Compensatory sprouting of uninjured magnocellular neurosecretory axons in the rat neural lobe following unilateral hypothalamic lesion

EXPERIMENTAL

NEUROLOGY

111,

g-24

(1991)

Compensatory Sprouting of Uninjured Magnocellular Neurosecretory Axons in the Rat Neural Lobe following Unilateral Hypothalamic Lesion’ Department

JOHN A. WATT

AND CHARLES

M. PADEN

of Biology,

State

Bozeman,

Montana

University,

59717

mammals (5, 29, 48). Subsequent studies have shown that following hypophysectomy or neurohypophysectomy, the severed neurosecretory axons sprout to form new neurohemal contacts both at the proximal end of the transected infundibulum (26,30,38, 39) and within the zona externa of the median eminence (25, 39). Regenerative growth of neurosecretory axons across the severed infundibular stalk has also been described (1, 27), as has penetration of intrahypothalamic grafts of neural lobe tissue (7,lO) and of the ventral leptomeninges (11) by sprouts from severed magnocellular axons. Thus, regenerative responses of magnocellular neurons following axotomy have been demonstrated in a variety of situations. However, the potential ability of uninjured magnocellular axons to undergo compensatory sprouting has not been previously investigated. Compensatory sprouting of intact contralateral parvocellular neurosecretory axons into a denervated region has been demonstrated within the zona externa of the median eminence following unilateral lesions of the paraventricular nucleus (PVN) in adrenalectomized rats (46). This finding led us to investigate whether intact magnocellular axons might possess a similar capability. We report here that compensatory sprouting of intact contralateral magnocellular efferents occurs within the partially denervated neural lobe following unilateral transection of the hypothalamo-neurohypophysial tract in young rats. This system provides a new model for further investigation of compensatory sprouting in central peptidergic neurons.

Axonal sprouting of intact neurons of the magnocellular neurosecretory system was investigated using a unilateral hypothalamic knife cut of the hypothalamoneurohypophysial tract to partially denervate the rat neural lobe (NL). Densitometric, morphometric, ultrastructural, and metabolic measures were utilized to demonstrate the compensatory response to denervation in this system. Densitometric analysis revealed a transient reduction in the intensity of vasopressin staining in the NL at 10 days postsurgery (PS) with a subsequent recovery by 20 days PS. There was a comparable initial reduction in the cross-sectional area of the NL followed by a more gradual recovery to normal by 90 days PS. Ultrastructural investigation revealed a reduction in total axon number in the NL at 10 days PS similar to the declines in vasopressin immunoreactivity and size of the NL. A subsequent partial recovery of axon number occurred, paralleling the return to normal NL size between 30 and 90 days PS. Hypertrophy of both somata and cell nuclei of magnocellular neurons in the supraoptic and paraventricular hypothalamic nuclei contralateral to the lesion was also apparent during this period. Daily measurements of urine osmolality revealed an initial transient hypoosmolality followed by a chronic hyperosmolality which persisted throughout the 90 day postsurgical period. There was a concomitant chronic decrease in both daily drinking and urine excretion volumes which began immediately following surgery. These results suggest that intact, contralateral magnocellular vasopressinergic efferents undergo compensatory sprouting as a result of partial denervation of the NL in the absence of a functional deficit in vasopressin. 0 1991 Academic Press, Inc.

METHODS

Animals. Male Holtzman rats (bred from stocks originally obtained from Charles River) were 35 days of age when hypothalamic lesions were performed. The sprouting efficacy of transected vasopressinergic neurosecretory axons has been shown to decrease with age (25,26), and this reduced propensity has been observed as early as 23 days postparturition (26). Therefore, we chose to use young rats to minimize any age-related impairment in the sprouting response. Animals were individually housed under a 12L:12D light cycle with ad lib

INTRODUCTION

The magnocellular neurosecretory system was one of the first sites in which presumptive sprouting by the severed axons of central neurons was demonstrated in

1 Some of these data have been previously published form (J. A. Watt and C. M. Paden, Sot. Neurosci. Abstr. and ibid. 15: 91, 1989).

Montana

in abstract

14: 116,1988; 9

All

Copyright Q 1991 rights of reproduction

0014.4886/91$3.00 by Academic Press, Inc. in any form reserved.

10

WATT

AND PADEN

access to tap water and lab chow throughout the investigation.

Hypothalamic lesions. Hypothalamic lesions were routinely performed under sodium pentobarbital anesthesia (50 mg/kg ip) (Nembutal, Abbott Laboratories). Animals also received methoxyflurane (Metofane, Pitman-Moore) as needed to deepen the plane of anesthesia. A wire knife constructed of HTX-33-gauge tubing was used to unilaterally transect the entire length of the hypothalamus through which the hypothalamo-neurohypophysial tract passes (lesion coordinates: AP -2 to +5 mm, ML -0.5 mm, approximate DV -10.2 mm, with the incisor bar in the horizontal position; bregma was used as stereotaxic zero, and depth was measured from the skull surface). The knife blade was positioned at AP -2 mm, lowered until it was felt to flex slightly upon touching the ventral surface of the cranial cavity, then raised 0.5 mm, and slowly brought forward to complete the lesion. Gelfoam (Upjohn) was used to inhibit bleeding, and nitrofurazone (TechAmerica) was applied as a topical antibiotic prior to suture of the scalp. Littermate sham-operated controls were prepared using identical surgical procedures except that the lesion extended only -5 mm below bregma to avoid damaging the hypothalamus. Tissue preparation for light microscopy. Tissue was collected from groups of experimental animals sacrificed between 10 and 90 days after surgery as well as from age-matched sham-operated and intact control animals. Animals were perfused intracardially with a mixture of 125 ml of formalin + 150 ml of saturated picric acid in 0.1 M phosphate buffer, pH 7.4, under deep ether anesthesia, and then decapitated, and the brain was removed with the neurohypophysis attached. The hypothalamus and attached neural lobe were excised, submersed in fixative overnight, then dehydrated, cleared in xylene, and embedded in paraffin. Ten-micrometer coronal sections were collected at 50-pm intervals throughout the extent of the supraoptic (SON) and PVN nuclei and serially through the entire NL. Hypothalamic sections were cleared and stained with cresyl violet for confirmation of lesions and morphometric analysis of magnocellular neurons. Immunocytochemistry for vasopressin was also performed on some sections of hypothalamus as described below. Densitometric determination of relative staining intensity. Fifteen sections of the NL per animal selected from approximately equivalent levels were deparaffinized and incubated as follows: rabbit anti-arginine vasopressin sera Nl-F (1:5000; gift of Dr. G. Nilaver) for 24 h (4’C); biotinylated goat anti-rabbit Ig’s (1:lOO; Cappel) for 1 h at room temperature; avidin-biotin complex (Vector laboratories) for 30-60 min; and 3,3’-diaminobenzidine with 0.003% hydrogen peroxide for 45 min. Because of the large amount of tissue to be examined,

immunocytochemistry was performed in batches composed of sections from each experimental and control group. Quantification of staining intensity and measurement of NL area were performed using an MCID image analysis system (Imaging Research, Inc.).

Cellular morphometry. Cross-sectional areas of cell nuclei and somata in the intact contralateral SON and PVN were measured by tracing microscopic images from sections stained with cresyl violet directly onto a digitizing tablet via a drawing tube. Stereometric calculations were performed using software developed by Dr. Steven Young of UCSD. Fifty individual magnocellular somata and their respective cell nuclei were measured in both the contralateral PVN and SON in each animal. Only neurons possessing a distinct cell boundary, a clearly defined nuclear envelope, and a distinct nucleolus were measured. Measurements in the PVN were restricted to neurons within the PVL and PVM magnocellular subnuclei known to project to the NL (3). Measurements in the SON were collected from within both the principle and retrochiasmatic divisions. Preparation of tissue for ultrastructural analysis of the NL. Animals were perfused intracardially under ether anesthesia with a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M NaPO, (pH 7.4) at 5,10,32, or 90 days postsurgery. Intact control animals were sacrificed at 35 and 125 days of age, corresponding to the age of experimental animals at time of surgery and 90 days postsurgery, respectively. The NL was removed and postfixed 4 h in 2% OsO,, dehydrated with ethanol and propylene oxide, and then embedded in Spurr’s resin using flat Beem capsule molds with the rostra1 tip of the NL positioned at the tip of the capsule for orientation. Blocks were trimmed so that ultrathin sections were collected only from the central region of the NL, the area predominately occupied by efferents from the SON (2). Sections were counterstained with uranyl acetate and Reynolds lead citrate for examination under a Zeiss EMlO/CR electron microscope.

Quantification of axon density in the NL. The number and density of axons in the NL were estimated by counting total axon profiles in each of 15 low-magnification electron micrographs per animal. Fifteen ultrathin coronal sections of the central NL were collected at random intervals. A single 5000X micrograph was then made at random from each section and photographically enlarged 2.4X for a final magnification of 13,500~. To exclude the possibility of experimenter bias, micrographs from all experimental and control groups were coded and mixed in random order by a third party prior to analysis. Every axon in each micrograph was counted using the presence of neurosecretory granules and/or neurofilaments as morphological criteria for distinguishing axon profiles from pituicyte and perivascular cell processes. The density of axons was deter-

COMPENSATORY

SPROUTING

mined by measuring the relative area occupied by axons, support cells (pituicyte and perivascular cell bodies and processes), and extracellular space (including capillary lumens) in each micrograph. Measurements were performed using a Zeiss interactive digital analysis system (ZIDAS). Metabolic analysis. Animals were individually housed in Nalgene metabolic cages throughout the experimental period. Daily drinking and urine excretion volumes were determined directly from graduated drinking and urine collection tubes, respectively. Urine samples were taken at a consistent time each day and then centrifuged to remove any precipitate prior to analysis using a Wescor vapor pressure osmometer. One-way analysis of variance Statistical analysis. was performed using the AVlW program in the MSUSTAT statistical package (developed by Dr. R. Lund of MSU), followed by testing of differences in group means using the MSUSTAT program COMPARE and the Newman-Keuls test. RESULTS

Effectiveness of the hypothalamic lesion. Complete unilateral transection of the hypothalamo-neurohypophysial tract by the knife cut was verified histologically in every animal. A large cavity was observed along the knife track, continuous with the severed lateral ventricle and extending from the dorsal to the ventral surface of the brain. The cavitation had no apparent effect on the ipsilateral SON but always resulted in medial displacement and compression of the ipsilateral PVN (Fig. 1). The contralateral SON and PVN were never directly affected by the cavitation. Cell death was apparent by 10 days postsurgery (PS) in the ipsilateral SON and was usually maximal by 30 days. The degree of SON cell death varied between animals ranging from partial to complete loss. However, there was no apparent relationship between the degree of cell death and the extent of the lesion, which appeared complete in every case. Extensive cell death occurred throughout the entire ipsilateral PVN due to the lateral displacement and compression caused by the cavitation. Immunocytochemistry for vasopressin (VP) was also employed to verify degeneration of the lesioned hypothalamo-neurohypophysial tract. Degenerating vasopressinergic fibers were apparent within the hypothalamus at 5 days postsurgery (Fig. 2A), and no immunoreactive fibers were seen entering the median eminence from the lesioned side of the hypothalamus at 90 days postsurgery (Fig. 2B). These data indicate that the hypothalamic knife cut was effective in permanently severing the ipsilateral hypothalamo-neurohypophysial tract. Lesion-induced changes in VP immunoreactivity and cross-sectional area of the neural lobe. In the initial ex-

OF

MAGNOCELLULAR

AXONS

11

periment, densitometric determination of relative staining intensity was used to estimate the time course of lesion-induced changes in the VP content of the NL. While such changes may reflect loading or release of VP from intact terminals and are therefore not a direct measure of axonal loss or sprouting, we reasoned that changes in VP immunoreactivity might indicate the most appropriate time points for ultrastructural analysis of the NL. As shown in Fig. 3, a decrease in VP immunoreactivity was quite apparent 10 days after the hypothalamic lesion (Fig. 3B), while an increase in VP immunoreactivity was visible at later times (Fig. 3C). These lesion-induced changes in VP immunoreactivity appeared to occur throughout the NL. Densitometric determination of relative VP staining density by digital image analysis revealed that a statistically significant reduction occurred at 10 days PS (P < 0.05; Fig. 4). VP staining density increased dramatically at 20 days PS to a level significantly above that of intact and sham controls (P < 0.05). Subsequent values fluctuated but were never statistically different from control levels. Sham surgery had no effect on VP staining intensity. Cross-sectional area of the NL was estimated by digital image analysis on the same sections used for densitometric analysis of VP staining density. A significant reduction in the cross-sectional area of the NL was observed at 10 days PS (P < 0.05; Fig. 5). While a subsequent trend toward recovery was apparent between 20 and 90 days PS, the largest increase occurred between 10 and 20 days PS with the result that none of the 20- to go-day values are significantly different from those of the intact controls. These changes in the size of the NL are apparent in the micrographs presented in Fig. 3. Sham surgery had no effect on the area of the NL. Lesion-induced changes in axon number and ultrastructure of the neural lobe. The similarity in the temporal pattern of changes in VP immunostaining density and NL cross-sectional area following unilateral lesion of the hypothalamo-neurohypophysial tract indicated that axonal degeneration was complete by 10 days PS if not earlier. These results also suggested that substantial sprouting of neurosecretory endings might be occurring between 10 and 90 days PS. We therefore examined the ultrastructure of normal and partially denervated NL at 5, 10,32, and 90 days PS to confirm the indications of axonal loss and subsequent sprouting observed at the light level as well as to look for other changes in neurohypophysial structure in response to partial denervation. The morphology of the intact neural lobe which we observed appeared essentially identical to that previously reported by others (28, 31, 34, 52). Axons were of varying caliber ranging from very small cylinders with numerous neurofilaments and few or no neurosecretory granules to large granule-filled axonal swellings containing numerous mitochondria but apparently

12

WATT

AND

PADEN

FIG. 1. cavitation persisted

A coronal section of the hypothalamus stained with cresyl violet to illustrate the ventral extent of the lesion and the exten lsive surrounding the knife cut at 10 days postsurgery. The cavitation occurred to varying degrees in all experimental animals and t .hroughout the experimental period. OT, optic tract; v, third ventricle; cav, cavitation; son, supraoptic nucleus. Bar = 400 pm

lacking neurofilaments (Fig. 6A). Axonal endings in close proximity to the perivascular space often contained small synaptic vesicles in addition to the larger neurosecretory granules. Synaptic vesicles were occasionally observed within axonal swellings as well. Occasional axonal swellings contained multilaminate bodies, swollen mitochondria, and vacuoles typical of degenerating fibers (8). Pituicyte ramifications were extensive and typically contained substantial amounts of lipid droplets, rough endoplasmic reticulum (RER), mitochondria, glycogen particles, and polysomal clusters. Axons were typically in close apposition to and sometimes completely enclosed within pituicytes. Perivascular cells, much more limited in number, displayed a more electron dense cytoplasm than pituicytes with extensive cisternae of RER and highly condensed chromatin, particularly in apposition to the inner membrane of the nuclear envelope. Perivascular cell ramifications were typically of smaller caliber and less extensive than pituicyte processes and were often in direct apposition to axons. Counts of the total number of axons and morphometric analyses of the area occupied by axons, support cells (pituicytes and perivascular cells), and extracellular space (including capillary lumens) were first performed on 15 low-magnification electron micrographs from each of three intact control animals at 35 days of age and three at 125 days of age to correct for any maturational changes occurring during the course of the exper-

iment. As shown in Table 1, the total number of axons did not increase significantly with age, and therefore the 35 and 125-day data were combined (grand mean of 1022 f 96, n = 6) for comparison to lesioned animals. While the number of axons did not change with age, the area occupied by axons increased 25%. This was offset by decreases in the area occupied by glia and by the amount of extracellular space. Lesioned animals sacrificed at 5 days PS showed a marked reduction in total axon population and in the area occupied by axons compared to intact controls (Table 1). In contrast, the area occupied by pituicyte and perivascular cells increased markedly over control values as did the amount of extracellular space. Surprisingly few degenerating axons were observed, suggesting that phagocytosis of degenerating axons was essentially complete by this time. The association between pituicytes and axons appeared normal, with no phagocytic engulfment of degenerating axons by pituicytes observed. There was a marked increase in the number of perivascular cells, and their processes were more extensive than previously observed. These cells typically contained dilated cisternae of RER filled with a granular amorphous substance and numerous granular inclusions indicative of phagocytic activity (Fig. 7). Axon number continued to decline at 10 days PS to 61% of the mean control value, concomitant with a continued decline in the area occupied by axons. At the same time extracellular space reached its peak value

COMPENSATORY

SPROUTING

OF

MAGNOCELLULAR

AXONS

13

FIG. 2. (A) Immunocytochemical demonstration of degeneratingvasopressinergic efferents in the ipsilateral hypothalamus 5 days following the knife cut. Arrowheads denote vasopressinergic axons proximal to the lesion (P) and dense globules characteristic of degenerating axons just distal to the lesion (D) and lateral to the median eminence. (B) Dark-field photomicrograph of vasopressin immunoreactivity in the median eminence 90 days following the knife cut. Arrows indicate vasopressinergic axons entering the median eminence from the contralateral hypothalamus. No axons are seen arising from the side ipsilateral to the lesion. V, third ventricle; ME, median eminence. Both micrographs are to the same scale: Bar = 100 Frn.

(Fig. 6B; Table 1). Total area occupied by support cells was less than that at 5 days PS but remained above normal. There was a decreased level of perivascular cell activity as indicated by fewer granular inclusions and picnotic nuclei and more constricted cisternae of RER. A substantial recovery in axon number was observed by 32 days PS. Total axons reached 76% of the mean control value, an increase of 25% over the lo-day PS

group. The area occupied by axons also increased. At the same time there was a further reduction in the area occupied by pituicytes and perivascular cells to an essentially normal value. The amount of extracellular space declined slightly but remained much greater than that of intact animals (Table 1). By 90 days PS the axon population increased to 83% of the mean control value, while the total area occupied

14

WATT

AND PADEN

COMPENSATORY

SPROUTING

OF

MAGNOCELLULAR

15

AXONS

N=5

Ln

c : P 0L c

A3 2 5

110

T

loo-

a, z

go-

80-

N=ll T

5 2

N=ll T

7060 50

0.50.

N=ll

0.40-

: 0.30& UI

INTACT INTACT

SHAM

10

20

30

SHAM

10

20

30

90

90

FIG. 4. Densitometric determination of relative VP staining intensity was performed on 15 coronal sections of the NL from each of five lesioned animals at 10,20,30, and 90 days postsurgery and from age-matched intact and sham-lesioned controls. There were no changes in staining density of either control group with time; therefore, only the grand mean is shown for each. A significant (*P < 0.05) 42% reduction in VP staining density is apparent at 10 days postsurgery. VP staining density increased significantly (‘P < 0.05) above control values at 20 days and then returned to near normal levels by 30 and 90 days postsurgery. Bars indicate SEM.

by axons returned to normal. The area occupied by pituicyte and perivascular cells continued to decline to a level substantially below that of age-matched intact controls. The amount of extracellular space also continued to decline but remained over twice that of agematched control animals (Fig. 6C, Table 1). Lesion-induced hypertrophy of contralateral magnocellular neurons. We performed a morphometric analysis of somal and nuclear size in magnocellular neurons in the SON and PVN contralateral to the hypothalamic knife cut to determine if hypertrophy occurred over a time course consistent with the axonal sprouting observed in the NL. Cellular hypertrophy has been correlated with the occurrence of axonal sprouting in intact neurons (15, 18, 19, 35, 36). However, changes in the distribution of Nissl substance may appear as increases in somal cross-sectional area in sections stained with cresyl violet (15). We therefore measured the cross-sectional area of cell nuclei as well, since the size of magnocellular nuclei has been shown to increase in response to heightened metabolic demand (4, 12, 30, 32, 37, 42,43). As shown in Fig. 8, significant increases in somal and nuclear cross-sectional areas occurred in both the con-

FIG. 5. Cross-sectional area of the NL was measured from the same sections analyzed for VP staining density (see Fig. 4). A significant reduction in NL size (‘P < 0.05) occurred at 10 days postsurgery, followed by a return to normal size at later times. There was no change in the area of the NL in either age-matched intact or sham-lesioned controls with time; therefore, only the grand mean is shown for each control group. Bars indicate SEM. X-axis indicates experimental groups in days postsurgery.

tralateral SON and the PVL and PVM subdivisions of the contralateral PVN. The enlargement of the somata and cell nuclei occurred in parallel in each case, but the extent and the time course of enlargement in the SON were different from those in the PVN. Increases were first apparent at 30 days PS in the SON, and the mean area of magnocellular somata and cell nuclei continued to enlarge at 90 days PS to 52 and 48% above control values, respectively. In contrast to the SON, magnocellular hypertrophy was delayed in the PVN, appearing only at 90 days PS. The degree of hypertrophy in the PVN was also smaller, with somal and cell nuclear areas showing increases of 32 and 33% above control values, respectively. Lesion-induced changes in urine osmolality, daily drinking, and urine excretion volumes. We explored the possibility of lesion-induced changes in circulating plasma VP levels indirectly by measuring urine osmolality as well as daily drinking and urine excretion volumes. Daily measurements of urine osmolality revealed an initial transient hypoosmolality followed within 48 h by a chronic hyperosmolality which persisted throughout the 90 day postsurgical period (Fig. 9A), suggesting that the lesion did not result in a functional deficit in circulating VP. There was a concomitant chronic de-

Immunocytochemical demonstration of changes in vasopressin (VP) content in coronal sections of the neural lobe (NL) following FIG. 3. unilateral hypothalamic lesion. Sham surgical controls are shown in (A) at 10 days following sham surgery and (D) at 90 days following sham surgery. No effects of sham surgery on either VP staining density or cross-sectional area of the NL were observed. (B) 10 days postlesion. Both the area of the NL and the density of VP staining are markedly reduced 10 days after unilateral hypothalamic lesion. (C) 90 days postlesion. Substantial recovery of both VP staining density and the area of the NL has occurred by 90 days after the lesion. The sections shown are representative of those employed in the quantitative analyses summarized in Figs. 4 and 5. IL, intermediate lobe. Bar = 500 Wm.

16

WATT

crease in both daily drinking tion (Fig. 9c) volumes which ing surgery.

AND

(Fig. 9B) and urine excrebegan immediately follow-

DISCUSSION The results of our studies indicate that axons of intact magnocellular neurosecretory neurons undergo a substantial sprouting response following partial denervation of the NL. The initial decline in the number of axon profiles in the medial region of the NL caused by unilateral sectioning of the hypothalamo-neurohypophysial tract was substantially reversed by 90 days PS. Histological and immunocytochemical analyses of the hypothalamic lesion site indicated that the recovery of axon number in the NL was not the result of regeneration of severed axons from magnocellular neurons in the ipsilatera1 SON or PVN. Ultrastructural analysis of the NL in age-matched intact control animals further revealed that the recovery in axon number was not the result of a normal developmental increase. We conclude that the increase in total axon profiles between 10 and 90 days PS is the result of compensatory sprouting by the uninjured magnocellular efferents from the contralateral SON and/or PVN. The observed changes in the ultrastructure of the NL, in the density of vasopressin immunoreactivity and cross-sectional area of the NL, and in the size of magnocellular somata and cell nuclei in the contralateral SON and PVN are all consistent with this conclusion. The rapid decrease in the relative area of the NL occupied by axons immediately following the lesion occurred concomitantly with increases in the relative area occupied by pituicytes, perivascular cells, and extracellular space. Conversely, as the number of axons increased between 10 and 90 days PS, the relative area occupied by support cells and extracellular space declined. However, while the volume of extracellular space remained greater than that of intact controls at 90 days PS, the area occupied by glia and perivascular cells fell below control values. This decline may stem from the gradual reduction in perivascular cells observed following the conclusion of the degenerative phase as well as from the reduced metabolic demand on the pituicyte population stemming from the reduced number of axons within the NL. The temporal pattern of these ultrastructural changes paralleled the shrinkage and subsequent enlargement of the NL seen at the light microscopic level.

PADEN

It is therefore most likely that the gross shrinkage of the NL seen at 10 days PS is the result of axonal degeneration, while the eventual recovery in size is primarily the result of axonal sprouting. Furthermore, there is a striking similarity between the time course of changes in the cross-sectional area of the NL and those of VP staining density and total axon population (Fig. lo), which suggests that each of these measures reflects the sprouting response of neurosecretory axons. It is important to note that the relative areas occupied by axons, support cells, and extracellular space were estimated within a sample of fixed size taken from the central region of the NL. No attempt was made to correct axon counts or other measures for the changes in the size of the NL observed in the light microscope, since the differences in tissue fixation and embedment used for light and electron microscopic analyses are likely to result in different amounts of tissue shrinkage (45). This is therefore a conservative approach to estimating the total axonal loss and recovery in the NL. Shrinkage of the gland is most likely to have increased the density of axons in the central portion at 10 days PS, causing an underestimate of the lesion-induced reduction in total axon number; conversely, subsequent enlargement of the gland may have resulted in underestimation of the extent of compensatory sprouting. The elimination of severed axons from the NL appeared to be essentially complete by 5 to 10 days PS, as evidenced by the infrequent occurrence of degenerating axon profiles and the increased volume of extracellular space, which presumably resulted from absorption of degenerating axons. It is notable that those examples of phagocytosis encountered involved exclusively perivascular cells. Although the pituicyte profiles contained higher numbers of lipid droplets and slightly dilated RER and Golgi apparati, no examples of phagocytosis by pituicytes were observed. These data are in agreement with similar observations of Zambrano and De Robertis (52) following destruction of the PVN efferents to the NL. Those investigators reported that perivascular cell activity peaked about 6 days following the lesion and that phagocytic activity was limited to the perivascular cell population of the NL. In contrast, infundibular stalk transection has been reported to result in the engulfment and complete disposal of degenerating neurosecretory axons by pituicytes rather than perivascular cells between 5 and 10 days after the lesion (9). The discrepancy between these studies may reflect a difference in the phagocytic response of pituicytes and

FIG. 6. Electron micrographs showing the central core of the neural lobe from intact and lesioned animals. (A) Intact 35day-old control. Axons ranging from small cylinders with numerous neurofilaments to large axon swellings with no neurofilaments and numerous neurosecretory vesicles can be seen. (B) 10 days following hypothalamic lesion. Note the increased volume of extracellular space, the pituicyte scaffolding, and the marked loss of axons. (C) 90 days following hypothalamic lesion. Note the recovery in axon number and the corresponding reduction in volume of extracellular space. Ax, axon filled with neurosecretory granules; P, pituicyte; L, lipid droplet; NF, neurofilaments. Bar = 4.0 pm.

COMPENSATORY

SPROUTING

OF

MAGNOCELLULAR

AXONS

17

18

WATT

EM

Group Intact control animals 35-day-old No. 1 No. 2 No. 3 Mean + SEM 125day-old No. 1 No. 2 No. 3 Mean + SEM Lesioned animals’ 5-day PS No. 1 No. 2 No. 3 Mean + SEM lo-day PS No. 1 No. 2 No. 3 Mean + SEM 32-day PS No. 1 No. 2 No. 3 Mean f SEM go-day PS No. 1 No. 2 No. 3 Mean + SEM

Stereometric

AND

PADEN

TABLE

1

Analysis

of the

Neural

Area” occupied by axons

Total number of axons

1661 970 876 969 + 53

1737.6 1995.4 1985.2 1906.1 + 84.3

817 934 1473 1075 + 202

2539.3 2379.1 2257.4 2391.9 k 81.6

480 653 775 636 + 86

Lobe

Area

occupied by gliab

Extracellular space’

1512.9 1200.2 967.5 1226.9 + 158.0 933.5 940.5 952.8 942.3 +

439.5 494.8 737.2 557.2 f 91.4

5.6

217.2 177.5 437.0 277.2 f 80.7

1740.2 1570.3 1428.3 1579.6 + 90.2

1252.0 1240.7 1919.1 1470.6 + 224.3

652.5 879.0 424.8 652.1 k 131.1

593 610 663 622 + 21

1417.7 1721.2 1407.9 1515.6 f 102.8

1288.7 1284.1 1365.6 1312.8 k 26.4

1007.4 684.8 916.5 869.6 k 96.0

598 975 761 778 f 109

1527.9 2062.4 2084.6 1891.6 k 182.0

1193.0 789.8 1035.4 1006.0 + 117.3

969.1 837.9 570.0 792.2 f 117.4

960 789 789 846 + 57

2624.8 2263.1 2397.1 2428.3 f 105.6

982.0 626.3 591.3 733.2 + 124.8

339.7 800.6 701.6 614.0 zk 140.1

a All areas measured in pm’; total area of NL measured b Includes pituicytes and perivascular cells. ’ Includes capillary lumens. d Lesions were performed at 35 days of age; thus 5-day

per animal,

postsurgery

perivascular cells under conditions of partial denervation versus total disconnection of the NL. A correlation between cellular hypertrophy and compensatory sprouting of intact afferents to a partially denervated site has been established in various CNS systems (15,18,19,35,36), and hypertrophy of SON neurons has been demonstrated to occur concomitant with the reformation of neurosecretory axon terminals in the proximal infundibulum following hypophysectomy (40). In the present study, hypertrophy of the magnocellular neurons in the contralateral SON coincided with the increase in axon number in the NL at 30 and 90 days PS, consistent with the conclusion that compensatory sprouting of intact neurosecretory efferents was occurring at this time. This hypertrophy probably reflects the increased biosynthetic activity necessary to maintain the enlarged axonal arbor of these cells.

3690 pm’.

(PS)

animals

were

40 days old at sacrifice,

etc.

Cellular hypertrophy within the magnocellular system has also been associated with heightened neurosecretory activity induced by dehydration or salt loading (12, 32, 37, 42, 43). For example, dehydration of 1.5, 3, and 5 days duration caused 25,28, and 86% enlargement of SON cells, respectively (42, 43), and hypertrophy as great as 180% of normal was seen following 12 days of dehydration (12). These acute effects occur rapidly, and somata return to normal size soon after rehydration or cessation of osmotic stimulation (12, 32). In contrast, the long delay observed in the present study between partial denervation of the NL and the onset of cellular enlargement in the contralateral SON suggests that increased secretion of VP is not the primary factor driving the hypertrophic response. Hypertrophy of magnocellular neurons within the PVL and PVM subdivisions of the contralateral PVN

COMPENSATORY

SPROUTING

OF

MAGNOCELLULAR

AXONS

19

FIG. 7. Electron micrographs showing evidence of axonal degeneration and phagocytosis by perivascular cells in the neural lobe 5 days after hypothalamic lesion. (A) Degenerating axon within the perivascular space of the NL, note the large multilaminate bodies within the axon. A perivascular cell foot process appears to be engulfing the axon. (B) Perivascular cell process within the perivascular space of the NL. Note the highly dilated rough endoplasmic reticulum (small arrowheads) and phagocytic debris (large arrowheads). Ax, axon; cap, capillary; MLB, multilaminate bodies; PVC, perivascular cell; P, pituicyte. Bar = 1.0 pm.

which are known to project to the NL (2, 3) was also observed, but this effect was only apparent at 90 days PS. The differential response of magnocellular neurons in the SON versus that in the PVN could be caused by

one or more factors. First, the lesser response of neurons in the PVN could be a reflection of the relative commitment of axons and terminals each nucleus makes to the NL. It has been reported that the PVN

20

WATT SON Cellular

AND

PADEN

Hypertrophy

Hypertrophy

275

325 300

250

* *

; 2 $ : z 5: .t

275

$:

100

250 225

f N=4

Ln

225

g

200

r

200 175 150 125

E ;

150

(g

125

.c

100

N=4 N=3

75 50

50 25

25

0 INTACT

c

SHAM

SON

10

Ceil

Nuclei

20

30

0 90

INTACT

Hypertrophy

SHAM

PVN Celi

D

120

10

Nuclei

30

90

Hypertrophy

120

110 100

N=4

175

hg

7.5

: e .L! -

PVN Cellular

B

350

110 i

4.

100

90

*

80 i

5

N=4

b ‘5 ?

ao70-

2

60-

g .c_ 8

50-

$

30-

N=4

40-

20loO-1 INTACT

SHAM

10

20

30

90

INTACT

SHAM

10

30

90

FIG. 8. The cross-sectional areas of the soma and cell nucleus of 50 magnocellular neurons were measured in the contralateral SON and PVN (PVL and PVM subnuclei) of each of four lesioned animals at 10,20,30, and 90 days postsurgery. Intact control animals were 35 days old, and sham controls were sacrificed 10 days after sham lesions. (A, C) Magnocellular somal (A) and nuclear (C) cross-sectional areas in the contralateral SON. Hypertrophy of both soma and cell nuclei occurred at 30 days and continued to increase at 90 days following surgery. (B, D) Magnocellular somal (B) and nuclear (D) cross-sectional areas in the contralateral PVN. In contrast to the SON, hypertrophy of PVN cell soma and nuclei did not occur until 90 days following surgery. Note the slight shrinkage of PVN soma and nuclei apparent at 10 days postsurgery. Statistically significant differences between lesion and control animals are indicated by *P < 0.05 and **P < 0.01. Bars indicate SEM. -

contributes approximately 20% of the axons and terminals in the NL while the SON contributes the remainder (33, 52). Second, Silverman et al. (47) have demonstrated that oxytocinergic afferent projections arise from either PVN and cross to contact the contralateral, equivalent PVL and PVM subdivisions of the PVN. The lesion employed in the present study would severe this afferent exchange between PVN nuclei, resulting in a partial deafferentation of the contralateral intact PVN. Neuronal hypotrophy resulting from deafferentation has been well documented (for review see (6, 15, 22)). Therefore the elimination of this reciprocal connection might have led to cellular shrinkage in the contralateral PVN which served to partially offset the hypertrophy associated with the sprouting response. In support of this hypothesis, cross-sectional areas of both somata and cell nuclei were reduced approximately 14% from

control values at 10 days PS in the contralateral PVN. Although this reduction was not statistically significant, it is suggestive given the absence of similar hypotrophy in the contralateral SON at this time. The marked decline and subsequent recovery of VP immunoreactivity within the NL closely paralleled the time course of axonal degeneration and sprouting, with the exception of the presence of intense staining at 20 days PS. This transient increase in immunoreactivity may reflect a buildup of peptide within neurosecretory terminals as intact vasopressinergic neurons respond to partial denervation of the NL with increased hormone synthesis and transport. One may speculate that this loading response might be further amplified if new axonal sprouts appearing around 20 days PS were not yet capable of neurosecretory activity. However, any hypothesis regarding changes in the rate of neurosecretion

COMPENSATORY Urine 2800 2550

SPROUTING

OF

MAGNOCELLULAR

Drinking

Osmololity 70 65

l Lesion o-•oSham

l

Urine

C

Excretion

Volume

l Lesion o-•oSham l -

60

T

21

AXONS

1

Volume

++ Lesion o-•oSham

0

5 $10 e n

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

FIG. 9. Effects of the unilateral hypothalamic lesion on fluid balance. (A) Urine osmolality decreased markedly within the first 24 h following surgery and then immediately increased to a hyperosmotic state which persisted throughout the experimental period. Daily drinking (B) and urine excretion (C) volumes were substantially reduced immediately following surgery and remained depressed throughout the experimental period. X-axis indicates experimental period in days. Experimental Days 1-15, n = 12 lesioned and n = 12 sham controls. Days 16-90, n = 6 lesioned and n = 3 sham controls. Bars indicate SEM.

must be tempered by consideration of the urine osmolality data which indicate that an adequate level of plasma VP was restored within 48 h of the hypothalamic lesion and was maintained continuously throughout the course of the experiment. In any case, the fact that VP immunoreactivity increased concomitantly with the number of axons in the NL suggests that vasopressinergic neurons were involved in the sprouting response. Further experiments are required to prove this hypothesis, as well as to determine the relative contributions of vasopressinergic versus oxytocinergic neurons. That oxytocinergic axons may be involved in this response is not unlikely. While possible differences in the extent or temporal patterns of regenerative sprouting from oxytocinergic versus vasopressinergic axons have not been extensively investigated, immunocytochemical analysis of the median eminence following hypophysectomy re-

vealed that oxytocinergic fibers invaded the zona externa over a time course similar to that of vasopressinergic fibers, albeit to a lesser extent (24). Chronic measurements of urine osmolality, urine volume, and drinking volume were undertaken with the expectation that any initial deficit in VP secretion following partial denervation of the NL might be reversed by subsequent compensatory sprouting of vasopressinergic axons. However, the complete absence of any lesion-induced deficit, other than a very brief postsurgical depression in urine osmolality, makes it impossible to correlate sprouting with functional recovery in these experiments. The absence of a functional deficit in VP secretion was not the result of an incomplete unilateral lesion, since animals receiving bilateral hypothalamic lesions using the same stereotaxic coordinates exhibited lowered urine osmolality as well as increased drinking

22

WATT

D 0 m

140 ? =

Y 0 L '; 6 "6 & 0 : Y d

Vasopressin staining density Cross-sectional area of neural Axon population

AND

lobe

120

100 80 60 40 20 0 INTACT

SHAM

10

30

90

FIG. 10. Lesion-induced changes in VP staining density, crosssectional area, and axon number in the neural lobe expressed as percentages of intact control values. The degree of loss and subsequent recovery to near normal values are very similar for each measure, supporting the conclusion that recovery of all three variables reflects the sprouting response of neurosecretory axons. Bars indicate SEM. X-axis indicates experimental groups in days postsurgery.

and urine excretion characteristic of diabetes insipidus (Watt and Paden, unpublished results). Rather it appears that the reserve capacity of the VP system is such that destruction of almost half of the magnocellular neurosecretory terminals is insufficient to compromise its normal function. This conclusion is consistent with the report of Jones and Pickering (23), who demonstrated that the normal daily requirement of VP in water-balanced rats comprises only 5% of the total neurohypophysial content. Thus, in retrospect, it is not surprising that hemisection of the hypothalamo-neurohypophysial tract failed to impair normal VP secretion. It remains to be determined whether any functional deficit in VP release might become apparent following partial denervation of the NL if subjects were subjected to hyperosmotic stress or water deprivation. The apparently permanent decrease in daily drinking and urine volumes which resulted from the unilateral lesion we employed was unanticipated. One possible explanation of these effects is that the lesion may have induced an increase in tonic vasopressin secretion by interrupting bilateral connections between the components of the magnocellular neurosecretory system. As discussed above, reciprocal oxytocinergic connections exist between the PVL and PVM subdivisions of the bilateral PVN (47), and electrophysiological evidence for polysynaptic connections between the bilateral SON has recently been reported (50). The functions of these reciprocal connections are not known, but it is conceivable that they serve to coordinate the activity of the magnocellular nuclei and that their disruption could cause disinhibition of vasopressin release, followed by increased urine osmolality and reduced volume. Reduced drinking would then occur as a compensatory response reflecting altered fluid needs.

PADEN

An alternative possibility is that the lesion directly affected mechanisms which control drinking. Comparison to the reported effects of other diencephalic lesions on drinking and urinary volume suggests that our results could have arisen from disruption of drinking mechanisms associated with the lateral hypothalamus (LH) and/or zona incerta (ZI). Gray and Everitt (16) reported that unilateral electrolytic lesions of the LH caused prolonged decreases in both water intake and urine volume comparable in magnitude to those we observed, with only mild hypophagia and no adipsia or aphagia as have been associated with the much more severe effects of bilateral LH lesions. Bilateral electrolytic lesions of the ZI have been reported to result in permanent hypodipsia, decreased urine volume, and increased urine osmolality essentially identical to the effects of our lesion (13). The complete but unilateral disconnection of the medial hypothalamus caused by our parasagittal knife cut is very different from the bilateral but focal lesions typically employed by those studying the neuroanatomy of drinking and fluid balance, making it impossible to draw firm conclusions regarding possible mechanisms. While our knife cut did not directly affect either the LH or ZI, it may well have disrupted both their afferent and efferent connections, particularly in the case of the rostromedial ZI which lies just dorsolateral to the PVN (17). Whatever the mechanisms involved, it appears that the knife cut primarily affected secondary drinking (14) with no impairment of body fluid homeostasis because of compensation through reduced urine output and increased osmolality. This compensatory response is presumably due to VP secretion, strengthening the conclusion that the axonal sprouting we have observed occurs independently of any significant deficit in neurohypophysial function. The conclusion that a functional deficit in circulating VP is not required to induce compensatory sprouting of magnocellular neurosecretory axons also has implications with regard to understanding the role of neuronal activity in the sprouting response. Herman et al. (20) have hypothesized that activation of damaged neurons may play a role in determining their regenerative success following injury. These authors have demonstrated that continuous infusion of VP will selectively impair vasopressinergic cell survival following neurohypophysectomy (21), an effect which may be attributed to negative feedback of peripheral VP on vasopressinergic cell activity (21, 51). Likewise, sprouting of intact parvocellular vasopressinergic axons into the zona externa of the median eminence following contralateral lesion of the PVN was only apparent when hyperactivity of these neurons was induced by adrenalectomy (46). In this case increased activity of the sprouting neurosecretory cells and a functional deficit of the final hormone in the hypothalamic-pituitary-adrenal axis occurred together. In the present experiments it appears that reduced water

COMPENSATORY

SPROUTING

intake may have led to increased neurosecretory activity by intact vasopressinergic neurons, as reflected in increased urine osmolality. Thus, increased neuronal activity may be associated with the sprouting response in each case, whereas deficits in circulating hormone levels are dissociated from sprouting in our experiments. Taken together, these results suggest that increased neuronal activity may be directly linked to the sprouting response, while plasma hormone levels are only indirectly related to induction of the sprouting response through their feedback effect on the activity of neurosecretory neurons, either directly (51) or via osmoreceptive and/or baroreceptive elements (41, 49). This hypothesis is consistent with our results as well as with the findings of Silverman and Zimmerman (46) and those of Herman et al. (20,21). However, the possibility that uninjured magnocellular neurons will undergo axonal sprouting in the absence of any increases in electrical activity cannot be excluded by the existing data. Further experiments are necessary to address this question and to elucidate additional factors which may affect the sprouting response in these cells. We believe that this experimental paradigm will have application to broader issues in neuroplasticity as well. Because the magnocellular neurosecretory system has served as the premier model for studying central peptidergic neurons since the original descriptions of neurosecretion (44), there exists a vast and growing literature on the physiology, biochemistry, cell biology, and molecular genetics of these neurons. Thus it may prove possible to relate the extent of the sprouting response to many other aspects of neuronal function in this system. Indeed, numerous pioneering studies of central neuronal plasticity have been performed using the hypothalamo-neurohypophysial system (see Introduction). The principle innovation in the present study is that the use of a unilateral hypothalamic knife cut instead of stalk section, hypophysectomy, or neurohypophysectomy permits the sprouting response of uninjured neurons to be studied in the intact NL, without the complications arising from hemorrhage or scar formation. The NL in turn offers a highly simplified tissue, of CNS origin but lying outside the blood-brain barrier, in which to investigate problems such as the role of glia and vascular elements in the sprouting response of central neurons. Future experiments which exploit the unique attributes of this model should provide novel data regarding the mechanisms of central neural plasticity.

OF

MAGNOCELLULAR

REFERENCES 1. ADAMS,

2.

3.

4.

5.

J. H., P. M. DANIEL, AND M. M. L. PRICHARD. 1969. Degeneration and regeneration of hypothalamic nerve fibers in the neurohypophysis after pituitary stalk section in the ferret. J. Comp. Neural. 135: 121-144. ALONSO, G., AND I. ASSENMACHER. 1981. Radioautographic studies on the neurohypophysial projections of the supraoptic and paraventricular nuclei in the rat. Cell Tissue Res. 219: 525-534. ARMSTRONG, W. E., S. WARACH, G. I. HATI‘ON, AND T. H. MCNEILL. 1980. Subnuclei in the rat hypothalamic paraventricular nucleus: A cytoarchitectural, horseradish peroxidase and immunocytochemical analysis. Neuroscience 5: 1931-1958. BANDARANAYAKE, R. C. 1976. Localization of functions in the magnocellular neurosecretory nuclei of the mammalian hypothalamus by autoradiography. Actu Anat. 95: 408-420. BILLENSTIEN, D. C., AND T. F. LEVEQUE. 1955. The reorganization of the neurohypophyseal stalk following hypophysectomy in the rat. Endocrinology 56: 704-717.

6.

COWAN, W. M. 1970. Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In Contemporary Research Methods in Neuroanatomy (W. J. H. Nauta and S. 0. E. Ebbesson, Eds.), pp. 217-251. Springer-Verlag, New York.

7.

DELLMAN, H.-D., L.-F. LUE, AND S. BELLIN. 1987. Fine structural characteristics of neurophysin-positive perivascular plexus that develop in the rat hypothalamus following interruption of the hypothalamo-neurohypophysial tract. Cell Tissue Res. 247: 137-143. DELLMAN, H.-D., AND E. M. RODRIGUEZ. 1970. Herring bodies: An electron microscopic study of local degeneration and regeneration of neurosecretory axons. Z. Zellforsch. 111: 293-315.

8.

9.

DELLMAN, H.-D., M. E. STOECKEL, A. PORTE, TINSKY. 1974. Ultrastructure of the neurohypophysial following stalk transection in the rat. Experientia 1222.

AND

F. STUglial cells 30(10): 1220-

10.

DELLMAN, H. D., L.-F. LUE, AND S. I. BELLIN. tory axon regeneration into intrahypothalamic graphs: Neurophysin immunohistochemistry Exp. Brain Res. 67: 543-555.

11.

DELLMAN, H. D., L.-F. LUE, S. I. BELLIN, AND M. QUASSAT. 1988. An immunohistochemical and fine structural analysis of peptidergic hypothalamic neurosecretory axon regeneration into the leptomeninges of the rat. Brain Res. 450: 181-189.

12.

ENESTROM, S. 1967. Nucleus supraopticus. A morphological and experimental study in the rat. Actu Puthol. Microbid. Scar& fSupp1.) 186: l-99. EVERED, M. D., AND G. J. MOGENSON. 1976. Regulatory and secondary water intake in rats with lesions of the zona incerta. Am. J. Physiol. 230: 1049-1057.

13.

14. 15.

1987. Neurosecreneural lobe alloand fine structure.

FITZSIMONS, J. T. 1972. Thirst. Physiol. Reu. 52: 468-561. GOLDSCHMIDT, R. B., AND 0. STEWARD. 1980. Time course of increases in retrograde labeling and increases in cell size of entorhinal cortex neurons sprouting in response to unilateral entorhinal lesions. J. Comp. Neurol. 189: 359-379.

16.

GRAY, R. H., AND A. V. EVERI?T. 1970. dipsia induced by unilateral hypothalamic J. Physiol. 219: 398-402.

17.

GROSSMAN, S. P. 1984. A reassessment of the brain mechanisms that control thirst. Neurosci. Biobehav. Reu. 8: 95-104. HEADON, M. P., J. J. SLOPER, R. W. HIORNS, AND T. P. S. PowELL. 1985. Effects of monocular closure at different ages on deprived and undeprived cells in the primate lateral geniculate nucleus. Dev. Brain Res. 16: 57-78.

ACKNOWLEDGMENTS We thank Dr. G. Nilaver for his generous gift of rabbit anti-vasopressin sera and Dr. A.-J. Silverman for her helpful discussions during the early stages of the project. This research was supported by NIH Grant NS23642 and RCDA NS01318 to C.M.P.

23

AXONS

18.

Hypophagia and hypolesions in the rat. Am.

24

WATT

AND

19.

HENDRICKSON, A., AND J. T. DINEEN. 1982. Hypertrophy rons in dorsal lateral geniculate nucleus following striate lesions in infant monkeys. Neurosci. Lett. 30: 217-222.

of neucortex

20.

HERMAN, J. P., F. F. MARCIANO, AND D. M. GASH. 1986. Vasopressin administration prevents functional recovery of the vasopressinergic neurosecretory system following neurohypophysectomy. Neurosci. L&t. 72: 239-246.

PADEN 36.

PEARSON, R. C. A., J. W. NEAL, AND T. P. A. POWELL. 1987. Increase in immunohistochemical staining of GABAergic axons in the superior colliculus and thalamus of the rat following damage of the ipsilateral striatum and frontal cortex. Brain Res. 412: 352-356.

37.

PETERSON, R. P. 1966. Magnocellular neurosecretory centers in the rat hypothalamus. J. Comp. Neural. 128: 181-190. POLENOV, A. L., M. V. UGRUMOV, M. V. PROPP, AND M. A. BELENKY. 1974. The hypothalamo-hypophysial system of hypophysectomized rats. I. Ultrastructure of nerve fibers in “intact” and dehydrated animals. Cell Tissue Res. 155: 541-554.

21.

HERMAN, J. P., F. F. MARCIANO, S. J. WIEGAND, AND D. M. GASH. 1987. Selective cell death of magnocellular vasopressin neurons in neurohypophysectomized rats following chronic administration of vasopressin. J. Neurosci. 7: 2564-2565.

38.

22.

JACKSON, J. R. H., AND E. W. RUEIEL. 1976. Rapid transneuronal degeneration following cochlea removal in chickens. Anat. Rec. 184: 434-435.

39.

RAISMAN, G. 1973. Electron ment of new neurohaemel the rat after hypophysectomy.

23.

JONES, C. W., AND B. T. PICKERING. port and turnover of neurohypophysial Physiol. 227: 553-564.

40.

RAISMAN, G. 1977. An ultrastructural pophysectomy on the supraoptic Neurol. 147: 181-208.

24.

KAWAMOTO, K. 1985. Immunohistochemical study of vasopressin and oxytocin in the neurosecretory system during reorganization of the neural lobe in mice. Zool. Sci. 2: 371-380.

41.

25.

KAWAMOTO, K., AND S. KAWASHIMA. 1985. Plasticity of vasopressinand oxytocin-containing fibers in the median eminence of hypophysectomized young and old mice. Brain Res. 330: 189193.

42.

26.

KAWAMOTO, K., neurohypophysial tomized immature

43.

27.

KIERNAN, J. A. 1971. Pituicytes and the regenerative properties of neurosecretory and other axons in the rat. J. Anat. 109(l): 97-114.

28.

KUROSUMI, K., T. MATSUZAWA, AND S. SNIBASAKI. 1961. Electron microscopic studies on the fine structures of the pars nervosa and pars intermedia, and their morphological interrelation in the normal rat hypophysis. Gen. Comp. Endocrinol. 1: 433452. MOLL, J. 1957. Regeneration of the supraoptico-hypophysial and paraventriculo-hypophysial tracts in the hypophysectomized rat. Z. Zellforsch. 46: 686-709.

RAMSEY, D. J. 1985. Osmoreceptors subserving vasopressin secretion and drinking-An overview. In Vasopressin (R. W. Schrier, Ed.), pp. 291-298. Raven Press, New York. REINHARDT, H. F., L. C. H. HENNING, AND H. P. ROHR. 1969. Morphometrisch-ultrastrukturelle untersuchungen am nucleus supraopticus der ratte nach dehydration. Z. Zellforsh, 102: 172181. REINHARDT, H. F., L. C. H. HENNING, AND H. P. ROHR. 1969. Morphometrisch-ultrastrukturelle untersuchugen am hypophysenhinterlappen der ratte nach dehydration. Z. Zellforsh. 102: 182-192. SCHARRER, E., AND B. SCHARRER. 1954. Hormones produced in neurosecretory cells. Recent Prog. Horm. Res. 10: 183-240. SCHUZ, A., AND G. PALM. 1989. Density of neurons and synapses in the cerebral cortex of the mouse. J. Comp. Neurol. 286: 442455. SILVERMAN, A.-J., AND E. A. ZIMMERMAN. 1982. Adrenalectomy increases sprouting in a peptidergic neurosecretory system. Neuroscience 7: 2705-2714.

29.

30.

31.

32.

1972. Intra-axonal transhormones in the rat. J.

AND S. KAWASHIMA. 1987. Regeneration hormone-producing neurons in hypophysecrats. Brain Res. 422: 106-117.

of

MOLL, J., AND D. DEWIED. 1962. Observations on the hypothalamo-posthypophysial system of the posterior lobectomized rat. Gen. Comp. Endocrinal. 2: 215-228. MONROE, B. 1967. A comparative study of the ultrastructure of the median eminence, infundibular stem and neural lobe of the hypophysis of the rat. Z. Zellforsch. 76: 405-432. MORRIS, J. F., AND R. E. J. DYBALL. 1974. A quantitative of the ultrastructural changes in the hypothalamo-neurohypophysial system during and after experimentally induced secretion. Cell Tissue Res. 149: 525-535.

study

44. 45.

46.

47.

48.

49.

50.

hyper-

33.

OLIVECRONA, H. 1957. Paraventricular nucleus and gland. Acta Physiol. Sand. (Suppl. 136) 40: 1-185.

pituitary

34.

PALAY, S. L. 1958. The fine structure Prog. Neurobiol. 2: 31-49.

35.

PEARSON, R. C. A., J. W. NEAL, AND T. P. S. POWELL. 1987. Bilateral morphological changes in the substantia nigra of the rat following unilateral damage of the striatum. Brain Res. 400: 127-132.

51.

of the neurohypophysis. 52.

microscopic studies of the developcontacts in the median eminence of Brain Res. 55: 245-261. study of the effects of hynucleus of the rat. J. Comp.

SILVERMAN, A.-J., D. L. HOFFMAN, AND E. A. ZIMMERMAN. 1980. The descending afferent connections of the paraventricular nucleus of the hypothalamus (PVN). Brain Res. Bull. 6: 47-61. STUTINSKY, F. 1951. Sur L’origine de la substance Gomori-positive du complexe hypothalamo-hypophysaire du rat. C. R. Sot. Biol. 145: 367-372. SVED, A. F. 1985. Central neural pathways in baroceptor control of vasopressin secretion. In Vasopressin (R. W. Schrier, Ed.), pp. 443-452. Raven Press, New York. TAKANO, S., H. NEGORO, K. HONDA, AND T. HIGUCHI. 1990. Electrophysiological evidence for neural connections between the supraoptic nuclei. Neurosci. Lett. 111: 122-126. VAN TOL, H. H. M., J. Z. KISS, AND J. P. H. BURBACH. 1989. Differential responses in vasopressin and oxytocin gene expression in distinct hypothalamic nuclei after hypothalamoneurohypophysial disconnection and vasopressin substitution. Neuroendocrinalogy 49: 337-343. ZAMBRANO, D., AND E. DE ROBERTIS. 1968. The ultrastructural changes in the neurohypophysis after destruction of the paraventricular nuclei in normal and castrated rats. Z. Zellforsch. 88: 496-510.