Magnocellular Neuroendocrine Cells Research Articles

Chapter 9 Functional synaptic plasticity in hypothalamic magnocellular neurons

The hypothalamic-neurohypophysial system undergoes dramatic morphological plasticity in response to physiological activation during parturition/lactation and dehydration, including somatic swelling, decreased glial coverage and increased synaptic innervation of the magnocellular neuroendocrine cells. Recent in-vitro electrophysiological studies in hypothalamic slices have demonstrated that coordinate changes in the synaptic physiology of the magnocellular neurons also occur under these conditions. Thus, the synaptic release of glutamate and GABA onto magnocellular neurons is increased during lactation and with chronic dehydration, and changes in postsynaptic glutamate and GABAA receptor expression lead to alterations of the functional properties of the glutamate and GABAA receptor channels. The presynaptic noradrenergic facilitation of glutamate release and inhibition of GABA release is also markedly enhanced following chronic dehydration. Additionally, both parturition and chronic dehydration are accompanied by an increase in the tonic activation of presynaptic metabotropic glutamate receptors due to the higher ambient glutamate concentration caused by decreased glial coverage and the resultant reduction in glutamate reuptake. Together, these electrophysiological studies reveal profound functional plasticity in the synaptic physiology of magnocellular neurons at parturition and following dehydration. The plastic changes support an increase in the excitability of magnocellular neuroendocrine cells by increasing glutamate inputs, decreasing GABA inputs, enhancing excitatory noradrenergic modulation, and reducing synaptic glutamatergic noise.

Osmotic and glutamate receptor regulation of c-Jun NH(2)-terminal protein kinase in neuroendocrine cells.

Expression of a c-Jun NH(2)-terminal protein kinase (JNK), also known as stress-activated protein kinase (SAPK) in rodents, has been implicated in the ability of cells to respond to a variety of stressors. In nonmammalian cells, JNK participates in the regulation of cell volume in response to hyperosmotic stress. To explore the possibility that JNK may participate in the transduction of osmotic information in mammals, we evaluated the expression of JNK immunoreactivity in neuroendocrine cells of the supraoptic nucleus. Low basal expression of JNK-2 (SAPK-alpha) and JNK-3 (SAPK-beta) was seen in vivo and in vitro. During water deprivation, JNK-2 increased in the supraoptic nucleus but not in the cortex. Osmotic or glutamate receptor stimulation in vitro also resulted in an increase in JNK-2 that was tetrodotoxin (TTX) insensitive and paralleled by increased nuclear phospho-c-Jun immunoreactivity. A TTX-sensitive increase in JNK-3 was seen in smaller neurons. Thus different JNK pathways may mediate individual cellular responses to osmotic stress, with JNK-2 linked to osmotic and glutamate receptor stimulation in magnocellular neuroendocrine cells.

Differential effects of K + channel blockers on frequency-dependent action potential broadening in supraoptic neurons

Recordings were made from magnocellular neuroendocrine cells dissociated from the supraoptic nucleus of the adult guinea pig to determine the role of voltage gated K + channels in controlling the duration of action potentials and in mediating frequency-dependent action potential broadening exhibited by these neurons. The K + channel blockers charybdotoxin (ChTx), tetraethylammonium (TEA), and 4-aminopyridine (4-AP) increased the duration of individual action potentials indicating that multiple types of K + channel are important in controlling action potential duration. The effect of these K + channel blockers was almost completely reversed by simultaneous blockade of voltage gated Ca 2+ channels with Cd 2+. Frequency-dependent action potential broadening was exhibited by these neurons during trains of action potentials elicited by membrane depolarizing current pulses presented at 10 Hz but not at 1 Hz. 4-AP but not ChTx or TEA inhibited frequency-dependent action potential broadening indicating that frequency-dependent action potential broadening is dependent on increasing steady-state inactivation of A-type K + channels (which are blocked by 4-AP). A model of differential contributions of voltage gated K + channels and voltage gated Ca 2+ channels to frequency-dependent action potential broadening, in which an increase of Ca 2+ current during each successive action potential is permitted as a result of the increasing steady-state inactivation of A-type K + channels, is presented.

Immunolabeling reveals cellular localization of the N-methyl-D-aspartate receptor subunit NR2B in neurosecretory cells but not astrocytes of the rat magnocellular nuclei.

Previous studies suggest that activation of N-methyl-D-aspartate (NMDA) receptors facilitates phasic firing and spike clustering displayed by magnocellular neuroendocrine cells (MNCs) of the supraoptic (SON) and paraventricular nucleus of the hypothalamus (PVN). Osmotic stimulation produces similar activity patterns which, in turn, can lead to enhanced release of vasopressin and oxytocin from MNCs. Our laboratory has shown that dehydration regulates the expression of the NMDA receptor subunits, NR1 and NR2B, in the SON and PVN, suggesting their involvement in osmoregulation. In the present study, we examined the cellular localization of NR2B, one of the glutamate-binding subunits of the NMDA receptor, with an NR2B-specific antibody. Using double-label immunohistochemistry and three different detection methods with metallic, peroxidase, and fluorescence markers, it was found that both vasopressin and oxytocin-producing MNC populations synthesize NR2B. The incidence of NR2B colocalization with vasopressin-neurophysin in the SON and lateral magnocellular PVN (PVL) was 0.95 and 0.91, respectively. For oxytocin-neurophysin, the corresponding values were 0.97 and 0.95, respectively. Furthermore, the extent of colocalization in MNCs of the SON, PVL, retrochiasmatic SON, and accessory neurosecretory nuclei was similar. Astrocytes associated with the SON, and identified with antibodies targeting glial fibrillary acidic protein (GFAP) or vimentin, were not colabeled with NR2B. Our results demonstrate that NR2B protein is expressed by almost all MNCs and that it is equally prevalent in vasopressinergic and oxytocinergic populations of various magnocellular neuroendocrine nuclei supporting a role of NMDA receptors in MNC-mediated neurosecretory processes. Although NR2B may form part of functional NMDA receptors on MNCs, it is probably not present on astrocytes associated with nearby MNCs.

The surface density of glial fibrillary acidic protein immunopositive astrocytic processes in the rat supraoptic nucleus is reversibly altered by dehydration and rehydration

Ultrastructural and immunohistochemical studies of the supraoptic nucleus (SON) have provided evidence that retraction and extension of astrocytic processes from between magnocellular neuroendocrine cells (MNCs) likely plays a role in the release of oxytocin, and/or vasopressin, that accompanies parturition, lactation and dehydration. The present study estimates the surface density ( Sv) of glial fibrillary acidic protein (GFAP)-immunoreactivity, predominantly in astrocytic processes, in the SON of normally hydrated, dehydrated and rehydrated rats. The Sv of GFAP processes in dehydrated rats was significantly reduced compared with control levels. Rehydration returned Sv to control levels. The reversible reduction in Sv indicates that the previously observed reduction in optical density is due to a rearrangement of astrocyte processes in the SON which occurs at the same time as the selective functional activation of MNCs.

Osmotic activation of the hypothalamo-neurohypophysial system reversibly downregulates the NMDA receptor subunit, NR2B, in the supraoptic nucleus of the hypothalamus

NMDA receptor activation produces a characteristic pattern of neuronal firing in magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) which has been associated with greater hormone release in vivo and in vitro. In addition, i.c.v. administered NMDA receptor blockers suppress the dehydration-induced rise in plasma vasopressin and drinking. To investigate the role of NMDA receptor subunits in the neuroendocrine functions of the magnocellular neuroendocrine cells of the hypothalamus, we examined the effects of osmotic stimulation on the protein expression of the NMDA receptor subunits, NR1 and NR2B, important in binding glycine and glutamate, respectively. Homogenates of SON, paraventricular nucleus of the hypothalamus (PVN), cortex and lateral hypothalamus from control rats and rats given 2% saline water to drink for 4–10 days were subjected to SDS–PAGE and Western blot analysis. This saline water drinking regimen produced a significant rise in plasma osmolality levels. NR1 and NR2B immunoreactivity was detected in SON, PVN, lateral hypothalamus and cortex but not in liver homogenates using subunit-specific polyclonal antibodies and quantified using computer-assisted densitometry. Mean NR2B immunoreactivity was significantly lower in SON (29%) and PVN homogenates (23%) from saline-treated rats than in those from control rats. In addition, the effect of dehydration on NR2B was regionally specific since no significant changes in NR2B expression were observed in homogenates of cortex and lateral hypothalamus. Rehydration allowed recovery of plasma osmolality as well as NR2B protein levels in the SON. These results suggest that changes in NMDA receptor subunit expression contribute to the plasticity manifested by in magnocellular neuroendocrine cells in response to osmotic activation of the hypothalamo-neurohypophysial system. In addition, our results indicate that NMDA receptors on SON and PVN MNCs may contribute to neuroendocrinological functions associated with body fluid homeostasis.

Functional activation of punch-cultured magnocellular neuroendocrine cells by glutamate receptor subtypes

To provide a simple means to isolate and study the cellular functions of small groups of neurons, we developed a modified ‘punch’ culture procedure that facilitates acute and long-term in vitro physiological studies. Primary ‘punch’ cultures of magnocellular neuroendocrine cells (MNCs) from the supraoptic nucleus (SON) were established and the basic physiological effects of subtype-specific glutamate receptor agonists were characterized. MNCs from the punch cultures established a mature morphology in culture with extensive outgrowth of axons and varicosities. After 8 days, a single cultured SON punch produced an average of 10.0±2.1 pg AVP and contained an average of 222±53 vasopressin-neurophysin immunoreactive cells. Patch clamp recordings from MNCs demonstrated the presence of N-methyl- d-aspartate (NMDA)-sensitive and dl, α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA)-receptors. Stimulation of metabotropic receptors with 1 S,3 R ACPD induced acute or gradual increases in intracellular calcium. NMDA, AMPA and metabotropic receptors all contributed to the secretion of vasopressin from the punch cultures with an agonist rank order potency of: NMDA (10 μM)>AMPA (1 μM)=1 S,3 R ACPD (100 μM)>kainate (10 μM). This culture preparation should be useful for cellular studies of small groups of neuroendocrine and other cells.

Chapter 2.1.2 Neurophysiology of magnocellular neuroendocrine cells: Recent advances

Magnocellular neuroendocrine cells of the hypothalamic paraventricular and supraoptic nuclei are responsible for most of the vasopressin and oxytocin in the peripheral blood as well as for central release of these peptides in selected brain areas. As the principal component of the hypothalamo-neurohypophysial system, these neurons have been a subject of continual study for half a century. The wealth of solid information from decades of in vivo studies has provided a firm basis for in vitro, brain slice and explant investigations of neural mechanisms involved in the control and regulation of vasopressin and oxytocin neurons. In vitro methods have revealed the presence and permitted the study of monosynaptic projections to supraoptic neurons from the olfactory bulbs, the tuberomammillary nuclei of the posterior hypothalamus and from the organum vasculosum of the lamina terminalis. Such methods have also facilitated the elucidation of the various ionic currents controlling neurosecretory cell activity as well as the roles of calcium binding proteins and release of calcium from internal stores. This review summarizes recent advances in our understanding of the afferent inputs that impinge upon these two cell types, and the cellular and molecular mechanisms intrinsic to these neurons that determine their activity patterns and, in part, their responses to incoming stimuli.

Channel properties of NMDA receptors on magnocellular neuroendocrine cells cultured from the rat supraoptic nucleus

Application of N-methyl- d-aspartate (NMDA) to the supraoptic nucleus of the hypothalamus (SON) generates clustered firing that may be important in hormone release. However, synaptically evoked EPSPs recorded from SON neurons exhibit varying contributions from NMDA receptors. We used the high resolution of single-channel recording to examine the receptor and ion channel properties of NMDA receptors expressed by SON neurons in `punch' culture. Biocytin introduced into individual neurons during patch clamp recording revealed large (32.1±3.3 μm), oblong somas and bipolar extensions typical of magnocellular neuroendocrine cells (MNCs). Rapid application of NMDA (100–300 μM) in the presence of 10 μM glycine to outside-out macropatches resulted in openings with an average conductance of 46.9 pS and reversal potential of +3.9 mV. Increasing glycine from 0.03 to 30 μM increased the apparent frequency, duration and occurrence of overlapping NMDA-elicited openings. NMDA responses were inhibited by Mg 2+ in a voltage-dependent manner and by the NMDA-site antagonist, d-(−)-2-amino-5-phosphonovaleric acid (D-APV). Application of saturating NMDA or glycine alone with the glycine-site antagonist, 5,7-dichlorokynurenate (DCK) or with D-APV, respectively, did not result in agonist-induced openings. NR1 immunoreactivity was observed in large neurons (>25 μm) with MNC-like morphology. These single-channel and immunocytochemical data confirm the presence of functional NR1-containing NMDA receptors in MNCs.

Glutamate microstimulation of local inhibitory circuits in the supraoptic nucleus from rat hypothalamus slices.

The hypothesis of a local inhibitory input to the hypothalamic supraoptic nucleus was tested with combined glutamate microstimulation and whole cell patch-clamp recordings in slices from rat hypothalamus. Synaptic activity in supraoptic magnocellular neuroendocrine cells (MNCs) was monitored and glutamate microdrops were applied in the perinuclear region of the supraoptic nucleus to evoke firing of action potentials in putative presynaptic inhibitory cells. The effect of glutamate microdrops applied in the perinuclear region was tested on 57 supraoptic MNCs. In control conditions, spontaneous excitatory (EPSCs) and inhibitory (IPSCs) postsynaptic currents were observed at resting membrane potential in all MNCs tested. Glutamate microstimulation evoked an abrupt increase in the frequency and size of spontaneous IPSCs in eight MNCs. Forty-nine MNCs did not show any change in the inhibitory synaptic input. Microapplication of glutamate in the periphery of the supraoptic nucleus did not modify the amplitude or the frequency of spontaneous EPSCs in any of the 57 MNCs tested. In the group of eight MNCs that responded to glutamate microstimulation by an increase in inhibitory input, two types of responses were observed. Four MNCs showed an increase in both size and frequency of spontaneous IPSCs through the entire range of amplitude. In the other four MNCs, local glutamate stimulation produced a dramatic increase in the size of IPSCs and a lesser increase in the frequency of the smaller IPSCs. The potential effect of the glutamate-evoked increase in inhibitory input on the firing activity of MNCs was tested in current-clamp conditions. Intracellular current injection was applied to evoke firing of action potentials in six MNCs that had responded to local glutamate microstimulation by an increase in inhibitory input. Glutamate microdrop applications inhibited the evoked action potential firing in all six cells. These results suggest 1) that local inhibitory interneurons are present in the periphery of the supraoptic nucleus, 2) that they contain functional glutamate receptors, 3) that they form inhibitory synapses with supraoptic MNCs, and 4) that activation of these interneurons inhibits firing in MNCs. These results support the hypothesis that local inhibitory interneurons play a important role in the firing activity of supraoptic MNCs.

Plasticity of astrocytes of the ventral glial limitans subjacent to the supraoptic nucleus.

We present evidence of gross morphological changes in astrocytes of the ventral glial limitans (VGL) associated with a well-known model of central nervous system (CNS) plasticity: the hypothalamic supraoptic nucleus (SON). Activity of SON magnocellular neuroendocrine cells (MNCs) was stimulated in experimental rats by substitution of 2% saline for drinking water for 2 or 9 days. Light microscopic measures revealed that a significant decrease in VGL thickness, by 34%, occurred with 9 days of stimulation. Astrocyte nuclei of 9-day dehydrated animals were also found to be 39% closer to the pial surface when compared with controls. Electron microscopy revealed a reorientation of individual astrocytes from a direction perpendicular (vertical) to the pial surface, to one parallel (horizontal) to this region. Vertically oriented astrocytes were found to be greater in the control group, by 49%, when compared with the 9-day dehydrated group, where cells were predominantly horizontal in orientation. Vertically oriented cells were further analyzed as to the direction of their vertical projections. Control, 2-day dehydrated and 9-day rehydrated animals, had more vertical cells which were oriented toward the pial surface when compared with 9-day dehydrated animals, where the relatively few vertically oriented astrocytes were significantly more likely to project toward the dendritic zone. In animals allowed to rehydrate for 9 days following a period of dehydration, these changes returned toward control levels. We conclude that astrocytes in vivo are capable of reversible gross morphological changes over a relatively short time.

Plasticity of astrocytes of the ventral glial limitans subjacent to the supraoptic nucleus

We present evidence of gross morphological changes in astrocytes of the ventral glial limitans (VGL) associated with a well-known model of central nervous system (CNS) plasticity: the hypothalamic supraoptic nucleus (SON). Activity of SON magnocellular neuroendocrine cells (MNCs) was stimulated in experimental rats by substitution of 2% saline for drinking water for 2 or 9 days. Light microscopic measures revealed that a significant decrease in VGL thickness, by 34%, occurred with 9 days of stimulation. Astrocyte nuclei of 9-day dehydrated animals were also found to be 39% closer to the pial surface when compared with controls. Electron microscopy revealed a reorientation of individual astrocytes from a direction perpendicular (vertical) to the pial surface, to one parallel (horizontal) to this region. Vertically oriented astrocytes were found to be greater in the control group, by 49%, when compared with the 9-day dehydrated group, where cells were predominantly horizontal in orientation. Vertically oriented cells were further analyzed as to the direction of their vertical projections. Control, 2-day dehydrated and 9-day rehydrated animals, had more vertical cells which were oriented toward the pial surface when compared with 9-day dehydrated animals, where the relatively few vertically oriented astrocytes were significantly more likely to project toward the dendritic zone. In animals allowed to rehydrate for 9 days following a period of dehydration, these changes returned toward control levels. We conclude that astrocytes in vivo are capable of reversible gross morphological changes over a relatively short time. © 1996 Wiley-Liss, Inc.

Temporal changes of c- fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress

The present experiments were undertaken to examine c- fos expression in magnocellular neuroendocrine cells (MNCs) of the rat hypothalamus with restraint stress using dual immunohistochemistry for c- fos and oxytocin. Restraint stress induced c- fos expression in oxytocinergic MNCs in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Quantitative immunohistochemical analysis revealed that percentages of c- fos—positive cells to oxytocin-immunoreactive MNCs in the SON and PVN maximally increased at 2 h after restraint stress had started, and began to decline in spite of the fact that the restraint of animals were continued. Similar results were obtained from time course of c- fos expression in parvocellular neurons of the PVN. When animals were released to move freely in their home cages following the 3-h restraint, the plasma levels of oxytocin declined to reach basal levels within 30 min and c- fos immunoreactivity in the hypothalamic MNCs and parvocellular neurons disappeared faster than those of the continually restrained. These results demonstrate that restraint stress induces c- fos expression in oxytocinergic MNCs in the SON and PVN, and that time course of c- fos expression is transient even in the continuation of restraint stress.

Effects of continuous environmental illumination on the albino rat hypothalamo-neurohypophysial system

Continuous environmental illumination or constant light (LL) exposure causes a suppression of daily water intake, and long-term exposure of greater than 19 days produces a hypertrophy of magnocellular neuroendocrine cells (MNCs) in the hypothalamus. These findings led Glantz to hypothesize that LL increases the secretion of vasopressin (VP). We wanted to determine whether LL could trigger morphological changes within the hypothalamo-neurohypophysial system (HNS) seen with other manipulations that result in enhanced hormone release. The posterior pituitary of male albino rats that were exposed to LL for 24 or 48 h were examined ultrastructurally for evidence of enhanced hormone release. In addition, water intake, plasma VP levels, and MNC size within the supraoptic nucleus (SON) were measured. After LL exposure, the posterior pituitary morphology was different, suggesting enhanced hormone release. LL exposure did not affect plasma VP or the size of SON MNCs, but did suppress drinking behavior. These data show that posterior pituitary morphology is affected rapidly by LL exposure. The HNS response to LL exposure may consist of changes within the first 24 h of LL found within the posterior pituitary followed later by hypertrophy of the SON MNCs.

Kindled seizures induce a long-term increase in vasopressin mRNA

Neuroendocrine disturbances are among the significant problems associated with animal and human seizures. To investigate the mechanisms for these disturbances, we examined changes in the expression of vasopressin (VP) mRNA in the hypothalamic magnocellular neuroendocrine cells of rats after amygdala kindled seizures, a model for temporal lobe epilepsy. A prominent increase in VP mRNA was found in the supraoptic nucleus of kindled animals by one week after the last seizure which persisted for at least 4 months. The increase occurred bilaterally in the SON and remained unchanged despite the absence of further stimulation, seizures or change in body fluid homeostasis. Since the VP mRNA change after kindling correlated with the duration of afterdischarge but not the number of amygdala stimuli the change appears to be an effect of the seizure. This chronic increase in VP mRNA appears to reflect a change in neuroendocrine gene expression and may identify an important new mechanism of plasticity that contributes to the neuroendocrine disturbances accompanying epilepsy.

Systemic signals contribute to induced morphological changes in the hypothalmo-neurohypophysial system

A systemic injection of hypertonic saline causes morphological changes in the supraoptic nucleus (SON) of the hypothalamus and the posterior pituitary, including withdrawal of astrocytic glial processes from among magnocellular neuroendocrine cells (MNCs) in the SON and increases in neural apposition with the basal lamina (BL) in the posterior pituitary. This study investigated whether systemic adrenalin provides a signal for these changes. Three groups of rats were given intraperitoneal injections of hypertonic (1.5 M) or normal (0.15 M) NaCl and were sacrificed 5 h after injection. One group was given no additional treatment, one group was anesthetized from prior to injection through perfusion, and one group was bilaterally adrenal-medullectomized several days prior to injections. Morphometric analysis of electron micrographs revealed rats given 1.5 M NaCl with no additional treatment showed expected changes in the SON and pituitary. Rats given 1.5 M NaCl and anesthetized showed diminished responses in the SON, and no changes in the pituitary compared to anesthetized rats given 0.15 M NaCl. No changes in withdrawal of glial processes from among MNCs or in neural apposition with the BL were seen in medullectomized animals. Thus, both anesthesia and adrenal-medullectomy interfere with signals for glial retraction and suggest that these responses are mediated by adrenalin.

Not only osmotic stress but also repeated restraint stress causes structural plasticity in the supraoptic nucleus of the rat hypothalamus

Magnocellular neuroendocrine cells (MNCs) in the supraoptic nucleus (SON) of the hypothalamus have been known to undergo dramatic structural changes during chronic stimulation such as osmotic stress. In the present study, we examined whether this anatomical neural plasticity is associated with an another stress, such as restraint. Rats were chronically stimulated by either dehydration with 2% saline drinking instead of water or daily restraint with leg immobilization. The structural reorganizations of MNCs in the SON were analyzed morphometrically with use of light and electron microscopy. The results were compared to control animals that had free access to water and food. In restraint rats, the soma size of both oxytocin (OXT) and arginine vasopressin (AVP) neurons was enlarged, and the percent of soma-somatic/dendritic membrane contact (juxtaposition) was elevated significantly. The number of total synapses per 100 μm soma membrane was not changed, although soma profiles were enlarged. However, the number of multiple synapses (which contacted with more than one postsynaptic element) per 100 μm soma membrane was significantly increased. Similar structural changes were observed in dehydrated animals, and the degree of morphological changes was stronger than the restraint one. These findings indicate that MNCs undergo structural plasticity during not only osmotic stress but also restraint stress.

Structural dynamics of neural plasticity in the supraoptic nucleus of the rat hypothalamus during dehydration and rehydration

It has been known that magnocellular neuroendocrine cells (MNCs) of mammalian hypothalamus show structural plasticity in response to chronic osmotic stimulation. In this study, we investigated the relationships among plasma osmolarity and several structural changes such as alterations of soma size, juxtaposition, and synapses of the supraoptic nucleus (SON) in the rat hypothalamus during dehydration and rehydration. Male rats were osmotically stimulated by supplying with 2% Nad solution instead of tap water for 10 days, and then they were rehydrated with tap water. Plasma osmolarity was gradually elevated with progress of salt loading and returned to control level on the seventh day of rehydration. Both the percentage of membrane contact (juxtaposition) and the soma size of MNCs were increased in response to the rise of plasma osmolarity, and decreased to control level on the seventh day of rehydration. The number of synapses including both single synapses and multiple synapses per 100 μm soma membrane was lower than control on the fifth day of dehydration, but it was not different from controls on the tenth day of dehydration, and on the seventh and fourteenth day of rehydration. The total number of synapses per 100 μm soma membrane, the synaptic density, was maintained relatively constant, although soma size was progressively changed during dehydration or rehydration. This synaptic reorganization seems to be mainly regulated by synaptic sprouting during dehydration and by degradation of synapses during rehydration. The number of multiple synapses per 100 μm soma membrane and the percentage of multiple synapses to total synapses were significantly higher than those of controls on the fifth and tenth day of dehydration and on the seventh day of rehydration, and they returned to control level on the fourteenth day of rehydration. Furthermore, most of the multiple synapses was observed to contact preferentially with soma and dendrites at increased stages of multiple synapses, indicating the special interaction of multiple synapses to soma by acting via adjacent dendrite.

Developmental expression of vasopressin in the human hypothalamus: double-labeling with in situ hybridization and immunocytochemistry.

The developmental expression of arginine vasopressin (VP) and VP mRNA in human hypothalamus was studied using combined immunocytochemistry (ICC) for VP-neurophysin II and in situ hybridization (ISH) for VP mRNA. Routine formalin-fixed autopsy material was used from 22 cases ranging in age from 18 wk of gestation to 21 yr. VP-neurophysin II immunoreactive cells were detected in the supraoptic, accessory supraoptic, and paraventricular nuclei of all brains examined. The average size of the immunocytochemically labeled cells increased until birth and remained constant thereafter. VP mRNA was first detected in cells at 21 wk gestation; 3 wk after the first detectable VP by ICC. After 27 wk of gestation, consistent and strong signals were obtained from the specimens that were double-labeled for both immunoreactive VP-neurophysin II and VP mRNA. Three populations of double-labeled cells were identified: type 1, intensely positive for both ISH and ICC (most magnocellular neurons); type 2, positive for ISH and weak or negative for ICC (rare and generally found in younger fetuses), and type 3, positive for ICC and weak or negative for ISH (mostly scattered in accessory nuclei). Thus, double-labeling techniques can be routinely used on frozen or paraffin sections of human autopsy material for the simultaneous assessment of both message and peptide. In the human fetus, the relatively late appearance of adult-like levels of VP mRNA in the magnocellular neuroendocrine cells suggests an association with the development of functional synaptic interactions in this system.

Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus

Although the hypothalamus is generally regarded to have low levels of glutamate receptors, anatomical and physiological studies have provided consistent evidence implicating glutamate as a potential transmitter for the control of neuroendocrine cell activity. To clarify the extent of the contribution of synapses utilizing glutamate for control of vasopressin/oxytocin neuroendocrine cells, we mapped the density and location of glutamate immunoreactive terminals in the supraoptic nucleus and surrounding hypothalamus. Colloidal gold particle densities in presynaptic terminals were measured from electron micrographs of: (1) the magnocellular neuroendocrine cell perikarya (main body of the supraoptic nucleus), (2) the dendritic field of the magnocellular neuroendocrine cells (ventral dendritic neuropil) and (3) the hypothalamic perinuclear zone dorsal to the supraoptic nucleus. In addition, serial sections were stained, alternately, for glutamate or GABA to determine glutamate staining in GABA cells. Terminals with high glutamate immunoreactivity were clearly distinguished from the glutamate precursor staining found in GABA terminals and were abundant at all rostral-caudal levels within each region. The number of glutamate terminals identified in each region was similar but represented a very high proportion of all terminals in the ventral dendritic neuropil (38%) vs. the main body of the supraoptic nucleus and the perinuclear zone (20–22%). The regional variation in the relative proportion of glutamate terminals was determined largely by differences in the number of non-glutamate terminals within each region. Glutamate and GABA terminals together accounted for over two-thirds of the innervation of vasopressin/oxytocin neuroendocrine cells. No systematic relationship was observed between excitatory and inhibitory inputs on the same cell. These results suggest that glutamate is the predominant excitatory transmitter used for control of vasopressin/oxytocin cells. The relative contribution of glutamate neurotransmission to a particular region will depend, in part, on the number and type of competing non-glutamate terminals.