A QUANTETATEVE CYFOARCHETECTONK ANALYSIS OF THE. ENTEMEQUNCLAR COMPLEX; Qt? WEE ALMNG .RAT Thesis EM €56 Dogma 05 M‘ A. MlCHlGAH ETHIE UNIVERSITY; Wiifiam Robert Ives £968- ABSTRACT A QUANTITATIVE CYTOARCHITECTONIC ANALYSIS OF THE INTERPEDUNCULAR COMPLEX OF THE ALBINO RAT By William Robert Ives The interpeduncular complex (IPN), situated on the medio- ventral surface of the midbrain, is universally present in all vertebrates including man. Experiments on the functional signif- icance of this structure, thus far unproductive, have generally assumed cytoarchitectonic homogeneity although this is contra- indicated by a number of qualitative studies. Because of the lack of quantitative data on the structure of the IPN the pre- sent study has undertaken an analysis of this structure using measures of cell size and packing density as measures of sub- nuclear differentiation. Cell size (area) and density werenwap sured in 4 spatially separate cell groups: pars lateralis (PL), pars medialis (PM),pars dorsalis parvocellularis (PDP), and pars dorsalis magnocellularis (PDM). The IPN of 3 normal, Nissl- stained, albino rats, sectioned in the horizontal, sagittal, and coronal planes of section served as material. 60 cells were randomly sampled from each of the h subnuclei for each of the 3 planes of section and their areas determined by plan- imetry. Density was measured within each subnucleus by count- ing the numbers of cells contained in several sample volumes of tissue. PDM had the largest cell size and the lowest den- sity of any of the 4 subnuclei while PDP had the smallest cell size and the highest density.£fl, and PM were intermediate in William Robert Ives both cell size and packing density and were not significantly different from one another..The differences were tested by a 2 way analysis of variance and Newman—Keuls tests at the .01 level of confidence. The absence of any quantiative differen- ces between PL and PM casts doubt upon earlier distinctions based on qualitative studies of these 2 subnuclei. The dis- tinction between PL and PM is a spatial one and not cytoarchi- tectural while PDM and PDP present both spatial and cytoarchi- tectural differences. These quantitative differences in cyto- architecture may imply functional differentiation within the IPN which may help to explain the variety of behavioral def- icits observed after gross lesions of this structure. ”/4 // “1%.”. A QUANTITATIVE CYTOARCHITECTONIC ANALYSIS OF THE INTERPEDUNCULAR COMPLEX OF THE ALEINO RAT By WILLIAM ROBERT IVES A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1968 If: I“:fi 3 ACKNOWLEDGEMENTS The author wishes to express his gratitude to the following professors whose guidance during my education has been invaluable; Dr. G. I. Hatton who first aroused my inter- est in the nervous system, Dr. T. W. Jenkins who provided me with an invaluable introduction to neuroanatomy,IHHJ}I.Johnson who furthered my interest in the nervous system, and Dr. M. Balaban who introduced me to develOpmental neuro~ anatomy. To my wife, whose efforts in reaching this stage in my education have been far greater than mine, I dedicate this thesis. This research has been supported by research grant MB 05982 to Dr. J. I. Johnson and NIH training grant NH 10611. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS....................................... ii LIST OF TABLES........................oo............... iv LIST OF FIGURES..o.o................o..oo..o....o...... v ABBREVIATIONS USED IN PLATES 1-6....adooooo......no.... vi INTRODUCTIOIJOO0.0.00...OOOOOOOOOOOOI.0.00.00.00.00.00.. 1 METHOD................................................. 8 RESULTS................................................ 10 DISCUSSION............................................. 23 REFERENCES............................................. 31 PLATES...so00000000Oo00.00.0000.00.000.000.000000000000 33 APPEDIDIX AOOIOOIOOOOO0.000.000.0000.0000000COOOOOOOOOOO 39 iii Table 1. LIST OF TABLES Summary of the results of the analysis of variance conducted on the cell size data.......... Summary of the results of the Newman- Keuls tests conducted on the cell size dataOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOI0....O... Cell size ratios computed as the ratio between a given subnucleus and PM................. Summary of the analysis of variance conducted on the density results.................. Summary of the results of the Newman- Keuls teSthggggg..........‘...cocoooooooooooooooo Density ratios computed as the ratio between a given subnucleus and PM................. iv Page 12 12 12 19 19 19 Figure 1. 9. 10. 11. Plate 1. LIST OF FIGURES page A schematic representation of the gener- alized fiber connections of the vertebrate IPNOOOOOOCOO-.0...OOOOIIOOOOOOCIOOOOOOOO0.0.0.... 3 A schematic representation of the subnuclei of the cat IPN after Berman and Bowers (1967).... 5 A schematic representation of the subnuclei Of the rat IPNoooooooococoon-00000000000one...coo 7 Results of cell area measurements for PM......... 13 Results of cell area measurements for PL......... 14 Results of cell area measurements for PDP........ 15 Results of cell area measurements for PDM........ 16 Composite of the histograms of Figures 4-7 for purposes of comparison....................... 17 Results of density measurements for PL and PIY‘IOO0.00.00.00.00.OOOOOOOOOOOOOOIOIOOOOO0.0.0.... 20 Results of density measurements for PDM and and PDP-0000oucooooobooooooooooooooo000000ooooooo 21 Composite of the histograms of Figures 9 and 10 for purposes of comparison.................... 22 page Caudal section through the midbrain at the level of the decussation of the superior cerebellar peduncle (DBC)oooooooooooooooooococoon 33 Caudal section through the midbrain at the level Of the trochlear nuCleus (IV)oooooooooooooo 3n Coronal section through the midbrain at the level of the caudal extent of the superior COlliCUlUS (COL. SUP.)ooooooooooooooooooooooooooo 35 Coronal section through the midbrain at the level of the oculomotor nucleus (III)............ 36 Coronal section through the midbrain at the level of the red nucleus (N. RUB.)............... 37 Coronal section through the midbrain at ros- tral levels through the IPNoooooooooooooo00000000 38 ABBREVIATIONS USED IN PLATES 1-6 Clcooooooooo.000.00.00.00.ooooooooooooooooo. commissure 0f the inferior colliculus COL. SUP. 0.00.0.0...oooooooooooooooooooooo. Superior COlliCUluS COB. PIN. 0.00.0000...cocoooooooooooooooooo. pineal body DBcoooooo..0.a.....o.0.....oocoooooooooooooo decussation Of the superior cerebellar peduncle DEC. TEG. DOES. 0......oooooooooooooooooo... dorsal tegmental \ decussation DEC. TEG. VENT. ............................ ventral tegmental decussation FLM.......o.....oo...cocoooooooooooooooooooo medial lOngitudi- nal fasciculus GR. CENT. .o.oooooooooooooooooooo...o....... central gray GR. POT‘IT. OO0.....0.‘......‘O............... pontine gray LEM. MED. o.00...cocoooooooooooooooo..000... medial lemniscus N. INTERPED. PARS DORSALIS PARS MAGNO...........o..oooooooooooooooooooo interpeduncular nucleus pars dor- salis pars magno- cellularis N. INTERPED. PARS DORSALIS PARS PABVO. 0...0000.oooooooooooooooooooo... interpeduncular nucleus pars dor- salis pars parvo- cellularis N. INTERPED. PARS LATERALIS...ooooooooooooo. interpeduncular nucleus pars lat- eralis N. INTEBPED. PARS MED. ...o...........o..oo. interpeduncular nucleus pars med- ialis N. LIN. ....0....0...-ooooooooooooooooooo0.. lifiéar DHC1euS N. OCULO. ..........0...o.........ooooooa... OCHlomOtor nuc- leus (III) vi ABBREVIATIONS USED IN PLATES 1-6 (continued) NO RUB. OOOIOOOOOOOOOOOCOCOOOOOOOOOOOCOOOOOOOO N's TBOCHO 0.00.00.00.00.00.0.0000...I...00.... PED. GER. .0...0.000000000000000000.0.00....0. PEDO P‘ZAI‘IIO 0......O...0......OOOOOOOOOOOOOOOOOO PLAD. PEYO OOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00... SUB. NIG. 0....0......OOOOOOOOOOOOOOOOOOOOOOO. TR. CORT. SP. TR. HAB. PED. IIIOOOOCOOOOOOOOIOO00......OOOOOOOOOOOOOOOOOOO vii red nucleus trochlear nuc- leus (IV) cerebral pe- duncle mammillary peduncle radiations of Meynert substantia ni- gra corticospinal tract habenulope- duncular trac t oculomotor nerve INTRODUCTION GeneralfiAnatomy Because of the juxtaposition of the interpeduncular nuc- leus (IPN) and the posterior perforated substance Forel (1877) first termed this nucleus das Ganglion der lamina perforata pesterior. Gudden (1881) later termed this cell group das Ganglion interpedunculare because of its position between the cerebral peduncles in mammals. Subdivisions of the nucleus were not described by these early investigators although Cajal (1952) described a stratification of the nucleus into two layers in the rabbit. The outer layer (i.e. dorsal) con- sisted of large multipolar cells (peripheral zone) while the inner (i.e. ventral) layer was described as consisting of small to medium sized cells (plexiform layer). These early descriptions for mammals were accepted until the work of Huber, Crosby,Woodburne,Gillian,Brown, and Tamthai (1943) in which it was noted that the IPN of a number of mammals (mink,rabbit,cat dog,sheep,pig,rat, and armadillo) appeared to consist of two spatially and cytoarchitectonically distinct subnuclei; a large celled lateral subnucleus and a smaller celled medial subnuc- ‘leus. According to the latter authors the IPN reached its greatest development in the rabbit where it consisted of three 2 subnuclei; a compact dorsal portion, a large celled lateral portion, and a small celled medial portion. Work on the submammalian IPN has been done by Herrick (1917) in the mudpuppy, Necturus, and by Jansen (1930) in the hagfish, Myxine. In the hagfish the nucleus consists of a rostrally located mesencephalic division which consists of large multipolar cells and a caudally located rhombencephalic division which contains large and small spindle shaped cells. A similiar subdivision of the nucleus into rostral and caudal components has been described for Necturus. A schematic drawing of the generalized fiber connections of the vertebrate IPN is shown in Figure l. The connections are general in the sense that all vertebrates minimally have them. The IPN receives a large contingent of fibers from the habenulae as the habenulopeduncular tract (HPT) which con- tains fibers from both the lateral and medial habenular nuc- lei (Kappers,Huber, and Crosby,1936). The IPN is also affer- ently connected with the mammillary bodies via the mammillo- peduncular tract. The efferent outflow of the nucleus is di- rected to the dorsal tegmental nucleus of the central gray via the pedunculotegmental tract. The dorsal tegmental nucleus contributes its fibers to the dorsal longitudinal fasciculus which synapses with several visceral efferent cranial nerve nuclei (VII,IX,X,XI)(Zeman and Innes,l963).« Several general features-of the vertebrate IPN need to be noted. That the nucleus is divided into at least two subnuc- lei in many vertebrates is quite evident. In Myxine and Necturus \JJ HAB. DTN \ ,1 \ ¥ \ I *\ ‘f \ I ‘\‘H P T ’l’ P T T \‘ ’I \ I MAM. BOD. ">-------------> MPT Figure 1. A schematic representation of the g neralized fiber connections of the vertebrate IPN. Arrows indicate the direction of conduction of nerve impulses. HAB.= habenula, DTN: dorsal tegmental nucleus, MAM. BCD.= mammillary body, IPN: interpeduncular nucleus, HPT: habenulopedunculwr tract, PTT: pedunculotegmental tract, MPT: mammillOpeduncular tract. h these divisions are rostral and caudal and as we ascend the phyletic scale they become medial and lateral. This shift in position of the subnuclei follows the shift in the position of the habenular subnuclei from a rostral-caudal subdivision to a medial-lateral one. The HPT also has been described as consisting of contributions from both the habenular subnuclei (Kappers,Huber, and Crosby,1936). The general existence of a divided input (EFT)to a nuc- leus that also exhibits divisions indicates that there may be a certain specificity of projection upon the IPN. A given habenular subnucleus might project to a given IPN sub- nucleus. One might also hypothesize that there exists some specificity of output through the pedunculotegmental tract although there is no experimental evidence to corroborate either of these points. The Problem A recent study by Berman and Bowers (1967) has renewed interest in the cytoarchitectonics of the IPN. They described five subnuclei of the cat IPN which differ from one another on the basis of cell size, staining characteristics, and packing density. These subnuclei are the posterior, apical, central, paramedian, and intrafascicular (see Figure 2). "The posterior nucleus comprises an outer division which forms a cellular cup surrounding the caudal part of the complex, and a bifurcated inner division separated from the outer by a cell sparse zone. The apical nucleus is embedded in the dorsal aspect of the outer division. The paramedian nuclei are paired columns of cells on either side of, but clearly separated from the inner division of the posterior nucleus. The central nucleus is an elongated structure rostral to, and partly fused with the inner SA£5 Figure 2. A schematic representation of the subnuclei of the cat IPN after Berman and Bowers (1967). In a, b, and c the nucleus is shown in the horizontal, sagittal, and coronal planes ofsection,respmctive1y. A: apical, C: central, P: posterior, L=_paramedian, and IF: intrafascicular. 6 division of the posterior nucleus. At the rostral end of the complex a fifth structure, the intrafascicular nucleus, forms a cap over the central nucleus, but extends beyond it as a narrow wedge in the median raphe between the two habenulopeduncular tracts.” Berman and Bowers, 1967, p.213. These authors stated that it was not possible to tell whether the intrafascicular nucleus was a part of the complex or a part of the central linear nucleus with which it merged. My own work in the rat (see plates and Figure 3) has indicated the presence of four subnuclei which differ from one another on the basis of location and/or cell size. These are the pars dorsalis magnocellularis (PDM), pars dorsalis parvocellularis (PDP), pars lateralis, and pars medialis. PDM consists of large cells which lie in the dorsocaudal third of the complex. PDP, located immediately rostral to PDM, con- sists of small spherical cells with lightly staining nucleoli. In sections through the red nucleus PDP turns ventral and comes to occupy the medial portion of the nucleus as well. PL consists of two well defined groups of cells which lie at the lateral extremities of the nucleus. PM is located just ventral to PDM and PDP and medial to PL. It is separated from all the above subnuclei by areas of lower cell density. The purpose of the present study was to quantitatively assess the nature of the aforementioned IPN subnuclei through measurement of cell size and packing density within the sub- nuclei. The values obtained for each of these measures was then compared with similiar measurements for the other subnuc- lei through the use of appropriate statistical tests. 8.) _ ' SAG PDP “a (33R h) N ‘ IJN PDP PDM HOR [ PM! 1 l iCOR c.) r—lPoM — h—J 'HOR ! PM] SAG Figure 3. A schematic representation of the subnuclei of the rat IPN. In a, b, and c the nucleus is shown in the hor- izontal, sagittal, and corbnal planes of section, respective- ly. PL: pars lateralis, PM: pars medialis. PDP: pars dorsalis parvocellularis, PDM: pars dorsali: magnocellularis, and N LIN: linear nucleus. METHOD Specimens Three albino rats were perfused intracardially with .87% saline solution followed by a .87%-10% formalin mixture and their brains removed and photographed. The brains were embedded ‘ in celloidin and sectioned a 25 microns in three planes of sec- tion, horizontal, coronal, and sagittal. Alternate sections were stained with iron hematoxylin for fibers (Sanides Haidenhain method) and thionin for cells bodies (Nissl method). Refer to appendix.A for the details of the histological pro- cedure. Cell Size Measurements Cells of the four subnuclei were magnified 2800 times via a camera lucida attachment on a Zeiss microscope. Ten cells were randomly sampled from each subnucleus for six sections through the nucleus such that a total of 60 cells were sam- pled from each subnucleus for each plane of section. Only cells whose nucleoli were visible were sampled. The areas of the cells were determined by planimetry with a Keuffel and Esser compensating polar planimeter. Three area measurements were taken for each cell and the mean of these measurements used as an estimate of the true area. Packing Density Measurements Densities were measured by counting the number of cells contained in a grid placed over the nucleus. The grid was a Whipple-Hauser disc which was placed directly in the ocular of the microscope. Knowing the thickness of the section and the area of the grid, volume could be determined by multi- plying thickness times area. Dividing the number of cells in a sample volume by the sample volume gives the density of cells within a given subnucleus. Thickness of a given section was measured in the following manner. The slide was placed on the microscOpe stage and the fine focus dial adjusted until the first cell came into focus. At this point the number on the focus vernier was read. The fine focus knob was then turned until the thick- ness of the section had been focused through and the last cell was in focus. The vernier was then read again and one reading subtracted from the other. The resulting number was the thick- ness of the section in microns. Thickness was measured three times for a given section and the mean of the measurements used as an estimate of the true thickness of the section. In order to avoid errors due to differential thickness within a given section measurements were only conducted upon the IPN and on no other portion of the section. RESULTS Descriptive Anatomy Plates 1-6 show the nuclear configuration of the IPN and surrounding midbrain structures in a caudal to rostral sequence. In caudal sections through the midbrain (Plates 1&2) the IPN shows a division into three subnuclei, pars lateralis (PL), pars medialis (PM), and pars dorsalis magnocellularis (PDM). PL and PM are particularly well developed at this level in terms of size and in fiber preparations fibers can be seen issuing from the lateral borders of PM. The cells of PDM, while being sharply segregated from the rest of the nucleus, appear to trail off into the overlying nucleus centralis superior. In middle and rostral sections through the midbrain (Plates 3-6), PDM can be seen to be replaced by pars dorsalis parvocellularis (PDP) on the dorsal aspect of the nucleus. In the most rostral sections (Plates 5&6) PDP turns ventral to replace PL and PM. Cell Size The results of the cell area measurements are presented in Figures 4-8. On the abscissa of each figure is plotted 2 X 10, while the ordinate the value for cellular area in microns plots the frequency of occurrence of a given cell size. A two-way analysis of variance revealed a significant difference in mean cell areas between the three planes of section (F:33.33, 10 11 2/708 df, p<:.01), a significant difference in mean cell areas between divisions (F:236.80, 3/708 df, p<:.Ol) and a significant interaction (F=4.25, 6/708 df, p‘<.01). The results of this analysis are presented in tabular form in table 1. a) 2 (decimal fraction of the variance accounted for by the treatments) for planes was .04, for divisions .46, and for the interaction .01. Comparisons between all pairs of means by the method of Newman and Keuls (Table 2) re- vealed that the mean cell sizes within all subnuclei were significantly different from one another with five exceptions: PDPsag:PDPcor, PDPsag=PDPhor,PMcor:PLcor, PMsag=PLsag, PMhor: PLhor. In order to ascertain whether or not the differences in cell size between planes (within a given subnucleus) could be attributed to shrinkage differences or differences in the actual orientation of the cells, ratios were computed. Three ratios for each plane were computed, PL/PM, PDM/PM, and PDP/PM. The values for these ratios are presented in table 3. It can easily be seen that the ratios remain es- sentially constant across planes which at least strengthens the argument for the cause of the differences being shrinkage. No statistical tests were conducted on these data. Density The results of the density measurements are presented in figures 9-11. The abscissa of each histogram plots the density as cells/micron3 X lO'LP and the ordinate plots the frequency of occurrence of a given density. A two-way analysis 12 Table 1. Summary of the results of the analysis of variance conducted on the cell size data. 4" : p< .01 sou—Ros SUM OF SQUARES df @AN SQUARE F 03 2 Planes of 282.69 2 141.34 33.33* .04 section Subnuclei 3,012.17 3 1,004.05 236.80* .46 Interaction 120.07 6 20.01 4.71* .01 Error 3,011.63 708 4.25 ------ --- Total 6,426.56 719 -------------- --- Table 2. Summary of the results of the Newman-Keuls tests con- ducted on the cell size data. Lines beneath two subnuclei in- dicate that they are p23 different from one another at the .01 level of confidence. PDP PDP PDP PM PL PM PM PL PL PDM PDM PDM c s c c s h s c s h h h 4(-************************************************************* Table 3. Cell size ratios computed as the ratio between a given subnucleus and PM. CORONAL SAGITTAL HORIZONTAL PL/PM:1.00 PL/PM:1.09 PL/PM:1.05 PDM/PM:1.63 PDM/PM:1.34 PDM/PM:1.44 PDP/PM:.53 PDP/PM:.52 PDP/PM:.57 20 IO 3O 20 IO 0 L. 0 I2 24 AREA “H10 $522133; 3:23 '. Coronal section X 103 Figure 4. Results of cell area measurements for PM. In this and the following figures the results are based on a random sample of 60 cells from each of the three planes of section.The means and standard evi- ations for the respective planes are 69 microns + 20 for the coronal plane, 85 microns2 t 24 for the sag- ittal plane, and 89 microns“ t 25 for the horizontal plane. Comparisons of the means by the Newman-Keuls method revealed PM i PM and PM i PM at the .01 level of confidence. The sectfon is approxymately at the level Of plate 2 . FREQUENCY 5253mm. A} . — r —2m a 2x mu .A LT E O 0 O O O 0 00 0 O 00R 3 2 .l 2 .I la 2 .I A 1h Coronal section f . 0 nlh.e 39b 1 non f.e .vnudnsdnuv Lglr.nnue e Ptoaolll ncc Snae roe flocvoV OnPnsEriVnoh fShnaelt Btfipr ST 1m 1t t rPOdOa noso C.u. ofnril .n y mt fiotol e Zlbpceh.e viroatnum+ot unu {la a c§1i818tm a nap1iail mesa S u x mans lie ho oneaKrLr aslohut .D.p e+cr+tnun P riane num__a aalinxz s vmmo«iwrus 18 frePl 1ldny on” e AZVh“ flan nod 2 enuo vie Ehuaqi fnar.th+u +o Dada +u to n 2 y EE asanusaibrcs it+vonuo P 1lssloris e “a +cr n/Fh cfdnccéua CI ene12 AWL pnafism Uni. +. no .Sfi) 8 cc 0 191 £d.‘ .39., ‘2) s.d.- 38 5 u 3 5 O I“ K O t 0 HUGO 54.45.10“ . "202 . 7 0.63.13 10" 5 5 ”—r 0 ' ‘ O . ° ° n o o m 3 .4 DENSITY (no/p mo DENSITY (Wynne‘ Figure 9. Results of density measurements for PL and PM. In this figure and Figures 10 and 11 the abscissa repre- sents the density as cells/micron3 X 10' while the ordi- nate represents the frequency of occurrence of a given density. In the upper right hand corner of each histogram is the number of cells counted and the standard deviation of the sample. The coronal plane is represented by the upper histogram, the sagittal plane by the middle histogram, and the horizontal plane by the lower histogram. Mean densities for the respective planes were: Coronal PL 1.638 x 10': 1,u69 X 10- cells/micron3 PM ” Sagittal PL 1.789 x 10'” " PM 1.663 x 10‘ " Horizontal PL 1.731 x 10"LF " PM 1.622 x 10‘ " Within planes comparsions of the means by the Newman-Keuls method revealed no significant differences between is and PM. FREQUENCY A 3": ‘T ' ‘2‘. . ,, {‘3‘ .7 .1".__é‘ . .~ ~, . . . . . . 71 i:\ '\ \ fisfl;.rr’- flfatyfil ”1“ ‘2 t ' "‘4 1:. 4 . . . 4..- ,‘_ t3}: ' ’fig‘ .153 £- 1 L i'gx’" 3.. 5 .‘.. 16" ~ \_~ -9 A“. . g ‘ ~ .g ' - . . . .:... . .. .- ' . ,0 0 $1“ - . . v s ‘ . ’E ‘ V§3 ' . a 0 ‘§.“.s* ‘1. N 05 "&".q >- m 1 3° —‘ 5L 78:1 #4:)? ‘1 :2: . \ :w . . . ' \. ~. 8 '..o . .‘. . U o 0' ofi ’3‘. a... u . ‘ E v53‘1vr- - ~ 9 :L , a. ' “r 3. fi.‘- 1 3- - ' ~ 5 ' o '4' \ ‘ ‘ . J . . - 5.. "\,‘ '. .. ‘4 \ ' ' ..- " c .- . ' .‘. I .'. f“. .. a '3':- " J. y . ‘ ~. " ‘- .. I- ' o , . : . ~.m 8.3.. :,wr~ t‘ ~~‘** °° o I? 5+} -..,..-,.... .~ M” 3k ‘1 £3- 53” -~" 0‘0 . .2 . . ' _. '. . .-,-‘ .‘ '..-.l -’.,-'.-.',’~‘TA ‘."" .-"".-.'.. 1‘ DENSITY mflm (If-3p 9&wfin fir, , .. A, '. ~, ~ I. . «A ~ .1...» DENSITY (no/yum" eagfttal section Figure 10. Results of density measurements for PDM and PDP. Ordinate and abscissa are the same as in Figure 9. In the upper right hand corner of each histogram is the number of cells counted and the standard deviation of the sample.The upper histogram represents the coronal plane, the middle his- togram represents the sagittal plane, and the lower histogram represents the horizontal plane, The means for the respective planes were: Coronal 5.938 X 10-: cells/micron3 1.979 X 10- " PDP PDM Sagittal PDP PDM 5.935 x 10:: 2.072 X 10 Horizontal 8.171 x 10:: 3.1h8 X 10 fl PDP PDM Within planes comparsions of the means by the Newman-Keuls method revealed PDP significantly greater in density than PDM at the .01 level of confidence. PM ‘. ll189 171 0.6.! .19! tdIM 5 5 ‘ 0 O ). N 215 > 191 g m .3911 ‘2’ ad». I] II 3 5 3 5 8 8 I: I: IL I. O 0 NI196 N act-1511M “a .13110'4 5 5 I 0 o o 12 0o o 12 DENSITY Inc/y’mO" DENSITY (wwmo‘ N-oo 4- .84110 5 > o N 27 > c t) . 2 s .d.‘ 36 ‘ g = = 2 E c I. t o N 00 dn.72l10' 5 I o o o u o o n DENSITY th" DENSITY (mam-b" PDM PDP Figure 11. Composite of the histograms of Figures 9 and 10 for purposes of comparison. Refer to Table 5 for results of between planes comparisons of the mean cell densities. ‘ DISCUSSION An Overview Generally, in the rat IPN, it has been found that the constituent cells are grouped into 9 spatially distinct sub- nuclei. These subnuclei are PL,PM,PbM,and PDP. While the lat- ter two subnuclei exhibit marked differences in both cell size and packing density the former two are not significantly dif- ferent from one another in terms of these measures. Because of the quantitative cytoarchitectonicsimilarity between PL and PM one might hypothesize that they are functionally sim- iliar subnuclei which have been separated from one another by two intrinsic fiber bands. That quantitatively different cytoarchitectonic divisions exist within the IPN is assumed to indicate a functional differentiation of the nucleus. Cell Size The high value for'U)2 for subnuclei (.46) and its rel- atively low value for the interaction and planes effects in- dicates that a knowledge of the subnuclei is a more efficacious predictor of cell size than is a knowledge of planes of sec— tion or both subnuclei and planes of section. This measure may be interpreted as indicating that our uncertainty about the cell size is reduced by 96% if we know the division from which the cell was sampled. This is compared with a reduction 23 24 of uncertainty of only 9% if we know the plane in which the nucleus was sectioned. Given both planes of section and sub- nucleus the uncertainty in predicting cell size is only re- duced by 1% over that given by the other two effects in- dependently. The virtual constancy of the ratios(Table 3) across planes (for a given ratio) indicates that differences which occurred between certain divisions aqmoss planes are prob- ably due to shrinkage. Thus, for example, PLcor was sig- nificantly different from PLsag and PLhor, although the ratio,PL/PM, was essentially constant for the respective three planes. We can conclude that within animal comparisons (i.e. within a given plane of section) of mean cell size in dif- ferent subnuclei can be done directly. This is opposed to between animal comparisons (between planes of section) which' must take the form of ratios or some other similiar technique which effectively'cancels ‘\ N \ \ \ 1 S Iv": ‘f ‘ \ .'. { \ o ‘. ,- . O \ ‘1 ‘J, \ I \ r‘ . .‘. I l {f a I ’9: .x‘... . s...\\u.... . .. . \ h... .D ... \ a. . v. v I. .“.... V .\o o... p .t . JG. If)! a ..b . . 14. .v \. IN.‘ .:..... 0.3.1.2.-. Y. . . ,. , ..-............. t” ..n . V . . M}. n . n J .. .. I .I ~15".....v s D\ o ..u\ I PS 6 I uvu ‘V. ’l‘“! J. D'IHI .. s. 1.. I . 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D ..:. v ’ E; '0‘! . ., .‘ft .‘.-0" . ., ‘_ r ‘0‘. a Ta 0 v d L.‘ I Q ,\ .’ I -at:- ; 'vd \. ’II '1 1 \ 5 l. .’ .. . , r.‘. , ' v.41: . 33 NINTERPED PARS LATERALIS Plate 1. Caudal section through the midbrain at the level of the decussation of the superior cerebellar peduncle (DBC). In this and the following plates the midbrain is magnified 30 times. For a further description refer to the text. sacoase casscmwfimm 39 \ NINTERPED, . . I. C.TEG.V NT. PARS MED. V °« 120A @127 ‘37 L m'fis'Tffi'EERIs _ .. .__ A Plate 2. Caudal section through the midbrain at the level of the trochlear nucleus (IV). For a description of the IPN at this level refer to the text. . .I’I K ‘llr‘3‘; . .. .‘.. .. . . 3..... . 0;»..th I .:..... massage SHSSCmUHQm 35 €3LIW! NIMIRPCD 'ARS m N. N. INTERPED. UN- PARS DORSALIS ue. PARS PARvo. IG. ./ w 0 135 NJNTERPE0,PARS LATERALIS _ I Plate 3. Coronal section through the midbrain at the level of the caudal extent of the superior colliculus (COL. SUP.). 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