WTIIMIIIIIIIE Iplm'hm9mnw ;, W; 1;." I; mm IIIIIIIII' III!I‘FIII:'?IIIT'IIIIWIII{!II IIII,§3."I'§III'II'MIIII [III EIE'II 'I"'I;I'I'III -‘ 7M gfII'IIIIWIIIIINIII WI .HIIfMII...” uh; .; .,I I; I '5'" I I I 'j‘II'II} Y :lII'. 'I' n I. :,;:;' ;, MI ”I '.J- 'II'I‘.‘ ‘ I| '4 ‘ ;..'12 ml: l“ 'I' II I "III SI. IIIIJ. IIIIIIIIIIII .II'” “III” " III .1.‘~‘IIIIIII~"I III III: II . III In; I'“ III. I! EII III. III 'I'. IIIJIIIQ I I141: III I IIIII‘II‘I" I‘II‘I I II’IIII‘I'II'I .4 .. .. ' I 0. . 'H L' 'L‘ ,. "'I..‘,' ' ;.I 1.; H ' I I IFI'III 'I"'I II 'I" ""5 " "“II'IvIr WWII .I‘I "I-"'I' ".II I'III' III.I"I;. 'II "fI'I'I 'II'IIIII'I""II'I IIII'I'IIII'I"'II" I' " " . _ '2'“ 'II'III 992;“ . ”III" 1'1. " I""I"’I"";:",\'.t']:I':I I. “'Ir'I' . [t:i"III}—'; I; (ELI?! "H; W? 'I.' . I‘IIIIIIIIIII' IIII ' W :Is..."IIIIIII;.1'.I“III';I;;II7I:'II1'I'""III'IIII II II: Ij‘ I‘IIuI III-II IIIII IIIEIII'I~III:II§~ I“ ' I" I“ E ' 3.1”I‘uI’II”? fl" I' I'JII‘I'h MI "WWW 3n “1‘? LIIIII ‘i:;I.I( W' Fig; " "”'"II{' ”WWW-'1 ' ' 'I'2'2'III' . - v ‘ I I . ; . ('1 1‘” ». q . I“. I. IIIIIH..miI.II‘I""'IWI" IIIIII I'II‘I ZIIHI; I;II.'I'$' IjJI'W :"I IS" IEIII’ '{IIII‘J‘T-I. . IIIIIT'IIInIsZIi: w; I ,I'.” F111,“ . ' _ '1 £22]: ‘giilh’ufl; 'W'I I ”(1%”; Qiiifii ""f’;';‘tn : :Efirx 34);!P'": ‘HJ i: . :3 -I in III" III; I IIIIIII‘I'IIIII‘IIJ. II’I'“ "II,.IS:I' O 5 0 )— Z i“: * tr: I.“ / ...?"‘:.;:§:§:§:1:7" 0 a SINGLE MULTIPLE Figure 4 - The percentage of sanatic menbrane contacted by terminals which formed single and multiple synapses. (Means and S.E., * significantly different) 25 3.0 CI counnots I] oenvonmss 2.0 nu “ (.9 < I: Lu ~ * > 8 I- z ......... nu ..... o .~.;‘.§L.'_.;;fig"._'..'.;.;_ m ..... w / o a. spme penrommso Figure5-Thepercentageof sanaticmembranecantactedbyteminals mociated with “tic spines and perforated postsynaptic deaities (men and 8.8., ‘ significantly different.) 26 significant difference in coverage by single terminals was still apparent (controls, 3.23 t 0.95; dehydrates 4.57 :t 0.47, p< 0.01). When multiples were compared there was no significant difference between the two groups (controls, .481 0.11; dehydrates 0.65 i0.16, p > 0.05). when singles and multiples appwed to smatic nanbrane with psdswereexpressedasapercentageofallpsdassociatedterminalsa significant increase in the proportien which were multiples was founnd in dehydrates (14.7 t 2.2) compared to controls (6.03 :t 1.3, p < 0.007). Figure 6 illustrates the relative contribution of singles annd multiples to the total axcnal contact with MR: sonata. Dehydrates had a significantly higher percentage of their total axcnal coverage made by terminals which formed multiple synapses ccmared to controls (dehydrates, 15.15 i 1.79: cantrols, 5.34 i 1.79, p< 0.0027) with a corresponding decrease in percentage made by single terminals. The percentage of terminals which were multiple, spinne and perforated is shown in Figure 7. Although nno significant differences were found in spinne (controls, 5.88 i 1.47, dehydrates 7.27 :t 1.89) or perforated synapses (control 12.00 1 1.89, dehydrates 7.33 r 1.70) a significant increase in the percentage of terminals which formed multiple synapses was seen in dehydrates (17.37 t 2.02) canpared to controls (5.36 r 1.66, p< 0.001). Stereolggical Measures The calculated mean disk diannneter (A) for controls was 1.43 nm annd 1.36 pm for dehydrates. Thus the mean surface area of an inndividual contact betweena terminal anndcell some for controlswas 1.60 m2 for 27 100 CONTROLS ._ it [:1 / DEHYDRATES ’_ _/ 2 n... H 2 / O H/ 0 _n _/ 5° < 5 h x < _ '2 _/ * Lu 0 _. V an: 111 / 0 ‘L SINGLE MULTIPLE Figure 6 - The percentage of total axonal contact with nade by terminals which formed single annd multiples synapses. (Mean annd S.E., * significantly different . ) 28 / * CONTROLS DEHYDRATES MULTIPLE SPINE PERFORATED 20 15 10 Figure 7 - Percentage of synapses which were multiple, perforated. (Mean annd S.E. , * significantly different.) PERCENT SYNAPSES spinneand 29 controls annd 1.46 m2 for dehydrated animals. It appears that the contact area fran dehydrated aninals is seller tnan controls, hmever, a statistical comparison requires that a meann dianneter be calculated for each aninal frann approxinately 300 syname lengths. The sampling in this study was nnot tnat extensive. The number of terminals which formed single synapses per 100 m2 of sanatic surface area wu significanntly higlar in conntrol aniinals (7.19 t 0.63) cannpared to dehydrates (4.63 r .44, p< 0.006) while the number of terminals which formed mltiple synapses per 100 m2 was significantly lower in conntrols (0.36 r 0.10) compared to dehydrates (0.85 10.16, p<.03). Cell Parmeters Tablelliststhegroupamaansandstandarderrors foreachofthe shape naasures obtained. There were nno significant differences between controlsanddehydratesinannyof theseparaneters. FigureSShOwe the size frequency distribution of cell surface area (W) for all of the measured cells. The distribution of cell surface areas for dehydrated aninnalsshifted totheright. AsshowtninFigure9themeansurface area of cells in dehydrated anninnals (2694 r 116) was significantly larger thann controls (1570 .t 83, p< 0.0001). TABIEl-CEILSHAPEPARW GROUP ASPECT SHAPE FDCEN- RATIO Fm TRICITY Control Bean 0 . 6785 0 . 7965 0 . 7143 8.3. 0.0063 0.0105 0.0058 Dehydrate Mean 0.6869 0.7882 0.7073 8.8. 0.0193 0.0145 0.0194 3O ""7 CONTROLS D Y VVVVV [:1 EH DRATES 25 ....... ....... ...... ...... ....... ...... ....... 2O 15 1O ............ NUMBER OF CELLS 700 1514 2328 3142 3956 4770 SURFACE AREA [pmzl Figure 8 - Size frequency distributiun of MC sunatic surface area for control and dehydrate animals. 31 3000 a): F“ -—( 2000 En" - E .3 < E 1000 < DJ 2 LI. m D U) / 7° CONTROLS DEHYDRATES Fignne9-11nemeansurfaceareaofnaurcnswithineachenperimental group (than and 8.3. , * significantly different). 32 Mr of was per W CelliBodY Figure 10 illustrates that the number of terminals which formed single synapses per cell body was not significantly different betmen the two groups (controls 111 r 9, dehydrates 125 i 14). Themmber of nmnltiple synapses per cell body was significantly higher in dehydrates (22.82 t 4.5) than cantrols (5.8 i 1.8, p< 0.009). The total number of synapses per cell body did not differ between the two groups (controls 120.0 t a), dehydrates (143 r 17). DISCUSSION mtitative wethods Sinncethegoalofthisstudywastoobtainuaasuresofthesynaptic input to bins, several asemnptions made in the qnantioative analysis deserve cumnt. The morphological criteria used to measure synapses varies considerably in morphomatric literature. The strict morphological definition of a synapse requires trat a presynaptic terminal with vesicles is amosed to postsynaptic membrane with a well defined postsynaptic de'aity. Many investigators assume that any apposition between a terminal and postsynaptic nenbrane eventually formaconventiualsynapseandtlunsusetheappositiunbetweena terminal and postsynaptic naubrane, regardless of pads, as an indicator of synaptic input. This study followed this model and measured the trace lengths of all terminals which centacted m0 sunata. These legthawerethenusedtocalculatethenumberofsynapsesperlOOan (Na) and the number of synapses per suna. Althongh the possibility exists ttat the above measures were overestimated since all contact legths were used (i.e., perhaps not all terminals form conventional 33 150 CONTROLS Q C] DEHYDRATES / %é§:§:;5if§5fi'1 10° _I _l LLI O 50 m I.“ O. U) I.” (I) Q < Z >. 0 (D SINGLE MULTIPLE Figure 10 - The number of single and multiple somatic synapses per cell (man and 8.3. , * significantly different). 34 was with psds) , the extent the cverestination is probably quite small since over 97% of the terminals followed in serial section formed synapses. Not all the terminals investigated in this study conformed to the assumption that appositiona zones between terminals and cell sanata were disks. The percentage of terminals which were associated with acetic spines in serial sections analysis was 22% in controls and 31% indehydratedaninals. T‘hesepercentagesarenuichhigherthanthose obtained in single sections where the percentages ranged fran 1.5% to 11.1% in control aninals and O to 13% in dehydrated anninals. Althongh slaps assnmticna do not influence the surface density (Ss) measures. they could affect the calculation of number of synapses per m2 (Ns) andthenumberofsynapsespersma. Theextenttowhichthissl'ape assumption influences these calculations is unknncm, although the extent of the bias probably remains constant for both groups . Methods fordeterminingthenuunberofsynapsespernmit volumeof tissueare available which do not require shape assumpticxa (see de Groot & Biernuan, 1986), however whether or not these procedures are also applicable to the number of centacts per unit surface area is not known. As mticned earlier, tissue volume was not used in this study since differential changes in tissue volmne are likely under the preent experimental canditions. Typically estimates of cell surface area assnme ttat neurons are either shaped like spheres (e.g. We & Matthew, 1985) or prolate sflneroids (e.g. Kaisernan—Abcramoff & Peters, 1972). Since previous research showing increases in SON cell size have used a prolate 35 spheroid as a mdel stape for neurons (e.g Ito. Iijina & Kawada, 1986; Kalimo. 1975) and since the measured shape parameters did not suggest ttatDmswerespheres, thesnn'faceareaofMICswascalculatedusing the fornmrla for a prolate spnercid. 'nne sanpling technique used to select cells (i.e. onnly cells with a praninent nuncleolus) may have biased the measured surface area of m since multiple nucleoli are not uncamn, and nucleoli are often eccentrically plead. Thus some ofthecellsmaynotravebeensectionedthrcughtheirlargestextent and their surface area would have been underestiuated. This is particularly true of the dehydrated aninals where multiple nuncleoli are rare m than in well hydrated aninals (Hatton & “alters, 1973). The extent of the underestimation is not 1cm, although calculation of aprogressivemaenfor lcngandshortFeretdimtersfranmcells frauacantrolanimlandzocellsfrauadehydrateeninalresultedin less ttan 10% variatian in these neans. Multiple Syngees and mo Sonata In the first report of synapse formation in the SON after chronnic dehydration, Tweedle and flatten (1984) reported an increase in the percentage of uagnocellular neurens contacted by multiple synapses frcn 0% in well hydrated aninals to 20% in chronically dehydrated anninals. These percentages are probably underestinates since the morphological criteria of a synapse included the presence of a synaptic thickenings, and measures were unis on single thin sectiens. The present has further documented multiple synapse formation in the SON. Virtually every measure pertaining to these nmiltiple synames substantiates this claim. The increase is reflected not only in the number of multiple 36 synapsespercellbody, butalsointhesubstantialincreaseinthe relative number of terminals which formed multiple contacts as well as the relative contribution of nunltiple terminals to the total anneal contact with Me. That the percentage of sauatic menbrane covered by multiples with an associated psd was nnot higher in dehydrated animals does not necessarily contradict this conclusion. Given tnat a large inncreaseincell surfacaareaoccurs, anequalincreaseinagiven structure must occur to maintain equal coverage of the sciatic meubrane. This is clearly denunnstrated in the signnificant decrease in the percentage of aquatic membrane covered by single terminals in dehydratedaniuals. Thisresultdcesnot reflectadecreaseinthe numberofsingleterminalsbutratheranincreaseinthereference surface. Thus. lumingthatthesurfaceinncreasesanrithatthepercent membrane coverage by psd-associated multiple synapses did not signnificantly decrease, onne could infer tnat these contacts did. in fact, increase. The signnificannt increase in the percentage of psd— associated terminals which were multiple synapses in dehydrated animals provides further evidence for this conclusion. The lack of a significant difference in the total number of terminals per sea is surprising given the significant increase in the numberofmnltiplesynapsespersanaanrincckangeinthenumberof single synapses per neuren. There are two very different but perraps nnot exclusive explanatias for this paradox. First. the inncrease in the number of nultiple synapses per cell, although significannt nay be "lost" in the variability in the total number of sanstic synapses. Seccmd, the fornatien of multiple synapses is thought to be due in part 37 to the connversion of singles into multiples. If a single terminal which contacts a cell because a nultiple by forming a new contact with adjacent cell sonata or dendrites then the number of total terminals per sane would nnot dange. Although this offers a partial explanation for not detecting an increase in the total number of terminals per neuron, the data presented here do not support the canclusion tkat terminals which form miltiple contacts are constructed fran existing sciatic singles. If tnat were the case, a decrease in the number of single synapses would be expected in the dehydrated group. Since there was no signnificant difference in the number of singles per neuron, there is probably sane proportion of synaptic input to Mine which is newly formed. It shouldbannoted tratttenumberof sanaticsynapsesperneuron derived in this study is much larger than those reported in the literature (111, controls; 125, dehydrates). An average of 57 smatic synapsespernem'anhesbeencalculatedbasedonavolnmetricstudy (Leranth, Zaborszky, Marten & Pallnovits, 1975). This method derived the number of synapses based an a morphological criteria which required postsynaptic densities, nnot terminals, which may partially account for the observed difference. Itch, Iijima & Kowada (1986) using a morphcnetricmthodsimilar totheonneusedinthis studyreported49 salaticsynapeesperneuren. TleaveragesurfaceareaofMICsanuitle percentageofscuaticnembreeconcactedbyterminalsreportedbythis groupwesapprmninatelytresameasthosemeasnnedinthisstudy. The discrepanncy between the number of synapses per cell is directly attributable to a different mean surface area of individual terminal 38 contacts wlnich was 1.60 m2 for controls and 1.46 m2 for dehydrates in this study compared to 2.56 m2 reported by Itoh et a1. (1986). The data pertaining to perforated contacts annd contacts onto somatic spines must be regarded as preliminary since they were collected using a sample size which was optimized for all axonal terminals not for spine or perforated synapses. As might be expected there was a great deal of variability within the groups in these measures. Onecould infer thatthelackofadecreaseinthepercent MNC somatic membrane covered by axonal terminals associated with aquatic spines reflects an increase in these terminals or their size. However, given ttat tre percent coverage by spine associated terminals is low in both controls and dehydrated aninals annd the apparent discrepancyintheirpercentage founndbetweentheserial sectioningand morphcmetric data, this conclusion awaits a more comprehensive investigation. The experimental evidence accumulated to data indicates flat the synaptic ctaracteristiss of the SON are altered in very specific ways under conditions of increased hornene demand. In this study the specificity of SON 's response to activatian is reflected in the observation trat only the number of multiple synapses per cell body increased in chronically dehydrated aninals. Furtl'er evidence for specificity in SON's plastic responses to activation has accunm1lated fran ultrastructural stuiies of tie deriritic region (located ventral to the cell bodies) and electrophysiological experiments. Multiple synapsesintl'edeririticregionhavealsobeenfoundtoincreasein frequency but only immediately following parturition, not during 39 lactation or chronic dehydration (Perlmutter, Tweedle & Hatton, 1984; 1985) . Perhaps the formation of multiple synapses is related to an increase in the activity of specific afferent inputs to this system which vary under the different experimental conditions. This hypothesis is supported by recent i_n_ gig; electrophysiological enperimentswhichreveal tnatdyecouplingamongSONneuronsincreases after electrical stimulatien of tlne lateral olfactory tract, but only unnder rather specific experinental conditions. Lateral olfactory tract stimllation results in incrmed dye coupling in lactating animals annd virgin fenales which have been induced to behave naternally by exposure to rat pups (Kidney, Yang annd I-Iatton, 1987; Yang & Hatton, 1987). Such stimulation does nnot affect dye coupling in uale or untreated virgin fenele rats. Thne, the experimental literature related to plasticity intteSONrevealstlatthisnulcleusisindeedcapableofrearkable dangeaduringincrasesinhormonedeand, butthatthenatureand extent of its reorgannization are dependent on both physiological and environmental conditions. Given that multiple somatic synapses incrme during chronic dehydration, tne question still reusine as to tie mechaniau througln which this occurs. As mentioned earlier, previous research has suggestedtratmlltiplesynapsesintheSONareformedthroughtle cenversion of single contacts into multiples. The observatian trat only nultiple synapses inncrease in frequency certainly supports this hypothesis. Since tlere was nnct a concannitalnt decrease in tne number of single synapses per sans, the formation of multiples must nnct occur exclusively through tte conversion of aquatic singles into multiples . 40 Of course it is entirely possible ttat existing dendritic singles are converted into sona—dedritic multiples. This hypothesis has not yet been tested. Also, the possibility that anneal sprouting participates in the reorganization of the synaptic cnaracteristies of SON, through either tie formation of mlltiple synapses or in naintaining the number of single synapses, cannnot be eliminated. Previous ultrastructural studies of the can have denonstrated ttat increases in sma-sanatic and sauna dedritic direct meubranne apposition occur during dehydration (Chapmn, Theodcsis, Mantagnese, Poulain & Morris, 1986; Perlmutter, Tweedle & Hatton, 1984; T‘weedle & Hatton, 1976, 1977). A similar significant increase in direct membrane apposition was found in this study. Tnat increases in membrane appositionn occur before significannt increases in cell size occur led to tie canclusion that astrocytic processes, which are normally interposed between adjacent neural elemnts, retract frau this position and allow these appositionns to occur (Tweedle & Hatton, 1977) . Glial retraction has also been postulated to participate in tl'e formation of multiple contacts by allowing axonal terminals access to cell sanata and dendrites. In this stndy, tre percentage of cell menbrane contacted by glial processes was not significantly different in control and dehydrated animals indicating tl'at pernaps glial conntact per increased after 10 days of dehydration. A similar finding l'as been reported by Chapman et al. (1986). Indeed there is report tlat astrocytes proliferate during dehydration in young aninals (Patterson and LeBlond, 1977) . The canbination of results fran acute and chronnic dehydration snggest that the role of astrocytic processes thronghout -s 41 dehydration is more complex than a permanent retractionn of glial processes. Perhaps an initial glial retraction allows for increased neural contact (between neurons, dendrites and terminals) and as cells increase in size glial processes cover the "new" portion of the sciatic neubrane. Tnat a decrease in glial processes is associated with the initial stages of synaptogenesis has recently been demonstrated in the ventral posterior nucleus of the rat thalamns (Wells & Tripp, 1987a; 1987b) . In this nucleus, lesionns of the dorcal column nulclei result in reactive synaptogennesis which does nnot begin until 30 days post-lesion. A study of the time course of this reactive process revealed flat the initial stages of synapse formation were associated with a decrease in the area of neuropil occupied by glial processes canpared to nonnlesioned controls. As synmses were replaced, glial process area again increased such that at the canpletion of synaptogenesistnerewasnodifferenceintheareaoccupiedbyglial processes between lesioned animals and conntrols. A similar process oouldbeenvisionedintheSONwderetheinitialstagesofsynapse formation are associated with a decrease in glial contact. After chronnic dehydration (i.e. 10 days of saline drinking) perhaps synapse formation has been completed and glial processes again cover an equivalent proportionn of the sanatic surface. Since there is currently no information about when multiple synapses form during chronic dehydratian this hypottesis renains to be tested with a time course study that incorporates all of the various paraneters which are affected by dehydration, i.e. cell size, glial coverage and number of contacts . 42 .. The precise functional significance of multiple contacts in the SON is, at present unknown. Little is known about the nenrotranenitters contained in the terminals which form multiples or the peptide content of the cells they cannot. Immocytochemical electron microscopic studies have shown that terminals which form multiple contacts may contain dopamine (Buijs, Geffard, Pool & Hoornenan, 1984) or GABA (Theodosis, Paut & Tappaz, 1986) which suggests flat the formation of multiples nay not be transmitter specific. There is sure evidence flat the alterations which occur during chronic dehydration nay effect primarily oxytocinergic neurcms, (Chapman, et. al, 1986) but this result has not been replicated. that multiple synapses contribute to the overall excitability and serve to coordinate the activity of the groups of SON during periods of increased homnone demand likely, especially considering the alterations in the electrophysiological characteristics of MNCs during activation of the systen. Perhaps future studies will better delineate the tine course for the appearance of these synapses, annd the nature of their contribution to the excitability of SON neurons during periods of chronic homone release. LIS'I‘OF m LISTOFWES Armstrong, W. 3., Gregory, W. A. & Hatton, G. I. Nucleolar proliferation and cell size changes in rat supraoptic neurons following osmotic and volenic clallenges. Brain Research Bulletin, 1977, g, 7-14. Arnnold, A. P. Gonnadal steroid-induced organization and reorganization of neural circuits involved in bird song. In: Sygaptic Plasticity, edited by Geri W. Cotnan, New York: Guilford Press, 1985, pp. 263-286. Bandaranayake, R. C. Karyunetric study of hypotlalamic neurosecretory neurons under different conditions. Acta Anatomica, 1974, 9_Q, 431- 461. Bicknnell, R. J. & Lenng, G. Relative efficiency of neural firing patterns for vasopressin release in vitro. Nenroendocrinolggy, 1981, 3_3_, 295-299. Buijs, R. M., Geffard, M., Pool, C. W. & I-bornean, E. M. D. The dopaminergic innervation of the supraoptic and paraventricular nucleus. A light and electron microscopical study. Brain Mon, 1984, 32;, 65-72. Case, C. P. & mtthews, M. R. A quantitative study of structural features , synapses anri nearest-neighbor relationships of enll, granule-containing cells in the rat superior cervical sympathetic ganglion at various adult stages. Neuroscience, 1985, ;_5_, 237-282. Castel, M., Gainer, H. & Dellmann, D. H. Neutral secretory systm. International Review of Gytglpgy, 1984 , §_8_, 303-359 . Chapman, D. 8., Theodosia, D. '1'., antagnme, C., Poulain, D. A. & Morris, J. A. Osmotic stimulation canmes structural plasticity of neuron-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus. Neuroscience, 1986, 11, 679-686. Cobbett, P. & Hatton, G. I. Dye coupling in hypothalamic slices: dependence can in vivo hydration state and oenolality of incubation medium. Journal of Neuroscience 1984 , 1, 3034-3038 . de Groot, D. M. G. 8: Biernan, E. P. B. A critical evaluation of methods for estimating the numerical density of synapses. Journal of Neuroscience Methods, 1986, 18, 79-101. 43 44 Ellnan, G. L. & Gan, G. L. Responses of the cells of the supraoptic nucleus: Kinetic aspects. MEAN Research, 1971, 14, 1-8. Enestroln, S. Nucleus Supraopticus: a morphological and experimental study in the rat . Acta Patholggica et Microbiolggical Scandinavia m 1976, m. 1—96. Forsling, M. L. Anti-diuretic hormne (Vol. 2). Montreal: Eden Press, 1977. Gainner, H. Precmors of vasopressin and oxytocin. Progress in Brai_r_n_, Research, 1983, 6_g, 205-216. Gill, J. L. Design; and Aralsgis of garments in the Aninal and Medical Sciences. Ames: Iowa State University Press, 1978. Greencmgh, W. T. & Clarg, F. L. F. Synaptic structural correlates of information storage in namlian nervous systm. In: my; Plasticigy, edited by Carl W. Conan, New York: Guilford Press, 1985. 311-334. Hatton, G. I . Reversible synapse formation and modulation of cellular relationships in the adult hypothalamus under physiological corriitions. In: Mtic Plasticity, edited by Carl W. Cotnan, New York: Guilford Press, 1985, pp. 373-404. Hatton, G. I. & needle, 0. D. Magnocellular neuropeptidergic neurons in hypotnalamus: Increases in membrane apposition and number of specialized synapses from pregnancy to lactation. Brain Research Bulletin, 1982, a, 197-204. Hatton, G. I. & Vhlters, J. K. Induced multiple nucleoli, nucleolar mergination, and cell size changes in supraoptic neurons during denydratien and rehydration n the rat. Brain Research, 1973, §_9_, 137-154. Hatton, G. L, Yang, Q. 2. 8: Cobbett, P. Dye coupling among immocytochenically identified nneurons in the supraoptic nucleus of lactating rats. Neuroscience, 1987, 2]_1_, 923-930. Itch, Y., Iijima, K. & Kowada, M. Ultrastructural and morphometric studies of noraecretory neurons of the rat supraoptic nucleus. Acta_Apatg§0.05 p>0.05 p>0.05 p<0.0001 df=7 df=6 57 APPENDIX.C (Cont'd.) TABLE 6 - PERCENTAGE OF'MNC SOMATIC MEMBRANE COVERED BY VARIOUS CELLULAR ELEMENTS ANIMAL GLIA AXONAL MNC OR NOT DENDRITE IDENTIFIED CONTROL 029 85.97 11.38 0.00 2.65 052 83.06 12.16 0.76 4.02 065 82.87 13.09 0.89 3.15 069 87.67 9.66 0.44 2.27 089 83.84 10.92 1.32 3.91 099 80.67 15.83 2.58 0.93 Mean 84.01 12.17 0.99 2.82 SE 1.01 0.87 0.36 1.16 DEHYDRATE 012 55.25 12.65 9.34 22.76 D17 76.09 6.55 11.23 6.48 D47 81.03 8.56 5.06 5.35 D54 79.26 6.79 8.83 5.12 D56 82.23 6.38 7.62 3.76 D83 85.10 7.73 6.10 1.03 D95 77.97 7.47 7.32 7.23 Mean 76.70 8.02 7.93 7.40 SE 3.74 0.82 0.78 2.67 t-tests Glia t'= 1.88 p > 0.0500 df=6 Axonal Contacts t = 3.45 p < 0.0050 MNO or Dendrites t = 7.58 p < 0.0001 unidentified t'= 1.55 p > 0.0500 df=6 58 APPENDIX 0 (Cont'd.) TABLE 7 - PERCENTAGE OF MNC SOMATA COVERED BY TERMINALS (BY TYPEL, ANIMAL SINGLE MULTIPLE SPINE 0.44 0.30 0.56 1.25 1.89 1.11 1.00 0.62 9_:_7_6_ 1.24 CONTROL 029 10.93 052 11.90 065 12.50 069 8.40 089 10.10 C99 M Mean 11 . 58 SE 1.01 DEHYDRATE 012 10.36 017 5.50 047 6.67 054 5.70 056 5.38 083 7.20 095 6.70 man 6 . 79 SE 0.65 t-tests Single Multiple Spine Perforated 4.12 I 2.23 I 0.76 = 2.69 PERF 2.05 2.12 0.61 1.74 1.24 3.30 0.94 1.48 0.40 0.90 new). 0.89 2.16 0.28 0.10 0.00 1.43 1.23 0.42 0.56 0.95 1.26 0.36 0.33 0.52 0.30 1.45 Q;§§_ 1.68 0.64 0.97 0.18 0.21 p < 0.002 p < 0.047 p > 0.050 p < 0.020 APPENDIX 0 (00nt'd.) TABLE 8 - PERCENTAGE OF TOTAL AXONAL COVERAGE MADE BY’SINGLES & MULTIPLES CONTROL, DEHYDRATE ANIMAL SINGLE MULTIPLE ANIMAL SINGLE MULTIPLE 029 96.10 3.90 012 81.99 18.07 052 96.86 2.01 017 84.32 15.68 065 95.68 4.32 047 78.01 22.05 069 87.02 12.98 054 83.50 16.43 089 92.24 7.76 056 84.32 15.68 099 98.96 1.01 083 92.10 8.01 __ __ 095 83:99. 10_-1-_5. Mean 94.64 5.34 84.88 15.15 SE 1.79 1.79 1.78 1.78 t-tests Single t = 3.822 p < 0.0028 Multiple t = 3.347 p < 0.0027 60 APPENDIX C (Cont'd.) TABLE 9 - PERCENTAGE OF STNAESES FORMING SINGLE, MULTIPLE,_ SPINE AND PEREORATEQ CONTACTS ANIMAL SINGLE MULTIPLE SPINE PERF CONTROL 029 98.15 1.85 11.11 11.11 052 96.10 3.90 3.90 9.09 065 96.00 4.00 6.67 20.00 069 89.13 10.87 8.70 10.87 089 90.00 10.00 3.33 6.67 099 §§;4;_ 1.59 1.59 14.29 khan 94.63 5.37 5.89 12.00 SE 1.66 1.66 1.47 1.90 DEHYDRATE 812 79.41 20.59 0.00 7.35 817 82.00 18.00 12.00 4.00 847 80.43 19.57 4.35 8.70 854 78.38 21.62 13.51 2.70 856 77.78 22.22 7.41 3.70 383 90.91 9.09 3.03 9.09 895 §2122. 10.53 10.53 15.79 Fheun 83.62 17.23 7.26 7.33 SE 2.03 2.03 1.90 1.70 t-tests Single t = 4.48 p < 0.009 Multiple t = 4.48 p < 0.009 Spine t = 0.55 p > 0.050 Perforated t = 1.83 p > 0.050 61 APPENDIX C (00nt'd.) TABLE 1£>'- PERCENT CINERAGE (Hi MNC SOMATA.WITH POSTSYNAPTIC DENSITIES CONTROL Singles Miltiples Total 029 7.97 0.44 8.41 052 9.03 0.26 9.29 065 7.58 0.57 8.15 069 5.20 0.98 6.18 089 7.33 0.48 7.81 C99 1.29.2.9 $1.5. 121$ hisun 8.23 0.48 8.71 SE 0.96 0.12 0.85 DEHYDRATE 012 6.84 1.49 8.33 017 4.10 0.68 4.78 047 4.31 0.77 5.08 054 3.17 0.43 3.60 056 3.81 0.64 4.45 083 5.58 0.28 5.86 D95 4_-21 M LL-fi Fheun 4.57 0.65 5.22 SE 0.47 0.18 0.58 t-tests Single t = 3.598 p < 0.0042 Multiple t = 0.8487 p > 0.0500 Total t = 3.469 p < 0.0052 62 APPENDIX C (Cont'd.) TABLE 11 - NUMBER.OF SINGLE AND MULTIPLE SYNAPSES PER 100 pg; 0F MNC SOMQEIC MEMBRANE Multiple t = 2.46, p < 0.031 CONTROL DEEYDRATE ANIMAL SINGLE mLTIPLE ANIMAL SINGLE MILTIPLE C29 6.80 0.27 012 7.08 1.57 052 7.40 0.19 017 3.76 0.68 065 7.78 0.35 047 4.56 1.29 069 5.23 0.78 054 3.90 0.76 089 6.22 0.50 056 3.68 0.68 099 9.75 0.10 083 4.92 0.42 __ __ 095 5.19.8. 91.5.2. Mean 7.19 0.36 4.64 0.95 SE 0.63 0.10 0.44 0.16 t-tests Single t = 3.39, p < 0.006 63 APPENDIX 0 (Cont'd.) TABLE 12 - NUMBER.OP SYNAgSES PERJMNC SOMATA CONTROLg DEHYDRATE ANIMAL SINGLE MULTIPLE TOTAL ANIMAL SINGLE ‘MULTIPLE TOTAL 029 116.48 4.69 121.17 012 198.84 44.14 242.99 052 113.20 2.85 116.05 017 117.24 21.32 138.56 065 122.19 5.47 127.66 047 118.63 33.61 152.24 C69 96.87 14.41 111.28 054 87.34 17.01 104.34 089 77.89 6.23 84.13 - 056 100.04 18.59 118.63 099 146.19 1.49 147.69 083 146.19 12.59 158.78 095 110.02 12.48 122.50 menl12.14 5.96 118.00 125.47 22.82 148.29 SE 9.46 1.85 8.53 14.03 4.46 17.36 t-tests Singles t a 0.77 p > 0.050 Multiples t = 3.29 p < 0.007 Tbtal t = 1.43 p > 0.050 ")I'iififiiijhilflw(Riflijifliflifilflfliflfiflll’fl