Cellular and Molecular Neurobiology, Vol. 12, No. 2, 1992
Postnatal Development of the Hypothalamic Ventromedial Nucleus: Neurons and Synapses Lucas D . P o z z o Miller 1'2 and Agustin A o k i 1 Received July 10, 1991; accepted July 19, 1991 KEY WORDS: differentiation; neurocytology; rat; transmision electron microscopy; ultrastructure.
SUMMARY
1. In this report the postnatal differentiation of the hypothalamic ventromedial nucleus (VMN) was studied. The main maturational changes detected at the fine structural level occurred between 10 and 20 days of postnatal life. 2. In 5-day-old rats the majority of neurons was undifferentiated, with rudimentary cytoplasmic organelles. Dendritic profiles presented an empty appearance due to an electron-lucent matrix and scarce content of organelles. 3. At 10 days there was a significant proliferation of cytoplasmic organelles in the perikaryon, mainly of those involved in protein biosynthesis as the rough endoplasmic reticulum (RER) and the Golgi complex. 4. After 20 days of age the VMN neurons acquired the cytological appearance of adult neurons, with well-organized RER, Golgi complexes, and pleomorphic mitochondria. Concurrent with these changes, there was a marked development of other organelles in the neuropil, which was accompanied by an increase in synaptic density and differentiation of their subsynaptic structures.
INTRODUCTION The hypothalamic ventromedial nucleus (VMN) is a critical component of the hypophysiotropic area of the hypothalamus. It plays a key role in the control of a Centro de Microscopia Electronica, Universidad Nacional de Cordoba, Casiila Postal 362, (5000) Cordoba, Argentina. 2To whom correspondence should be addressed at Department of Neurosciences, School of Medicine, E-604, Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106-4901. 121 0272-4340/92/0400-0121506.50/0© 1992PlenumPublishingCorporation
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series of autonomic, neuroendocrine, and behavioral functions (Colpaert and Wiepkema, 1976; Goy and McEwen, 1980; Nance, 1976; Panksepp, 1971; Pfaff, 1980). The majority of these functions is dependent upon sex hormones (Carrer and Aoki, 1982; Pozzo Miller and Aoki, 1991). The VMN also exhibits a well-defined regional differentiation of sex steroid concentrating neurons between its dorsomedial and its ventrolateral subdivisions (Pfaff and Keiner, 1973; Stumpf, 1970). The majority of the morphological studies performed on the structural differentiation of the hypothalamus has been directed toward the development of the median eminence and medial preoptic nucleus (Euremius and Jarskar, 1971; Fink and Smith, 1971; Reier et al., 1977). In the present report a fine structural study was performed on the postnatal differentiation of neurons and synapses of the ventrolateral subdivision of the VMN (VMN-VL). The main purpose of this investigation was to characterize the changes occurring in this region during neuronal development and synaptogenesis, as a baseline for future investigations on the morphophysiology of the VMN. Preliminary results of this study have been presented in abstract form at the American Association of Anatomists' 103rd Annual Meeting, Philadelphia, PA (Pozzo Miller and Aoki, 1990).
MATERIALS AND METHODS
Thirty Wistar rats of both sexes, raised in this laboratory and maintained in a light (14 hr light/10 hr dark cycle)- and temperature (22 + 3°C)-controlled animal room, were used in the present study. The hypothalami of 5, 10, 20, 45, and 100 days old rats were processed for electron microscopy, as described previously (Pozzo Miller and Aoki, 1988). Briefly, rats 5 and 10 days old were anesthetized with an aqueous urethane solution (1.2g/kg body weight) injected intraperitoneally (i.p.), while older animals were injected i.p. with an aqueous chloral hydrate solution (600 mg/kg body weight). The brains were fixed by cardiac perfusion with 1.5% glutaraldehyde, 1.5% formaldehyde, and 0.1M cacodylate buffer, pH 7.2. The fixed brains were excised and placed in fresh fixative for an additional 4 hr at room temperature. The hypothalamus was diced carefully with two frontal cuts, one at the posterior border of the optic chiasma and the other just in front of mammillary bodies. A horizontal section was made at the level of the fornices and the blocking was completed with two parasaggital cuts immediately outside the fornix at each side of the hypothalamus. To facilitate the orientation of the blocks for sectioning, the rostral face was trimmed smaller than the caudal one. The hypothalamus was divided into right and left halves with a razor blade through the third ventricle and the median eminence. Each half was treated with 2% osmium tetroxide for 2 hr, stained en bloc with 2% uranyl acetate for 2 hr, washed in buffer, dehydrated in a series of graded cold acetones, and embedded in Araldite. One whole half of the hypothalamus was cut serially into 1-/lm-thick sections in a frontocaudal progression and stained with an aqueous 1% toluidine blue and 1% sodium borate solution to map the VMN. Ultrathin sections for electron microscopy (80 nm, silver interference color) were
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cut with a diamond knife from the ventrolateral subdivision of the VMN. Then the sections were stained with uranyl acetate and lead citrate and photographed at 4000× in a Siemens Elmiskop 101 electron microscope operated at 80 kV.
RESULTS Postnatal Development of VMN-VL Neurons Comparative qualitative analysis of the male and female VMN-VL did not reveal striking differences in either neuronal cytology of the time course of developmental events. Therefore, the specific details of VMN-VL maturation described here apply equally to both sexes. The majority of neurons of the VMN-VL of 5-day-old rats presented a rudimentary organization (Fig. 1). A large, spheroidal, or slightly indented nucleus occurred excentrally within the perikarion. A prominent nucleolus and finely dispersed euchromatin were the main components of the nuclear matrix. The cytoplasm contained abundant free ribosomes and polyribosomes, scarcely developed Golgi complexes, small and vacuolated mitochondria, and few cisternae of rough endoplasmic reticulum (RER). The cisternae of R E R exhibited a canalicular profile, with numerous branches and segmentary dilatations decorated with ribosomes (Fig. 2). The neuropil was poorly differentiated and the scant organelles yielded an empty looking appearance.
Fig. 1. Neuronal cell body in the 5-day-old female rat VMN-VL. The nucleus is surrounded by a pale cytoplasmic rim, and the processes in the neuropil present an emptylooking appearance. ×11,300; reduced 50% for reproduction.
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Fig. 2. Neuronal cell body in the 5-day-old male rat VMN-VL. The cisternae of the RER show a canalicular profile, and the Golgi complex is poorly differentiated, x23,000; reduced 47% for reproduction.
Pozzo Miller and Aoki
Fig. 3. Nucleolar extrusion in the perikaryon of a 5-day-old male rat VMN-VL. Note the proximity of cisternae of the RER and free ribosomes. ×18,500; reduced 47% for reproduction.
A common feature observed in neurons of 5-day-old rats was round structures containing a fine granular material and one to three vacuoles with a lighter electron density. These structures were identified by others as nucleolar extrusions (Fig. 3) (Santolaya, 1973; Shimizu and lshii, 1965). The cytology of the VMN-VL neurons changed dramatically between 10 and 20 days of age. The nucleus and nucleolus showed only minor modifications, in contrast with the cytoplasmic organelles which developed remarkably. The most striking changes were found in the Golgi apparatus and in the RER. The cisternae of the latter were observed organized in large bodies of stuffed membranes decorated with ribosomes (Figs. 4 and 5). This enlarged ergatoplasm corresponded topographically to the Nissl bodies occurring at either marginal cytoplasmic regions or proximal dendritic processes. Between 20 and 45 days of postnatal life the VMN-VL there was a slowdown in the maturational changes. The nucleus acquired a lobulated contour, with deep
Fig. 4. Neuronal cell body in the 10-day-old female rat VMN-VL. A dramatic increase in the RER is evident. Cisternae are arranged parallel to each other, x14,600; reduced 50% for reproduction.
Fig. 5. Enlarged ergatoplasm in the VMN-VL of a 10-day-old male rat. x17,300; reduced 50% for reproduction.
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indentations of the nuclear membrane, and the neuropil increased the density of organelles, particularly the R E R and the Golgi complexes. From the 45th day to adulthood two distinct cellular types were recognized. The neurons most commonly seen were characterized by a rudimentary appearance due to the scarce of cytoplasmic organelles, with few and small mitochondria, long and isolated cisternae of R E R , a small Golgi complex, and an empty-looking cytoplasmic matrix. This seemed associated with a reduced metabolic activity (Fig. 6). In some neurons the nucleolus-like extrusions were also present. The other cell type exhibited a remarkable development of Nissl bodies, Golgi complexes, mitochondria, and large lobulated nucleus containing a conspicuous nucleolus. These features can be associated with an enhanced metabolic activity and resemble those found in the VMN neurons of 10-day-old rats.
Postnatal Development of VMN-VL Axodendritic Synapases Two types of axodendritic synapses were identified in the V M N - V L neuropil: (a) shaft synapses (Fig. 7) or axonal boutons on a dendritic shaft and (b) spine synapses (Fig. 8) containing through a dendritic spine. The latter were identified by the continuity of the spine with a dendritic shaft and by the lack of mitochondria and microtubules, distinctive features of dendritic shafts. Both types of synapses were present from the early postnatal periods to adulthood, however, shaft synapses occurred more frequently than spine synapses in the rat VMN-VL.
Fig. 6. Adult female rat VMN-VL neuron is characterized by a pale cytoplasm, with scattered mitochondria and large dense bodies. A few isolated costernae of RER are presented. )
Postnatal Development of the Hypothalamic Ventromedial Nucleus: Neurons and Synapses Lucas D . P o z z o Miller 1'2 and Agustin A o k i 1 Received July 10, 1991; accepted July 19, 1991 KEY WORDS: differentiation; neurocytology; rat; transmision electron microscopy; ultrastructure.
SUMMARY
1. In this report the postnatal differentiation of the hypothalamic ventromedial nucleus (VMN) was studied. The main maturational changes detected at the fine structural level occurred between 10 and 20 days of postnatal life. 2. In 5-day-old rats the majority of neurons was undifferentiated, with rudimentary cytoplasmic organelles. Dendritic profiles presented an empty appearance due to an electron-lucent matrix and scarce content of organelles. 3. At 10 days there was a significant proliferation of cytoplasmic organelles in the perikaryon, mainly of those involved in protein biosynthesis as the rough endoplasmic reticulum (RER) and the Golgi complex. 4. After 20 days of age the VMN neurons acquired the cytological appearance of adult neurons, with well-organized RER, Golgi complexes, and pleomorphic mitochondria. Concurrent with these changes, there was a marked development of other organelles in the neuropil, which was accompanied by an increase in synaptic density and differentiation of their subsynaptic structures.
INTRODUCTION The hypothalamic ventromedial nucleus (VMN) is a critical component of the hypophysiotropic area of the hypothalamus. It plays a key role in the control of a Centro de Microscopia Electronica, Universidad Nacional de Cordoba, Casiila Postal 362, (5000) Cordoba, Argentina. 2To whom correspondence should be addressed at Department of Neurosciences, School of Medicine, E-604, Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106-4901. 121 0272-4340/92/0400-0121506.50/0© 1992PlenumPublishingCorporation
Pozzo Miller and Aoki
122
series of autonomic, neuroendocrine, and behavioral functions (Colpaert and Wiepkema, 1976; Goy and McEwen, 1980; Nance, 1976; Panksepp, 1971; Pfaff, 1980). The majority of these functions is dependent upon sex hormones (Carrer and Aoki, 1982; Pozzo Miller and Aoki, 1991). The VMN also exhibits a well-defined regional differentiation of sex steroid concentrating neurons between its dorsomedial and its ventrolateral subdivisions (Pfaff and Keiner, 1973; Stumpf, 1970). The majority of the morphological studies performed on the structural differentiation of the hypothalamus has been directed toward the development of the median eminence and medial preoptic nucleus (Euremius and Jarskar, 1971; Fink and Smith, 1971; Reier et al., 1977). In the present report a fine structural study was performed on the postnatal differentiation of neurons and synapses of the ventrolateral subdivision of the VMN (VMN-VL). The main purpose of this investigation was to characterize the changes occurring in this region during neuronal development and synaptogenesis, as a baseline for future investigations on the morphophysiology of the VMN. Preliminary results of this study have been presented in abstract form at the American Association of Anatomists' 103rd Annual Meeting, Philadelphia, PA (Pozzo Miller and Aoki, 1990).
MATERIALS AND METHODS
Thirty Wistar rats of both sexes, raised in this laboratory and maintained in a light (14 hr light/10 hr dark cycle)- and temperature (22 + 3°C)-controlled animal room, were used in the present study. The hypothalami of 5, 10, 20, 45, and 100 days old rats were processed for electron microscopy, as described previously (Pozzo Miller and Aoki, 1988). Briefly, rats 5 and 10 days old were anesthetized with an aqueous urethane solution (1.2g/kg body weight) injected intraperitoneally (i.p.), while older animals were injected i.p. with an aqueous chloral hydrate solution (600 mg/kg body weight). The brains were fixed by cardiac perfusion with 1.5% glutaraldehyde, 1.5% formaldehyde, and 0.1M cacodylate buffer, pH 7.2. The fixed brains were excised and placed in fresh fixative for an additional 4 hr at room temperature. The hypothalamus was diced carefully with two frontal cuts, one at the posterior border of the optic chiasma and the other just in front of mammillary bodies. A horizontal section was made at the level of the fornices and the blocking was completed with two parasaggital cuts immediately outside the fornix at each side of the hypothalamus. To facilitate the orientation of the blocks for sectioning, the rostral face was trimmed smaller than the caudal one. The hypothalamus was divided into right and left halves with a razor blade through the third ventricle and the median eminence. Each half was treated with 2% osmium tetroxide for 2 hr, stained en bloc with 2% uranyl acetate for 2 hr, washed in buffer, dehydrated in a series of graded cold acetones, and embedded in Araldite. One whole half of the hypothalamus was cut serially into 1-/lm-thick sections in a frontocaudal progression and stained with an aqueous 1% toluidine blue and 1% sodium borate solution to map the VMN. Ultrathin sections for electron microscopy (80 nm, silver interference color) were
Development of Ventromedial Nucleus
123
cut with a diamond knife from the ventrolateral subdivision of the VMN. Then the sections were stained with uranyl acetate and lead citrate and photographed at 4000× in a Siemens Elmiskop 101 electron microscope operated at 80 kV.
RESULTS Postnatal Development of VMN-VL Neurons Comparative qualitative analysis of the male and female VMN-VL did not reveal striking differences in either neuronal cytology of the time course of developmental events. Therefore, the specific details of VMN-VL maturation described here apply equally to both sexes. The majority of neurons of the VMN-VL of 5-day-old rats presented a rudimentary organization (Fig. 1). A large, spheroidal, or slightly indented nucleus occurred excentrally within the perikarion. A prominent nucleolus and finely dispersed euchromatin were the main components of the nuclear matrix. The cytoplasm contained abundant free ribosomes and polyribosomes, scarcely developed Golgi complexes, small and vacuolated mitochondria, and few cisternae of rough endoplasmic reticulum (RER). The cisternae of R E R exhibited a canalicular profile, with numerous branches and segmentary dilatations decorated with ribosomes (Fig. 2). The neuropil was poorly differentiated and the scant organelles yielded an empty looking appearance.
Fig. 1. Neuronal cell body in the 5-day-old female rat VMN-VL. The nucleus is surrounded by a pale cytoplasmic rim, and the processes in the neuropil present an emptylooking appearance. ×11,300; reduced 50% for reproduction.
124
Fig. 2. Neuronal cell body in the 5-day-old male rat VMN-VL. The cisternae of the RER show a canalicular profile, and the Golgi complex is poorly differentiated, x23,000; reduced 47% for reproduction.
Pozzo Miller and Aoki
Fig. 3. Nucleolar extrusion in the perikaryon of a 5-day-old male rat VMN-VL. Note the proximity of cisternae of the RER and free ribosomes. ×18,500; reduced 47% for reproduction.
A common feature observed in neurons of 5-day-old rats was round structures containing a fine granular material and one to three vacuoles with a lighter electron density. These structures were identified by others as nucleolar extrusions (Fig. 3) (Santolaya, 1973; Shimizu and lshii, 1965). The cytology of the VMN-VL neurons changed dramatically between 10 and 20 days of age. The nucleus and nucleolus showed only minor modifications, in contrast with the cytoplasmic organelles which developed remarkably. The most striking changes were found in the Golgi apparatus and in the RER. The cisternae of the latter were observed organized in large bodies of stuffed membranes decorated with ribosomes (Figs. 4 and 5). This enlarged ergatoplasm corresponded topographically to the Nissl bodies occurring at either marginal cytoplasmic regions or proximal dendritic processes. Between 20 and 45 days of postnatal life the VMN-VL there was a slowdown in the maturational changes. The nucleus acquired a lobulated contour, with deep
Fig. 4. Neuronal cell body in the 10-day-old female rat VMN-VL. A dramatic increase in the RER is evident. Cisternae are arranged parallel to each other, x14,600; reduced 50% for reproduction.
Fig. 5. Enlarged ergatoplasm in the VMN-VL of a 10-day-old male rat. x17,300; reduced 50% for reproduction.
Development of Ventromedial Nucleus
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indentations of the nuclear membrane, and the neuropil increased the density of organelles, particularly the R E R and the Golgi complexes. From the 45th day to adulthood two distinct cellular types were recognized. The neurons most commonly seen were characterized by a rudimentary appearance due to the scarce of cytoplasmic organelles, with few and small mitochondria, long and isolated cisternae of R E R , a small Golgi complex, and an empty-looking cytoplasmic matrix. This seemed associated with a reduced metabolic activity (Fig. 6). In some neurons the nucleolus-like extrusions were also present. The other cell type exhibited a remarkable development of Nissl bodies, Golgi complexes, mitochondria, and large lobulated nucleus containing a conspicuous nucleolus. These features can be associated with an enhanced metabolic activity and resemble those found in the VMN neurons of 10-day-old rats.
Postnatal Development of VMN-VL Axodendritic Synapases Two types of axodendritic synapses were identified in the V M N - V L neuropil: (a) shaft synapses (Fig. 7) or axonal boutons on a dendritic shaft and (b) spine synapses (Fig. 8) containing through a dendritic spine. The latter were identified by the continuity of the spine with a dendritic shaft and by the lack of mitochondria and microtubules, distinctive features of dendritic shafts. Both types of synapses were present from the early postnatal periods to adulthood, however, shaft synapses occurred more frequently than spine synapses in the rat VMN-VL.
Fig. 6. Adult female rat VMN-VL neuron is characterized by a pale cytoplasm, with scattered mitochondria and large dense bodies. A few isolated costernae of RER are presented. )
