multiple synapse measures for the three incubation times.
The percentage of somatic membrane contacted by multiple synapses
did not reach the p < 0.05 significance level (p < 0.07). In contrast,
the number of multiple synapses per 100 pm of somatic membrane
did increase signficantly.

* p < 0.05 compared to 5.5 hour
# p < 0.05 compared to 2.5 hour

59
the presence of numerous 0’s and extreme variability. This is likely to be a
sampling problem since the number of multiple synapses from a given animal is
fairly small. Further partitioning into groups of this small number results in the
extreme variability. These data are presented in Figure 14 which is based on all the

multiple synapses within each time period, not the group means.

AREA(11m 2)

PERIMETERUtm)

8

 

 

 

 

 

 

 

 

 

 

 

[:1 1 HOUR
2.5 HOUR
2:1: 5.5 HOUR
350-
* #
fi—
*
3m-1
//
250- /
/2
70 ‘ *
“T—
fi—
V
60 /
9 fl

 

 

 

 

 

 

 

INCUB ATION TIME

Figure 13 - Comparisons of cell area and perimeter over the three
incubation times. Both measures decreased over time.

* P < 0.05 compared to 5.5 hour
# p < 0.05 compared to 2.5 hour

%%
DE

 

 

 

 

 

 

_

2.5 5.
INCUBATION TIME

 

 

 

 

 

 

mmmmmmm

napses associated with de

- The percenta e of multi 1e 8

Figure 14

8 P Y .
(pr) and coated vesicles (cv). No obvrous or consrstent
n the incubation times, erha s due to extreme

polyribosomes
differenc

es were 8 over
variability between animals.

DISCUSSION

An enduring question for neurobiologists concerns the mechanisms by which
specific neural connections are formed and modified over time. Among the
multitude of issues raised by this broad query are how, and under what conditions
synapses form? As with most questions in neuroscience, a variety of techniques
and approaches have been used to further our understanding of synaptogenesis. The
electron microscopic evaluation of neural tissue from cultures, developing and adult
animals has provided a wealth of information concerning the characteristics of, and
conditions under which new synapses form. An important general finding of the
present work is that hypothalamic slices are capable of initiating synapse formation
in a relatively short period of time. As with previous i_n m work, synaptogenesis
in the SON i_n flit) appears to be a specific response since multiple synapses
increase without concurrent increases in the single synapse morphometric measures.

In Experiment I the increased osmolality of the medium was clearly associated
with changes in MNCs that are similar to those seen with dehydration io v_iv_o.
Than MNCs alter their electrophysiological characteristics during i_n m osmotic
manipulations is well documented (Bourque, 1989; Bourque & Renaud, 1984; Hatton
et al., 1978; Mason, 1980). In the present study the 50% increase in firing rate
supports the contention that these cells can respond to osmotic challenges
administered i_n firm. This contention is further supported by the obtained increase
in somatic multiple synapses in high osmolality slices. The formation of multiple
synapses in response to osmotic challenges administered in yi_vo is well-established
(Chapman, et al., 1986; Modney & Hatton, 1989; Tweedle & Hatton, 1984a), this

experiment is the first report of synapses forming in m in response to osmotic

62

63

stimulation. In contrast to data obtained from the somatic region there was no
detectable increase in multiple synapses in the dendritic region of SON. This is in
contrast to the results of Tweedle et al. (1989) who found significant increases in
dendritic multiple synapses following transcardial perfusion with hypertonic
solutions. However, chronic dehydration produced by 2% saline drinking for 10
days does not result in the formation of dendritic multiple synapses (Perlmutter et
al., 1985). Thus the relationship between dendritic multiple synapses and osmotic
activation of MNCs is unclear. With regard to the present i_n_ m data, it is
possible that synapse formation requires an intact afferent input, i.e., the terminals
that form multiple synapses may have to remain connected to their cell bodies. In
this respect, input to the dendritic region may come from afferent systems that are
no longer intact in the slice, while input to the somatic region, which could come
from closer perinuclear areas, would remain intact. Until more information is
obtained concerning the precise termination of particular afferent systems, as well as
information concerning the ability of distal severed axons to form synapses, this
contention remains a hypothesis.

In Experiment 11 and 111, there was no detectable relationship between the
medium osmolality and the various dependent variables. In Experiment IH there
was a significant increase over time in several of the multiple synapses measures,
with no corresponding increase in the single synapse measures. These data support
a previous i_n_ yilo study (see below) that demonstrated that multiple synapses form

rapidly (within 24 hours) in response to deafferentation.

l ' n i ' 11
Wow — Since the primary goal of these experiments was to

analyze quantitatively the morphological characteristics of MNCs from slices, several
assumptions and issues should be considered when interpreting these data. Synapses
were inferred any time an axonal terminal apposed a post-synaptic structure
regardless of whether synaptic specializations were present. Serial section analyses
of SON synapses in m (Modney & Hatton, 1989; Smithson & Hatton, 1990) have
revealed that 90% of the terminals that contact a post-synaptic structure form a
conventional synapse. It is possible that a similar situation exists 'm y_it_ro. The
data collected here show an increase in the number of terminals apposed to more
than one post-synaptic element. This has been interpreted as supporting the
statement that multiple synapses have formed. A more extensive sampling
procedure, and/or serial sections would be necessary to make this statement
definitive.

Several of the measures made are sensitive to changes in the size of the
measured cellular element. With regard to terminals that form multiple synapses,
their size can alter their apparent frequency (larger terminals would appear more
often in random sections). In the present experiments no detectable changes in the
apposition length between terminals and somata for either single or multiple
synapses was found. Thus, the increased number of multiple synapses is not due to
changes in their size. A similar principle applies to cell size which could influence
the number and/or percentage of somatic membrane contacted by synapses. The
present results support the statement that multiple synapses are actually being

formed io v_igo regardless of decreases in cell size (which could lead to more

65

synapses/100 1.1m without actually increasing the number of synapses). Three
arguments support this conclusion: 1) in Experiment I there was not a detectable
change in cell size; 2) if cell size was influencing the number of synapses or the
percentage of somatic membrane contacted by synapses this should also be detected
in the single synapse measures. No changes were seen in these measures in
Experiments 1 and III. 3) In Experiment 111 there was no correlation between cell
size and the multiple synapse measures.

Exmriment I vs Experiment III - In Experiment I multiple synapses formed in
response to an osmotic manipulation while in Experiments II and 111 no osmotic
effect was detected. This discrepancy between the experiments is difficult to
explain. Methodologically experiments I and III appear identical, although there
were minor changes in the actual preparation and location of the Experiments.
Experiment I was done in an electrophysiology set-up in the basement of the
building while Experiments II and 111 used an electrophysiology set-up on the
second floor. In terms of the most of the experimental equipment (except the
Vibratome, see below) there were no significant variations between these two
electrophysiology setups. There may have been slight variations in the ambient
temperature and humidity between these locations, however, it is difficult to see
how these relatively minor changes could alter the pattern of results obtained. A
second difference between the experiments was the Vibratome that was used to cut
the slices. In Experiment I an old Vibratome that was often "finicky" was used.
While the slice thickness was nominally 500-600 pm the control for thickness did
not always raise the stage so that slices were often cut by visual approximation.

Slices in Experiment 11 and III were cut with a new Vibratome that cut and

66

advanced quite consistently (nominally 550 um). Any difference between the two
groups in slice thickness could alter the extent and/or pattern of deafferentation
between slices in the different experiments. At this point there is no way to
determine which, if any, of these possibilities resulted in the discrepancy among the
three experiments.

It is possible that the lack of an osmotic effect in Experiment III was due to
technical problems with the incubation medium which was clearly not accurate in
the 5.5 hour group. In general the measured osmolality of the medium for the 5.5
hour groups was lower than anticipated, however this was not universally true since
some measures were quite reasonable (e.g. 341 mOsm/kg). It may be that had the
medium really been 340 mOsm/kg for 5.5 hours multiple synapses would have
increased more than the low osmolality slices. While this is a distinct possibility,
the values from both the 2.5 hour groups are already quite high (percent synapses
multiple = 16.1) relative to values from Experiment I (high osmolality = 11.6) and
values from chronically dehydrated rats i_n \_Ii_\Lo (17.4, data from Modney & Hatton,
1989). Evidence from io v_ilo studies suggest that there is a ceiling for the number
of multiple synapses that form with prolonged lactation (Montagnese et al., 1987), it
is not known if a similar ceiling exists 'm mo. Since no significant increase in
multiple synapses occurred between the 2.5 hour and the 5.5 hour groups this
ceiling may have been reached by 2.5 hour io mo. The lack of significant
correlations between osmotic pressure and the multiple synapse measures also argues
against an osmotically induced increase in these synapses in Experiment 111. It
seem likely that whatever induced synapse formation in Experiment 111, most likely

a reactive process, prevented the detection of an osmotic effect.

67

In addition to the multiple synapse measures the cell size measure were quite a
bit lower in Experiment III compared to Experiment I and io yi_\;o values (5.5 hour
cell area (um’) 268 i 4.0; Experiment I low osmolality = 431 :1: 13; in mo control
males = 380 :l: 21). Since the size of post-synaptic elements in some neural
systems is clearly dependent on intact afferent input (e.g. Rubel, Hyson & Durham,
1990) the relatively smaller cells in Experiment III could mean that these cells
underwent more deafferentation that the slices from Experiment 1.

El 1 i l ' n f

While ultrastructural studies of brain slices are relatively rare compared to their
use for electrophysiological studies they have provided some important information
concerning the slice preparation. The extent to which these preparations correspond
to the i_n m situation can only be determined by continued investigations using
both in yiyo and io yjtgz approaches. In the case of the SON the present
experiments demonstrated that synapse formation can occur very quickly io m.
The notion that i_n v_itm preparations may be a useful tool for combining anatomical
and electrophysiological studies is certainly not new.

Most electron microscopic investigations of slices have emphasized the general
ultrastructural integrity of the tissue over varying incubation times and its
similarities to perfused tissue of the same brain region (see Table 5). Several
studies have attempted to correlate electrophysiological and/or biochemical measures
with the morphological characteristics of the slice. In general electrical activity is
associated with good ultrastructural preservation (Bak, Misgeld, Weiler & Morgan,
1980; Frotscher, Misgeld & Nitsch, 1980; Yamamoto, Bak, & Kurokawa, 1970).

Similarly, a good correlation between tissue preservation and stimulated cGMP

 

68

TABLE 5 - ELECTRON MICROSCOPIC INVESTIGATIONS
OF BRAIN SLICES

Sli E .

Cerebellum
Cortical Areas

Lateral Geniculate

Medulla/Pons

Hippocampus

Hypothalamus

° not explicitly given

Incubation

Time
2 hours
5 hours

5 hours

2 hours

10 hours

5 hours
10 hours
8 hours
4 hours
1.5 hours

2.5 hours’

2.0 hours'
8.0 hours
4.0 hours’

5.5 hours

12 hours

9 hours

0.5 hours

Source

Garthwaite, Woodhams, Collins &
Balazs, 1979

Bak, Misgeld, Weiler & Morgan,
1980

Yamamoto, Bak & Kurokawa,
1970

1 4,le
p

Pitkanen, Korpi & Oja, 1985

 

Crunelli, Leresch, Hyde, Patel & I
Pamaveles, 1987
Hinrichsen, 1980 i

Chang & Greenough, 1984
Frotscher & Misgeld, 1989
Frotscher, Misgeld & Nitsch, 1981
Jensen & Harris, 1989

Lee, Oliver, Schottler & Lynch,
1981

Meshul & Hopkins, 1990
Misgeld & Frotscher, 1982
Petukhov & POpov, 1986

Reid, Schurr, Tseng & Edmonds,
1984

Schurr, Reid, Tseng & Edmonds,
1984

Hatton, Doran, Salm & Tweedle,
1980

Pow & Morris, 1990

69
production was found in cerebellar slices (Garthwaite, Woodhams, Collins & Balaz,
1979).

Two general findings of some of the investigations listed in Table 5 were
also observed in the present study and deserve comment. First, qualitatively, there
is a general decline in the ultrastructural integrity of slices with increased incubation
time. In the studies presented here this is most clearly demonstrated in Experiment
111 where the percentage of slices used for electron microscopic analysis increased
with decreasing incubation time, particularly between the 1 hour and 5.5 hour
groups. This does not necessarily imply that the best preservation was in the
shortest incubation time, since excellent tissue preservation was obtained with a long
incubation time (e.g Figure 5A which is from a slice that were incubated for 5.5
hours). Perhaps the longer incubation times provide more time for things to go
wrong.

The second general conclusion reached by most slice studies is that tissue
quality is not uniform throughout the slice. When viewed in cross-section two outer
zones generally have poor tissue quality and an inner zone is well preserved. In
most cases these outer layers correspond to the cut edges of the tissue block, one of
which lies on the chamber floor while the other is exposed to air. In this study a
similar pattern was observed. The ventral surface of the brain, which was placed
on the net in the static bath or on filter paper in the perifusion chamber,
consistently contained vacuolated and/or swollen processes and cells. The
orientation of SON in the slice generally prevented the ventral surface of SON from
being placed directly on the chamber floor. Often, portions of the pia-arachnoid

remained attached ventral to SON and perhaps this prevented extensive damage to

 

70

the dendritic zone and glial limitans. Several studies suggest that hypoxia may be
responsible for extensive vacuolation in hippocampal slices (Misgeld & Frotscher,
1982; Reid, Schurr, Tseng & Edmonds, 1984), although controlled studies using
slices from other brain regions have not been performed. The second outer zone,
which corresponds to a cut surface which is usually exposed to air also exhibits
poor preservation, but it is not as extensive as the ventral or downward placed
tissue. This damage is presumably due to the cutting procedure itself and varies
somewhat from slice to slice. In this study the best slices used for morphometric
evaluation had Vibratome cuts that were dorsal to the SON and so the nucleus
proper was not included in the damaged region. Occasionally the most dorsal cells
in the nucleus were slightly damaged but this was not preferentially distributed
among groups nor were there detectable changes in the morphometric measures.
The third zone of the slice is the inner zone which exhibits the best tissue
morphology and corresponds to virtually the entire SON when horizontal slices are
prepared

In contrast to the general ultrastructural characterization of brain slices few
studies have quantitatively investigated changes in morphology with varying
experimental procedures. Using slices of cerebral cortex Pitkanen, Korpi & Oja
(1985) have demonstrated a significant depletion of synaptic vesicles in axonal
terminals when slices are incubated in Na’ free medium (choline was substituted for
Na’ so that medium was isotonic) compared to medium with Na‘. Interestingly, a
significant but not as extensive depletion was also observed in slices that incubated
in the normal Na+ containing medium relative to perfused tissue or tissue that was

immersion fixed immediately after decapitation. This suggests that morphometric

 

71
measures obtained from slices incubated in "normal" slice medium are not
necessarily identical to those observed i_n yiyo.

Several studies have investigated quantitatively morphological changes in
hippocampal slices following the induction of long-term potentiation (LTP). Two
studies (Chang & Greenough, 1984; Lee, Oliver, Schottler & Lynch, 1981) have
provided evidence that LTP induced by stimulation of the lateral-commissural
projection from hippocampal field CA3 to field CA1 results in synapse formation.
The increase in the number of synapses was detectable within 15 minutes of
stimulation, lasted for at least 8 hours, and was not present in control stimulated
slices (Chang & Greenough, 1984). Recently Meshul & Hopkins (1990) have used
this same system i_n mo (i.e. CA3-CA1) to demonstrate ultrastructural changes in
the synaptic vesicle population in terminals, presumably at the potentiated synapses.
In contrast, LTP induced by stimulating the mossy fiber projection from the dentate
to CA3 does not lead to the formation of new synapses but alters the morphological
characteristics (i.e. psd length) of existing synapses (Petukhov & Popov, 1986).

The results of the hippocampal slice studies together with the present
experiments support the view that 31 Geo slice preparations can be used to study
rapid synaptogenesis. In addition the present experiments suggest that i_n v_itro
preparations may be more dynamic than previously thought in terms of their ability
to reorganize in relatively short periods of time.

R 'v R i n F rm ' 11

As argued earlier the results of the present experiments demonstrate that

somatic multiple synapses form rapidly in the SON io m. Such rapid formation

of multiple synapses in SON is not unprecedented since dendritic multiple synapses

72

form within 20 minutes following intense osmotic activation "i_n yi_vo" (T weedle et
al., 1989). In Experiment I osmotic stimulation resulted in somatic but not
dendritic multiple synapses. There was an increase in the percentage of dendritic
multiple synapses associated with polyribosomes in slices incubated in high
osmolality medium. The significance of this is not clear since there was no
detectable increase in the dendritic multiple synapse measures. While the majority
of the literature supports the association between polyribosomes and synapse
formation, there is evidence that they may also be associated with modified existing
synapses (Hwang & Greenough, 1986). Perhaps dendritic multiple synapses were
being modified by the high osmolality medium.

In Experiment III multiple synapses increased over time. It seems likely that
in this experiment synapses were forming in response to deafferentation. A rapid
response to deafferentation in SON has been demonstrated 'm vi_vo. Removal of the
afferent projection from the subfomical organ to the SON by knife cuts or lesions
results in multiple synapse formation within 24 hours, the earliest time point studied
(Weiss et al., 1988). The results of the present experiments suggest that
deafferentation can result in reactive synapse formation even earlier than this 24
hour period, perhaps as early as 1 hour, certainly within 2.5 hours.

Reactive synaptogenesis in neural systems is ubiquitous (see Cotman, Nieto-
Sampedro & Harris, 1981). It is but one part of a complex compensatory
mechanism that is initiated following deafferentation. Synapse replacement, while a
general process, varies in its time course quite a bit from system to system. The
rate at which new synapses form may depend on a number of factors. The extent

of the lesion, the length of the severed distal axons and the rate at which the

 

“awn. .J- I. I!

73

degenerating axons are cleared by glia are thought to influence the rate of synapse
replacement. In most systems reactive synaptogenesis is much slower than the time
course demonstrated here (e.g. 5-7 days). Cotrnan et a1. (1981) postulate that a
major rate limiting step in reactive synaptogenesis is the time it takes to clear away
degenerating terminals. In several systems however new synapses form prior to
complete removal of the damaged fibers, or even before evidence of degeneration is
observed.

Chen and Hillrnan (1982) examined the time course for synapse replacement
following partial deafferentation of Purkinje cells in the cerebellum. As early as 10
hours after deafferentation Purkinje cell spines were often dually innervated, i.e. a
single spine was contacted by two terminal boutons, one of which was apparently
degenerating. Since dual innervation is extremely rare in control animals the
authors conclude that new synapses had formed within 10 hours of the lesion. Dual
innervation of spines was not seen at later time points suggesting that the second
terminal remains after the degenerating terminal is removed. Murray and
Goldberger (1986) report that reactive synaptogenesis after partial deafferentation of
Lamina II in the cat spinal cord was partially complete at the first time point
investigated (2.5 days). No evidence of degeneration was seen at this time
suggesting that it was cleared away rapidly. In a system that is remarkably similar
to the SON in terms of morphological changes Goshgarian and colleagues
(Goshgarian & Yu, 1990; Goshgarian, Yu & Rafols, 1989) have described the rapid
formation of multiple synapses (and dendritic bundles) in the phrenic nucleus.
Within 4 hours of spinal cord hemisection (the first time point studied) multiple

synapses were significantly elevated relative to control animals (Goshgarian et al.,

74
1989). In this study multiple synapses reached their maximum number by four
hours since no further increase for up to four days post-hemisection was detected.

Rapid synapse formation is not only a response to deafferentation since new
synapses can form rapidly in response to other manipulations. As mentioned earlier
synapse formation in hippocampal slices occurs within 15 minutes and depends on
the patterned electrical stimulation of LTP. Rapid formation of synapses i_n filo
occurs following induction of LTP by stimulation of the Schaffer collaterals to CA1
pyramidal cells (Lee, Schottler, Oliver & Lynch, 1980). (Note: synapse formation
apparently does not occur in the dentate gyrus of the hippocampus following
induction of LTP by stimulation of the entorhinal cortex, see Desmond & Levy,
1990.) New synapses also form in the hippocampal CA1 pyramidal cell layer
within 15-30 minutes following activation with kainic acid (Petit, LeBoutillier,
Markus & Milgram, 1989). Interestingly but unfortunately not quantitative,
Hinrichsen (1980) has reported the presence of growth cones in slices of the
medulla/pans (5 hour incubation time) suggesting that perhaps new connections are
beginning to form in this preparation. Such growth cones were not seen in
conventionally prepared perfused tissue from normal animals.

The literature reviewed above suggests that synapse formation in adult
animals can occur quite rapidly. The extent to which this statement applies to the
nervous system in general awaits further exploration. It could be that rapid synapse
formation is common but investigators have simply not "looked" for evidence of
synapse formation early enough. Rapid multiple synapse formation in SON has
been relatively easy to detect since somatic multiple synapses are relatively rare in

normal animals.

75

The precise stimulus for rapid synapse formation is not known. Clearly
synaptogenesis during LTP is dependent on patterned neuronal activity. Based in
part on the massive literature from the neuromuscular junction, Cotrnan et al. (1981)
consider neuronal inactivity as one of the major postulates governing axonal
sprouting following deafferentation.

In SON, multiple synapse formation during dehydration and
parturition/lactation is clearly associated with increases in MNC activity. A strong
case can be made that intravascular perfusion of hypertonic solutions increased
MNC activity which resulted in very rapid multiple synapse formation (Tweedle et
al., 1989). Multiple synapses also form during intracerebroventricular injections of
OX (Theodosis et al., 1986) a manipulation that may have increased MNC activity
since OX excites oxytocinergic neurons (e.g. Yamashita et al., 1987). In
Experiment I multiple synapses formed along with a detectable increase in the
average firing rate of MNCs.

The interrelationships between MNC inactivity, deafferentation and multiple
synapse formation are less clear. One study has shown that multiple synapses form
following deafferentation (Weiss et al., 1988). Given that the afferent projection
removed in this experiment (subfomical organ) is most likely excitatory (Jhamandas,
Lind & Renaud, 1989) it may be that MNC activity decreased. Synapse formation
in SON could conceivably occur in response to both increased and decreased MNC
activity.

M l' l n F rm ' 11
Multiple synapses as defined here are not unique to the SON. Certainly the

complex synaptic glomeruli characteristic of many brain regions (e.g. olfactory bulb,

 

76

cerebellum, and other, see Peters, Palay & Webster, 197 6) qualify according to the
definition "a single axonal terminal that simultaneously contacts 2 or more
postsynaptic elements". What makes multiple synapses in SON interesting and
worthy of special attention is their specific formation and disappearance in adult
animals. Although no one has yet demonstrated the exquisite matching of true
physiological condition and multiple synapse formation as occurs in the SON during
lactation, multiple synapses are present and malleable in several neural systems. A
selective survey of multiple synapse formation in other neural systems may provide
some clues as to how and when these synapses are formed.

1' l n 'n v 'n m Synaptogenesis in developing
animals is an extremely complex process that varies somewhat according to the
neural system being investigated (see Burry, Kniss & Scribner, 1984 and Vaughn,
1989 for reviews). In several systems the development of adult patterns of
innervation seems to involve both the formation and elimination of synapses. The
extent to which synaptogenesis in adult animals mimics the process that occurs
during development is unknown, but clearly some aspects are similar (e.g.
accumulations of polyribosomes and coated vesicles). The intent here is not to
review the developmental process extensively but rather to glean some insight into
the nature of multiple synapse formation as it occurs during development.

Few studies have addressed the issue of multiple synapses in developing
animals. As mentioned earlier glomeruli fit the definition of a multiple synapse and
their development is probably at the extreme end of multiple synapse formation in
terms of complexity. Methodologically the formation of multiple synapses during

development is difficult to assess since often increases in terminal number occur at

77

the same time that multiple contacts are being formed. This makes it impossible to
know whether pre-existing terminals are forming additional contacts or if the new
terminals are forming multiple contacts during their growth stage. Studies of
glomeruli formation have suggested that both of these processes occur depending on
the neural system involved.

There is evidence that the mossy fiber system in the developing cerebellum
establishes multiple synapses quite early since growth cones are seen forming
several contacts (Hamori & Somogyi, 1983; Mason & Gregory, 1984). In contrast,
studies of the mossy fiber system in the hippocampus (Amaral & Dent, 1981),
multiple synapses in the superior colliculus (Lund & Lund, 1972) and retino—
geniculate glomeruli (Mason, 1982; Brunso—Bechtold & Vinsant, 1988) suggest that
multiple synapses begin as relatively simple single synapses that develop over time
into more complex multiple synapses. This progression from single to multiple
synapses in the lateral geniculate requires action potentials since eye injections of
tetrodotoxin for 8 weeks postnatally prevents glomeruli formation. These inactive
terminals retain their single synapse configuration (Kalil, Dubin, Scott, & Stark,
1986).

While there is no evidence to suggest that glial process withdrawal
participates in the formation of multiple synapses during development the data
support the concept of a conversion from single to multiple synapses. Mason
(1982) has suggested that glial encapsulation of glomeruli acts to prevent terminals
from forming additional synaptic contacts. This is based on the observation that

synapse formation is complete at around the same time that glial processes

78
completely surround the glomerulus. It is the opposite of this, glial withdrawal, that
is thought to promote/permit multiple synapse formation in the SON.

Multiplesymosemeooeaeimes - Perhaps the first report of alterations in
multiple synapses in adult animals was given by Raisman (1969) who utilized the
now common multi-synaptic index ("the number of multiple contacts per 100 single
contacts"). Raisman has described in meticulous detail the reinnervation of the
septal nuclei by remaining intact fibers. Part of this reinnervation is accounted for
by an increase in the number of terminals that form multiple synapses (Raisman,
1969; Raisman & Field, 1973). Increases in multiple synapses following
deafferentation have also been reported in the spinal cord (Bernstein & Bernstein,
1977; Goshgarian et al., 1989), the hippocampus (Cotrnan, Gentry & Steward, 1977;
Matthews, Cotrnan & Lynch, 1976; Steward, Vinsant & Davis, 1988) and in the
dorsal lateral geniculate nucleus (Kalil & Behan, 1987). Interestingly, in some areas
of the spinal cord, deafferentation appears to result in a loss of multiple synapses
followed by synapse replacement by smaller terminals that make single contacts
(Murray & Goldberger, 1986).

Multiple synapses have been shown to respond to manipulations other than
deafferentation in areas other than SON. In the spinal nucleus of the
bulbocavemosus muscle testosterone maintains synaptic input to the sexually
dimorphic motor neurons (Matsumoto, Micevych & Arnold, 1988). The response of
these neurons to castration is the opposite of what occurs in SON with activation.
That is, multiple synapses and direct appositions between adjacent somata and
dendrites decrease. These decreases are prevented by testosterone replacement at the

time of castration. There is evidence for environmental regulation of multiple

79
synapses in the cat visual system. In this case, placement of cats in an enriched
environment is associated with significant decreases in the number of GABAergic
multiple synapses (Beaulieu & Collonnier, 1988). ‘

A few general conclusions can be reached concerning multiple synapses.
They are common and malleable in many neural systems (in fact they may be more
common than previously thought since not all studies of plasticity measure this type
of synapse). Their formation and disappearance can be influenced by several
different experimental manipulations. Following deafferentation multiple synapses
are often a unique synapse type that is not present or is rare in normal animals.

Few investigators have postulated a mechanism by which a single terminal
eventually contacts two postsynaptic elements. An exception is the mechanism
originally proposed for SON (Hatton & Tweedle, 1982) and postulated by
Goshgarian (1989) for multiple synapse formation, namely retraction of glial
processes from between adjacent neural elements. In the case of deafferented
systems it is often implied that part of the reactive process involves existing single
synapses converting to multiple synapses (e.g. Raisman, 1969), however in many
cases terminal proliferation occurs at the same time as multiple synapse formation
(Steward et al., 1988). In these systems strong statements regarding a conversion
process from single to multiple synapses are more difficult to make.

Whether multiple synapses serve a function that is unique to their anatomical
configuration, i.e. two postsynaptic elements that simultaneously receive an
excitatory or inhibitory signal, is not clear. In the case of deafferentation multiple
synapse formation is seen as a mechanism for maintaining synapse numbers and the

functional significance of multiple vs single synapses has been largely ignored. In

80
the phrenic nucleus the appearance of multiple synapses occurs concurrent with the
unmasking of a previously ineffective neural pathway (Goshgarian et al., 1989).
That is, a neural pathway which in normal animals does not evoke a postsynaptic
response is able to activate the postsynaptic cells following neural injury. It is not
known if this now effective pathway is the one that forms multiple synapses nor is
it clear if the anatomical configuration is significant.

In SON multiple synapses have often been postulated as one of several
potential mechanisms that serves to coordinate and/or synchronize MNC activity
(e.g. Hatton & Tweedle, 1982; Theodosis et al., 1981). Whether this is their
function in the deafferented SON is speculative. It may be that the conversion of
single to multiple synapses is simply a very efficient and rapid way to maintain the
synaptic input to MNCs following deafferentation.

i ' li 1

Previous io vi_vo studies have consistently found increases in direct
postsynaptic membrane apposition in both the somatic and dendritic regions of SON
during osmotic challenges. No increases in direct apposition were detected the any
of the present experiments. In addition, multiple synapses have always been
associated with increases in direct membrane appositions. The reverse is not
necessarily true since direct appositions can occur without increases in multiple
synapses, e. g. during acute dehydration. The present results show that multiple
synapses can increase without detectable changes in direct appositions. The lack of
a significant correlation between multiple synapses and direct appositions in
Experiment III suggest that the two changes occur independently. It may be that

very subtle changes in stimulus intensity (i.e. magnitude of osmotic increase) as well

81
as duration (number of days/hours of osmotic stimulation) produce different patterns
of these two morphological changes.

In the phrenic nucleus following spinal cord hemisection a similar
dissociation of membrane appositions and multiple synapses have been described.
Multiple synapses reach their maximum number by four hours while only the length
of dendrodendritic appositions increased at this time. At later time point (2 days)
the number of appositions and the dendritic bundling measures increased
(Goshgarian et al., 1989). In this system dendrodendritic appositions have also been
shown to increase without concurrent increases in multiple synapses following two
days of chronic hypoxia (Goshgarian & Yu, 1990).

While glial retraction has been postulated as the mechanism by which
morphological changes in SON and the phrenic nucleus occur, direct evidence for
this mechanism remains elusive. One of the few studies that has provided direct
evidence for glial changes (Salm et al., 1985) demonstrated a redistribution of
immunoreactive glial fibrillary acidic protein (GFAP) during lactation which is
consistent with glial retraction. Similar direct evidence for glial retraction has been
difficult to obtain at the electron microsc0pic level. Since glial processes cover the
vast majority of MNC somatic membrane, the small decreases in glial contact that
would occur with the addition of relatively small multiple synapses do not
significantly change this measure. Data collected on glial contact fiom chronically
dehydrated rats is complicated by the fact that cell size is increased dramatically in
these animals (Modney & Hatton, 1989). (In the phrenic nucleus glial contact has
not been measured.) In the present Experiment III there was a significant decrease

in the percentage of somatic membrane contacted by glial processes, with no change

82
in the extent of direct soma/somatic membrane apposition. This decrease can be
accounted for by the significant increase in coverage by unidentified elements. It is
likely that the increased coverage by unidentified elements is related to the general
decline in tissue morphology associated with increased incubation time. A cogent
argument can, and has been made (see Hatton, 1990) that direct appositions between
neural elements are most likely due to glial retraction since these changes occur
without concurrent changes in soma or dendrite size. This is still however, indirect
evidence for the postulated mechanism. Future morphological studies that continue
to measure glial elements may be able to provide more direct evidence for glial

retraction.

SUMMARY

It is clear from the present experiments that neuronal connections can be
modified rapidly io yi_tro. What remains unclear is the precise stimulus for this io
yimo reorganization. The results of Experiment I demonstrated that these synapses
could form in response to a well—characterized stimulus for SON neurons, namely
increased osmolality. In Experiment 111 there was no significant effect of
osmolality, however multiple synapses formed in both low and high osmolality
slices over time. The results of the third experiment support a previous i_n flyo
experiment that demonstrated reactive synapse formation in SON following removal
of an afferent input to MNCs. While reactive synapse formation in SON is not as
well established as synaptogenesis in response to osmotic stimulation, the data
presented here suggest that reactive synaptogenesis can be a rapid and robust
phenomena.

Given the discrepancy between these experiments it is clear that further To
yjyo and m v_itr_o studies must be done to reach a firm conclusion about the what
stimulates synapse formation in SON. The SON provides neurobiologists with a
model system to investigate how synapses in an adult mammalian system form.
Since synaptogenesis occurs in response to both physiological (e.g. dehydration) and
pathological (e. g. deafferentation) manipulations, a rare opportunity exists to compare

and contrast this process during these two different conditions.

83

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neurons receive peroxidase-labeled glutamate decarboxylase- or gold-labeled
GABA-immunoreactive synapses. ml N r in , 1985, 3, 2940-
2954.

Vaughn, J. E. Review: fine structure of synaptogenesis in the vertebrate central
nervous system. Synapse, 1989, 3, 255-285.

Weiss, M. L., Tweedle, C. D., Marzban, F., Modney, B. K. & Hatton, G. I. Rapid
formation of new double synapses in rat supraoptic nucleus (SON) in response
to interruption of subfomical organ efferent projections. Smiety for
Neueoscience Abstiacts, 1988, M, 1133.

Weiss, M. L., Yang, Q. Z. & Hatton, G. I. Magnocellular tuberomammilary nucleus
input to the supraoptic nucleus in the rat: Anatomical and it; _vitio
electrophysiological investigations. Neuroseienee, 1989, 3_1, 299-311.

Winer, B. J. ' ' Prin i l in Ex rim n l D i n. McGraw Hill: New
York, 1962

Yamashita, H., Okuya, S. Inenaga, K., Kasai, M. Uesugi, S. Kannan, H. & Kaneko,
T. Oxytocin predominantly excites putative oxytocin neurons in the rat
supraoptic nucleus ie v_iuo. Brain Researeh, 1987, 4_16, 364-368.

Yamamoto, C., Bak, I. J. & Kurokawa, M. Ultrastructural changes associated with
reversible and irreversible suppression of electrical activity in olfactory cortex

slices. Experimental Btain Reseatelr, 1970, _1_l, 360-372.

95

Yang, Q. Z. & Hatton, G. I. Dye coupling among supraoptic nucleus neurons
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Yang, Q. Z. & Hatton, G. I. Histamine and histaminergic inputs: Responses of rat
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Yulis, C. R., Peruzzo, B. & Rodriguez, E. M. Immunocytochemistry and
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Cell a_ngl Tissue Reseai'eh, 1984, 236, 171-180.

 

APPENDICES

.1

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APPENDIX A - TISSUE EMBEDDING PROTOCOL

 

APPENDIX A

TISSUE EMBEDDIN G PROTOCOL

General Notes
1) Spurr’s resin was obtained from Electron Microscopy Sciences (Low
Viscosity Embedding Kit). Follow manufacturer’s recipe when mixing the
resin components.
2) Osmicated tissue was typically stored in cacodylate buffer until the end of
the week and all the slices from a given week were embedded together.
Be sure that before staining with uranyl acetate that tissue is rinsed well
with distilled water.
DAY ONE
Slice Preparation - Fix by immersion overnight.
DAY TWO
Core and osmicate tissue. If embedding immediately stain overnight in
uranyl acetate, or store in buffer.
DAY THREE
1) Three water rinses (10 minutes each)
2) Stain tissue with saturated uranyl acetate (4%), filtered with a 0.22 um
Millipore filter, store overnight in refrigerator.
DAY FOUR - Infiltration

1) Water rinses (3 @ 15 minutes each)

96

97
APPENDIX A (Cont’d.)
2) 50%, 75%, 80%, and 95% alcohol (1 rinse each dilution for 10 minutes)
3) 100% alcohol (4 rinses @ 15 minutes each - OPEN A FRESH BOTTLE)
4) 2:1 mixture of alcohol to resin (3 hours)
5) 1:2 mixture of alcohol to resin (3 hours)
6) 100% resin mixture (overnight)
DAY FIVE - Embed
1) Embed tissue (molds or flat embed on release agent coated slides)

2) Polymerize overnight in an oven @ 65-70° C.

APPENDIX B - SUPPLIES & EQUIPMENT

APPENDIX B

SUPPLIES AND EQUIPMENT

EQU EMENT

Advantage Computer
SummaSketch Tablet

Diatome, Inc. (1987)
Diamond Knife (2.4mm)

EQS Systems Inc (1984)
Houston Instruments Hipad Digitizing Tablet

Gilson Medical Elec Inc (1985)
Peristaltic Pump

Jandel Scientific
Sigma-Scan Software

JEOL (Approximate)
JEOL CX-100 Electron Microscope

Kopf Vertical Pippette Puller
LKB Knifemaker 11

Logitech (1985)
Modula 2 compiler
Mouse (3-button)

Mager Scientific (1987)
Reichard Ultracut E ultramicrotome
" " section counter
Nikon Alphaphot microscope

Markson, Inc (1987)
Markson pH meter
pH electrode
Temperature probe

Millipore, Inc. (1987)
Milli-R0 4 (Water purification system)
Milli-Q (II M)
Cartridges for above system

98

465.00

2,500.00

828.00

1,136.00

465.00

300,000.00
2,910.00
4,875.00

560.00
1 19.00

23,160.00
657.00
1000.00

495.00
89.00
59.00

1,482.00
1,947.00
793.00

99
APPENDIX B (cont’d)

SUPPLIES & EQUIPMENT (cont’d)

MSU Computer Center (1985)
Zenith 150 Computer w/monochrome monitor

Olympus (1986)
C-2 Image Analysis System
(excluding microscope)

Polysciences - Vibratome

Technical Manufacturing Corp. (1987)
Micro-g air table

Ted Pella, Inc (1987)
Infiltron (approximate equivalent)
Sodium Vapor Safelight

Tektronix Oscilliscope

Thomas Scientific (1987)
Mettler Balance
Blue M Gravity Oven
Fiber Optic Illumination System
Sage Syringe Pump (approx replacement - 2 channel)
Refrigerator/Freezer
YSI Temperature probe & controller (replacement)

VWR Scientific (1987)
Stereozoom microscope (approx. replacement)

Wild Microscope
S U PPLIES

Electron Microscopy Sciences (1987)
Copper Grids (200 mesh-thin bar: 100/vial)
Forceps

3C
anticappillary, self-closin g

Electron Microscopy Sciences (cont’d.)
Glassine Envelopes for negatives (1000)
Glutaraldehyde (Biological Grade) 500 ml
Grid Boxes (12)

Uranyl Acetate (25 g)

1 ,499.00
8,000.00
3,750.00
2,340.00
577.00
398.00
9,865.00
2,795.00
669.00
2,795.00
1 ,550.00
1375.00
1005.00
1,336.00
5,000.00

13.50

8.50
19.50

34.50

6.50
32.00
12.00

 

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100
APPENDIX B (cont’d)

SUPPLIES & EQUIPMENT (cont’d)

Millipore, Inc. (1987)
GSTF filters (.22ttm)

MSU Stores (1987)
Assorted glassware (beakers, volumetrics, etc)
Assorted disposable plastics (syringes, etc)
D-19 developer
Desiccator (small glass)
Electron Microscopy Film (box w/ 100 sheets)
Kodabromide F4 Paper (8" x 10", 250 sheets)
Polycontrast Paper (11" x 14", 50 sheets)‘

Sigma Chemical Co. (1987)
Cacodylate Acid (500 g)
Paraformaldehyde (1 kg)

Stevenson Metal Supply (1987)
Osmium tetroxide (1 g)

Vets Ace Hardware (1987)
Red Devil Razor Blades (100)

VWR Scientific (1987)
Microscope Slides (case)

Wescor Osmolality Standard

15.00

100.00
60.00
2.89
74.23
35.70
97.50
37.1 1

182.00
12.75

35.00
9.40

1 10.62

37.25

 

 

APPENDIX C - DATA TABLES

EXPERIMENT I

APPENDIX C

DATA TABLES - EXPERIMENT I

TABLE 6 - CELLULAR ELEMENTS CONTACTING MNC SOMATIC MEMBRANE

 

Glial Axonal Direct Not Total
Processes Terminals Apposition“ Identified
um % um % um % um % pm
290 mOsm
Slice #
35 550.6 80.6 75.9 11.1 13.3 1.9 43.2 6.3 683.0
37 599.8 85.8 65.9 11.0 10.2 1.4 12.9 1.8 699.8
48 572.6 74.8 90.6 1 1.8 32.4 4.2 69.9 9.1 765.5
52 560.9 88.9 45.2 7.2 1.5 0.2 22.7 3.5 630.3
53 629.8 87.2 58.5 8.1 26.6 3.5 8.2 1.1 722.3
59 433.5 77.4 88.9 15.5 4.0 0.7 36.4 6.3 572.7
63 379.3 75.9 81.9 16.4 8.6 1.7 29.7 5.9 499.4
65 497.6 82.7 42.3 7.0 40.3 6.7 21.3 3.5 601.6
70 525.9 84.4 64.8 10.4 13.7 2.2 18.4 3.0 622.9
Mean 527.5 82.0 67.0 10.9 16.7 2.5 29.2 4.5 644.1
SE 26.5 1.7 6.1 1.1 4.4 0.7 6.3 0.8 27.4
340 mOsm
Slice #
38 488.1 82.7 60 5 10.2 15.7 2.6 25.9 4.4 590.3

43 517.4 73.7 117:6 16.7 11.3 1.6 55.4 7.9 701.6
46 398.1 67.9 100.3 17.1 13.6 2.3 73512.5 585.5

56 560.1 81.7 79.4 11.6 12.5 1.8 33.1 4.8 685.1
58 549.6 71.2 94.7 12.2 74.0 9.6 52.6 6.8 771.0
66 474.2 84.0 62.2 11.0 11.4 2.0 16.6 2.9 564.5
67 501.0 83.4 61.3 10.2 11.8 2.0 26.4 4.4 600.5
69 467.3 77.3 65.5 10.5 23.3 3.8 50.0 8.2 604.1
Mean 494.5 77.0 80.5 12.4 21.7 3.2 37.9 6.5 637.8
SE 18.1 2.2 7.7 1.0 7.6 1.0 8361.1 25.7
t = 1.53 -1.0 -0.62 -145
p = 0.14 0.33 0.55 0.16

* Direct apposition between adjacent somata and or dendrites

101

TABLE 7 - MULTIPLE SYNAPSE MEASURES

% Somatic
Membrane
290 mOsm/kg
Slice #
35 1.31
37 0.85
48 0.55
52 0.46
53 0.45
59 0.67
63 1.50
65 0.65
70 0.76
MEAN 0.80
SE 0.12
340 mOsm/kg
Slice #
38 1.41
43 1.62
46 1.82
56 1.33
58 1.72
66 0.84
67 1.48
69 0.66
MEAN 1.36
SE 0.14
t = -2.97

p = 0.01

102
APPENDIX C (cont’d.)

#/100

pm of
Somatic
Membrane

1.46
0.86
0.39
0.32
0.28
0.70
1.60
0.66
0.80

0.78
0.16

1.19
1.28
1.54
1.02
1.95
0.87
1.50
0.66

1.25
0.14
-2.16

0.05

%

Synapses

14.5
8.95
4.22
5.61
3.92
5.80

11.10
8.89
8.77

8.0

1.1

11.86
9.57
11.69
11.86
15.96
9.80
15.00
6.90

11.6
1.0
-2.30
0.04

%

Axonal
Contact

1 1.76
7.70
4.69
6.46
5.98
4.31
9.16
9.27
7.34

7.4

0.8

13.77
9.72
10.64
1 1.45
14.00
7.64
14.56
6.30

1 1.0
1.1
-2.73
0.01

103
APPENDIX C (cont’d.)

TABLE 8 - MEAN LENGTH (1.1m) OF APPOSITION BETWEEN
AXON TERMINALS AND MNC SOMATIC MEMBRANE

 

290 mOsm/kg 340 mOsm/kg

Slice # Length Slice # Length
35 1.10 38 1.02
37 1.14 43 1.25
48 1.27 46 1.30
52 1.28 56 1.34
53 1.14 58 1.01
59 1.28 66 1.22
63 1.13 67 1.02
65 0.96 69 1.09
70 1.14

MEAN 1.16 1.15

SE 0.04 0.05

t = 0.13

p = 0.90

104
APPENDIX C (cont’d.)
TABLE 9 - SOMATIC SIZE AND SHAPE MEASURES

Area Perimeter Shape
01m”) (um)
290 mOsm

Slice #
35 360.1 72.2 0.86
37 413.0 77.6 0.85
48 384.7 80.0 0.75
52 384.7 80.6 0.88
53 448.0 82.6 0.83
59 427.6 80.2 0.84
63 441.1 80.1 0.86
65 486.8 83.7 0.87
70 461.0 82.2 0.84
Mean 431.2 79.9 0.84
SE 13.3 1.1 0.03

340 mOsm

Slice #

38 522.3 88.9 0.87
43 386.9 74.7 0.88
46 396.0 76.3 0.85
56 389.1 73.4 0.90
58 405.8 77.7 0.84
66 499.0 83.2 0.89
67 367.5 73.1 0.86
69 442.4 79.0 0.88
Mean 429.9 78.2 0.87
SE 22.8 1.9 0.01
t = 0.05 0.76 -1.92

p = 0.47 0.46 0.07

105
APPENDIX C (Cont’d.)

TABLE 10 - AVERAGE FIRING RATES

290 mOsm/kg 340 mOsm/kg

Cell # Rate (Hz) Cell # Rate (Hz)
8 2.7 1 5.5
11 3.4 2 3.4
15 7.6 5 9.1
18 5.0 6 6.9
20 5.2 7 11.9
21 7.9 27 7.7
22 5.8 29 2.7
23 3.3 30 5.5
24 3.3
33 8.7
34 2.7
35 3.4

Mean 4.70 7.49

SE 0.71 1.30

106
APPENDIX C (cont’d.)

TABLE 11 - DENDRITIC SYNAPSES

290 mOsm/kg Total # Terminals Multiple Multiple
Terminals Apposed to Synapses Synapses
Slice # >1 Dendrite w/pr* w/cv#
35 238 43 3 5
52 328 43 7 9
53 231 46 4 4
59 307 79 7 3
65 279 56 5 12
340 mOsm/kg
Slice #
38 241 47 11 9
46 338 92 24 12
56 486 90 12 9
58 358 71 13 21
66 312 86 8 12

*pr - polyribosomes

#cv - coated vesicles

107
APPENDIX C (cont’d.)

TABLE 12 - DENDRITIC MULTIPLE SYNAPSES

290 mOsm/kg
Slice # % Terminals % Multiple % Multiple
Apposed to Synapses Synapses
>1 Dendrite w/pr w/cv
35 18.07 6.98 1 1.63
52 13.1 1 16.28 20.93
53 19.91 8.70 8.70
59 22.48 10.14 4.35
65 10.07 8.93 21.43
MEAN 18.73 10.20 13.41
SE 1.57 1.60 3.38
340 mOsm/kg
Slice #
38 19.50 23.40 19.15
46 27.22 26.09 13.04
56 18.52 13.33 10.00
58 19.83 18.31 29.58
66 27.56 9.30 13.95
MEAN 22.53 18.09 17.14
SE 2.00 3.10 3.34
t = -1.49 -2.26 -0.77
p = 0.17 0.05 0.47

108
APPENDIX C (cont’d)

TABLE 13 - PERCENTAGE OF DENDRITIC MEMBRANE
CONTACTED BY CELLULAR ELEMENTS

Single Multiple Adjacent
290 mOsm/kg Synapses Synapses Dendrites
Slice #
35 5.87 2.36 9.11
52 6.93 1.96 7.91
53 5.90 2.86 10.00
59 4.68 2.35 8.73
65 5.44 2.44 12.84
MEAN 5.74 2.39 9.72
SE 0.36 0.14 0.85
340 mOsm/kg
Slice #
38 5.73 2.42 8.07
46 5.45 4.48 10.78
56 7.23 2.50 8.32
58 5.31 3.50 10.11
66 6.32 2.03 10.69
MEAN 6.01 2.98 9.59
SE 0.35 0.44 0.58
t = 0.48 -1.27 0.12

p = 0.64 0.23 0.90

109
APPENDIX C (cont’d)
TABLE 14 - AVERAGE LENGTH (um) OF DENDRITIC SYNAPSES

AND PERCENTAGE OF DENDRITIC AXONAL CONTACT
MADE BY MULTIPLE SYNAPSES

 

Single Multiple % Axonal
Synapses Synapses Contact made
By Multiples
290 mOsm/kg
Slice #
35 0.86 0.74 28.67
52 0.89 0.78 22.05
53 0.92 0.71 32.57
59 0.90 0.71 33.43
65 0.80 0.75 30.96
MEAN 0.87 0.74 29.54
SE 0.05 0.01 2.04
340 mOsm/kg
Slice #
38 0.85 0.7 1 26.56
46 0.87 0.80 56.63
56 0.79 0.69 25.00
58 0.85 0.66 40.09
66 0.79 0.67 15.81
MEAN 0.83 0.72 32.82
SE 0.02 0.02 7.10
t = 1.60 1.16 -0.44

p = 0.14 0.28 0.66

APPENDIX D - DATA TABLE

EXPERIMENT II

APPENDIX D

DATA TABLE - EXPERIMENT H

TABLE 15 - PERCENTAGE OF SYNAPSES
THAT WERE MULTIPLE SYNAPSES

290 mOsm/kg 340 mOsm/kg
Slice # % Multiple Slice # % Multiple
Synapses Synapses

115 15 107 15

120 15 110 17

121 12 113 12
119 14
122 10

MEAN 14.33 13.60

SE 1.20 1.20

t = 0.40

p = 0.70

110

APPENDIX E - DATA TABLES

EXPERIMENT HI

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