Neuropeptide FF and neuropeptide VF inhibit GABAergic neurotransmission in parvocellular neurons of the rat hypothalamic paraventricular nucleus

Neuropeptide FF (NPFF) and neuropeptide VF (NPVF) are octapeptides belonging to the RFamide family of peptides that have been implicated in a wide variety of physiological functions in the brain, including central autonomic and neuroendocrine regulation. The effects of these peptides are mediated via NPFF1 and NPFF2 receptors that are abundantly expressed in the rat brain, including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we examined, using whole cell patch-clamp recordings in the brain slice, the effects of NPFF and NPVF on inhibitory GABAergic synaptic input to parvocellular PVN neurons. Under voltage-clamp conditions, NPFF and NPVF reversibly and in a concentration-dependent manner reduced the evoked bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in parvocellular PVN neurons by 25 and 31%, respectively. RF9, a potent and selective NPFF receptor antagonist, blocked NPFF-induced reduction of IPSCs. Recordings of miniature IPSCs in these neurons following NPFF and NPVF applications showed a reduction in frequency but not amplitude, indicating a presynaptic locus of action for these peptides. Under current-clamp conditions, NPVF and NPFF caused depolarization (6-9 mV) of neurons that persisted in the presence of TTX but was abolished in the presence of bicuculline. Collectively, these data provide evidence for a disinhibitory role of NPFF and NPVF in the hypothalamic PVN via an attenuation of GABAergic inhibitory input to parvocellular neurons of this nucleus and explain the central autonomic effects of NPFF.     

ANG II-induced excitation of paraventricular nucleus magnocellular neurons: a role for glutamate interneurons

The hypothalamic paraventricular nucleus (PVN) plays a critical role in cardiovascular and neuroendocrine regulation. ANG II (ANG) acts throughout the periphery in the maintenance of fluid-electrolyte homeostasis and has also been demonstrated to act as a neurotransmitter in PVN exerting considerable influence on neuronal excitability in this nucleus. The mechanisms underlying the ANG-mediated excitation of PVN magnocellular neurons have yet to be determined. We have used whole cell patch-clamp techniques in hypothalamic slices to examine the effects of ANG on magnocellular neurons. Application of ANG resulted in a depolarization of magnocellular neurons, a response that was abolished in TTX, suggesting an indirect mechanism of action. Interestingly, ANG also increased the frequency of excitatory postsynaptic potentials/currents in magnocellular neurons, an effect that was abolished after application of the glutamate antagonist kynurenic acid. ANG was without effect on the amplitude of excitatory postsynaptic currents, suggesting a presynaptic action on an excitatory interneuron within PVN. The ANG-induced depolarization was shown to be sensitive to kynurenic acid, revealing the requisite role of glutamate in mediating the ANG-induced excitation of magnocellular neurons. These observations indicate that the ANGergic excitation of magnocellular PVN neurons are dependent on an increase in glutamatergic input and thus highlight the importance of a glutamate interneuron in mediating the effects of this neurotransmitter.     

Angiotensin depolarizes parvocellular neurons in paraventricular nucleus through modulation of putative nonselective cationic and potassium conductances

Neurosecretory parvocellular neurons in the hypothalamic paraventricular nucleus (PVN) exercise considerable influence over the adenohypophysis and thus play a critical role in neuroendocrine regulation. ANG II has been demonstrated to act as a neurotransmitter in PVN, exerting significant impact on neuronal excitability and also influencing corticotrophin-releasing hormone secretion from the median eminence and, therefore, release of ACTH from the pituitary. We have used whole cell patch-clamp techniques in hypothalamic slices to examine the effects of ANG II on the excitability of neurosecretory parvocellular neurons. ANG II application resulted in a dose-dependent depolarization of neurosecretory neurons, a response that was maintained in tetrodotoxin (TTX), suggesting a direct mechanism of action. The depolarizing actions of this peptide were abolished by losartan, demonstrating these effects are AT(1) receptor mediated. Voltage-clamp analysis using slow voltage ramps revealed that ANG II activates a voltage-independent conductance with a reversal potential of -37.8 +/- 3.8 mV, suggesting ANG II effects on a nonselective cationic current. Further, a sustained potassium current characteristic of I(K) was significantly reduced (29.1 +/- 4.7%) by ANG II. These studies identify multiple postsynaptic modulatory sites through which ANG II can influence the excitability of neurosecretory parvocellular PVN neurons and, as a consequence of such actions, control hormonal secretion from the anterior pituitary.     

Vasopressin receptor distribution in adrenalectomized rats using vasopressin anti-idiotype

Following adrenalectomy, it has been demonstrated that parvocellular corticotropin-releasing factor-containing neurons in the paraventricular nucleus (PVN) of rat hypothalamus synthesize vasopressin. The present study examined whether putative vasopressin receptors are expressed in parallel with the appearance of vasopressin immunoreactivity in these parvocellular neurons. A vasopressin anti-idiotypic antibody which immunostains putative vasopressin receptors associated with magnocellular PVN neurons was utilized. Following adrenalectomy, antivasopressin immunostained neurons in parvocellular and magnocellular PVN, whereas the anti-idiotypic antibody immunostained magnocellular neurons only. We therefore conclude that the putative vasopressin receptor recognized by the anti-idiotype is not demonstrated in association with parvocellular vasopressin-producing neurons of the adrenalectomized rat.     

Intranuclear coupling of hypothalamic magnocellular nuclei by glutamate synaptic circuits

Magnocellular neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) display bursting activity that is synchronized under certain conditions. They receive excitatory synaptic inputs from intrahypothalamic glutamate circuits, some of which are activated by norepinephrine. Ascending noradrenergic afferents and intrahypothalamic glutamate circuits may be responsible for the generation of synchronous bursting among oxytocin neurons and/or asynchronous bursting among vasopressin neurons located in the bilateral supraoptic and paraventricular nuclei. Here, we tested whether magnocellular neurons of the PVN receive excitatory synaptic input from the contralateral PVN and the region of the retrochiasmatic SON (SONrx) via norepinephrine-sensitive internuclear glutamate circuits. Whole cell patch-clamp recordings were performed in PVN magnocellular neurons in coronal hypothalamic slices from male rats, and the ipsilateral SONrx region and contralateral PVN were stimulated using electrical and chemical stimulation. Electrical and glutamate microdrop stimulation of the ipsilateral SONrx region or contralateral PVN elicited excitatory postsynaptic potentials/currents (EPSP/Cs) in PVN magnocellular neurons mediated by glutamate release, revealing internuclear glutamatergic circuits. Microdrop application of norepinephrine also elicited EPSP/Cs, suggesting that these circuits could be activated by activation of noradrenergic receptors. Repetitive electrical stimulation and drop application of norepinephrine, in some cases, elicited bursts of action potentials. Our data reveal glutamatergic synaptic circuits that interconnect the magnocellular nuclei and that can be activated by norepinephrine. These internuclear glutamatergic circuits may provide the functional architecture to support burst generation and/or burst synchronization in hypothalamic magnocellular neurons under conditions of activation.     

Relationship of ACTH1-39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

Dual antigen immunocytochemical staining procedures were used in the same tissue section to determine the distribution of ACTH immunostained fibers and varicosities within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus and elucidate its anatomical relationship to vasopressin (VP) and oxytocin (OXY)-containing neurons. Double immunostained preparations using glucose oxidase-antiglucose oxidase complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue section were employed. ACTH-immunoreactive (ir) fibers were distributed throughout the periventricular stratum and the parvocellular component of the PVN; in the latter area fibers were particularly dense in the ventral medial portion of the medial parvocellular division. Dual immunostained sections revealed a close anatomical association between opiocortin fibers and oxytocin and vasopressin parvocellular neurons. ACTH immunostained fibers were present in the anterior and medial magnocellular component of PVN and in the ventral medial portion of the posterior magnocellular division; these immunoreactive fibers were in intimate proximity to oxytocin-ir perikarya. The very close approximation between the ACTH-ir fibers and oxytocin-containing cell bodies suggests potential cell to cell communication between the two peptidergic systems in PVN. Few ACTH immunostained fibers were seen in the dorsal lateral portion of the posterior magnocellular division in which vasopressinergic neurons predominate. The present anatomical study supports pharmacological and physiological studies which indicate that opioids can influence the activity of magnocellular PV neurons. This study also elucidates an anatomical relationship between opiocortins (ACTH1-39) and parvocellular PV neurons which suggests that the opiocortin system may play a role in the regulation of both the neuroendocrine and autonomic activities of specific PV neurons.     

Coexistence of CRF peptide and oxytocin mRNA in the paraventricular nucleus

Several studies have reported coexistences of peptides in parvocellular neurons of the paraventricular nucleus (PVN). However, the coexistence of peptides in the magnocellular PVN is less clear. Controversy exists in particular about the coexistence of corticotropin-releasing factor (CRF) and oxytocin (OX). Although these peptides are present in distinct areas of the PVN, some overlap may exist. This study investigated a potential coexistence of OX and CRF in magno- and parvocellular PVN. The data demonstrate with clarity that neurons containing both the mRNA for OX and the peptide CRF are present in subpopulations of magnocellular and parvocellular neurons of the PVN.     

Electron microscopic analysis of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80-120 nm dense core granules and 30-50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons contained 70-100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN. Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN. In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70-120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons. These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

Interleukin-1beta depolarizes magnocellular neurons in the paraventricular nucleus of the hypothalamus through prostaglandin-mediated activation of a non selective cationic conductance

Interleukin-1beta (IL-1beta) is involved in hypothalamic regulation of the neuroimmune response by influencing the synthesis and secretion of corticotropin releasing hormone (CRH), vasopressin (VP) and other stress-related mediators. VP secretion from magnocellular (MNC) neurons of the paraventricular nucleus (PVN) of the hypothalamus at the posterior pituitary and/or median eminence contributes to increasing adrenocorticotropin hormone (ACTH) output and ultimately glucocorticoid release, which then contributes to the stress response. In this study, using whole-cell patch clamp recordings from neurons in a slice preparation of the rat PVN, we show that MNC neurons are also influenced by IL-1beta. In response to 1 nM IL-1beta, 62% of MNC neurons tested depolarized (mean depolarization=10.9+/-1.4 mV); effects which were maintained in the presence of a sodium channel blocker, tetrodotoxin (TTX). The effects of IL-1beta on MNC neurons were blocked in the presence of a specific cyclooxygenase (COX)-2 inhibitor, NS-398, indicating a dependence on prostaglandins (PG) in mediating these effects. In response to direct application of 1 muM PGE2, 57% of MNC neurons depolarized, exhibiting a membrane potential change similar to that induced by IL-1beta (mean depolarization=7.8+/-1.1 mV). Voltage clamp experiments examining the effects of PGE2 on the currents evoked by slow voltage ramps revealed activation of a conductance characteristic of a non-selective cationic conductance (NSCC) (voltage-independent, with a reversal potential of -41.8+/-7.6 mV), suggesting that this prostanoid directly modifies cationic currents in MNC neurons. These data provide evidence that IL-1beta depolarizes MNC neurons in the PVN as a result of prostaglandin-mediated activation of a NSCC.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

Intracellular electrophysiology of mammalian peptidergic neurons in rat hypothalamic slices

The magnocellular neuropeptidergic cells (MNCs) of the paraventricular and supraoptic nuclei have been a model for biochemical and physiological studies of peptidergic neurons in the mammalian brain, but nearly all the electrophysiological studies of these vasopressinergic and oxytocinergic neuroendocrine cells are based on extracellular recordings. This paper reviews recent literature on electrophysiological properties of neurons in the magnocellular nuclei in which the rat in vitro slice preparation and intracellular recording were used. Spontaneously occurring action potentials and synaptic potentials (excitatory and inhibitory) have been observed in hypothalamic slices. The spike patterns have included slow and irregular firing, short rapid bursts of inactivating spikes, and slow phasic discharge with prolonged active and silent periods. Some studies have shown that increased osmolality causes neuronal firing, but this area is controversial. Intracellular injections of lucifer yellow have shown that some MNCs are dye-coupled and electron microscopic observations with the freeze-fracture technique have revealed occasional gap junctions, thus suggesting that some MNCs are electrotonically coupled. Both excitatory and inhibitory postsynaptic potentials have been evoked with extracellular stimulation. Therefore, action potentials, synaptic potentials, burst discharges, and probably electrotonic coupling have been found with intracellular recording in mammalian neuroendocrine cells. Future studies with intracellular recording and staining followed by immunohistochemical identification of cells should provide significant new information on the membrane physiology and synaptic pharmacology of vasopressinergic and oxytocinergic cells.     

Co-existence of CRF and vasopressin immunoreactivity in parvocellular paraventricular neurons of rat hypothalamus

New dual immunocytochemical staining procedures were used in the same tissue section to elucidate the distribution and co-existence of CRF and vasopressin in parvocellular neuronal perikarya in the paraventricular nucleus (PVN) of rat hypothalamus. CRF immunostained cells were for the most part concentrated in the medial parvocellular component of PVN. Few vasopressin-immunoreactive (ir) neurons were seen in this area in the normal and colchicine-treated animals. Vasopressin-containing neurons predominated in the magnocellular component of PVN. In the adrenalectomized and adrenalectomized-colchicine-treated animals, a dense accumulation of vasopressin-ir cells were observed in the medial parvocellular area of PVN; this region is normally vasopressin-ir poor and CRF-ir rich. The vasopressin immunostained cells appeared to have an anatomical distribution similar to that seen for CRF-containing cell bodies. Results of this study unequivocally establish the co-existence of vasopressin and CRF in the same parvocellular perikarya of PVN following pertubation of the pituitary-adrenal axis.     

Orexin depolarizes rat hypothalamic paraventricular nucleus neurons

Orexins, also called hypocretins, are newly discovered hypothalamic peptides that are thought to be involved in various physiological functions. In spite of the fact that orexin receptors, especially orexin receptor 2, are abundant in the hypothalamic paraventricular nucleus (PVN), the effects of orexins on PVN neurons remain unknown. Using a whole cell patch-clamp recording technique, we investigated the effects of orexin-B on PVN neurons of rat brain slices. Bath application of orexin-B (0.01-1.0 microM) depolarized 80.8% of type 1 (n = 26) and 79.2% of type 2 neurons tested (n = 24) in the PVN in a concentration-dependent manner. The effects of orexin-B persisted in the presence of TTX (1 microM), indicating that these depolarizing effects were generated postsynaptically. Addition of Cd(2+) (1 mM) to artificial cerebrospinal fluid containing TTX (1 microM) significantly reduced the depolarizing effect in type 2 neurons. These results suggest that orexin-B has excitatory effects on the PVN neurons mediated via a depolarization of the membrane potential.     

Genomic Effects of Cold and Isolation Stress on Magnocellular Vasopressin mRNA-Containing Cells in the Hypothalamus of the Rat

We assessed the effects of cold and isolation stress on arginine vasopressin (AVP) mRNA in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Vasopressin mRNA levels were determined by in situ hybridization histochemistry at the cellular level. In posterior magnocellular neurons of the PVN isolation stress for 7 or 14 days increased vasopressin mRNA levels 28 and 29%, respectively, compared to group-housed controls. No significant alterations in vasopressin gene expression were observed in the SON after 7 or 14 days of isolation stress. Scattered magnocellular AVP mRNA-expressing cells of the medial parvocellular PVN showed increases of 19 and 34% after 7 and 14 days of isolation, respectively. We also studied the effect of cold or combined cold and isolation stress on vasopressin gene expression in the PVN and SON. Cold stress for 3 h daily for 4 consecutive days increased AVP mRNA levels in the posterior magnocellular PVN by 15%. Cold-isolated animals showed an increase of 21%. No significant effect on AVP mRNA levels in the SON was observed. In contrast to the posterior magnocellular PVN, cold or cold-isolation stress increased AVP mRNA in magnocellular neurons of the medial parvocellular region of the PVN by 25 and 43%, respectively, relative to control rats. These results suggest that psychological and metabolic stress may be added to the list of stressors that activate the hypothalamo-neurohypophysial system.     

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei

The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Activation of adenosine A2A receptors alters postsynaptic currents and depolarizes neurons of the supraoptic nucleus

Supraoptic nucleus (SON) neurons secrete oxytocin or vasopressin in response to various physiological stimuli (e.g., lactation/suckling, dehydration). Released near fenestrated capillaries of the neurohypophysis, these peptides enter the blood and travel to peripheral target organs. The pervasive neuromodulator adenosine, acting at A1 receptors, is an important inhibitory regulator of magnocellular neuroendocrine cell activity. Another high-affinity adenosine receptor exists in this system, however. We examined the physiological effects of adenosine A2A receptor activation and determined its localization among various cell types within the SON. In whole cell patch-clamp recordings from rat brain slices, application of the selective adenosine A2A receptor agonist CGS-21680 caused membrane depolarizations in SON neurons, often leading to increased firing activity. Membrane potential changes were persistent (>10 min) and could be blocked by the selective A2A receptor antagonist ZM-241385, or GDP-beta-S, the latter suggesting postsynaptic sites of action. However, +/--alpha-methyl-(4-carboxyphenyl)glycine or TTX also blocked CGS-21680 effects, indicating secondary actions on postsynaptic neurons. In voltage-clamp mode, application of CGS-21680 caused a slight increase (approximately 8%) in high-frequency clusters of excitatory postsynaptic currents. With the use of specific antibodies, adenosine A2A receptors were immunocytochemically localized to both the magnocellular neurons and astrocytes of the SON. Ecto-5'nucleotidase, an enzyme involved in the metabolism of ATP to adenosine, was also localized to astrocytes of the SON. These results demonstrate that adenosine acting at A2A receptors can enhance the excitability of SON neurons and modulate transmitter release from glutamatergic afferents projecting to the nucleus. We suggest that adenosine A2A receptors may function in neuroendocrine regulation through both direct neuronal mechanisms and via actions involving glia.     

Actions of neuropeptide W in paraventricular hypothalamus: implications for the control of stress hormone secretion

Neuropeptide W (NPW) is produced in neurons located in hypothalamus and brain stem, and its receptors are present in the hypothalamus, in particular in the paraventricular nucleus (PVN). Intracerebroventricular (ICV) administration of NPW activated, in a dose-related fashion, the hypothalamic-pituitary-adrenal axis, as determined by plasma corticosterone levels in conscious rats but, at those same doses, did not stimulate the release of oxytocin or vasopressin into the peripheral circulation or alter blood pressure or heart rate. The ability of ICV-administered NPW to stimulate the hypothalamic-pituitary-adrenal axis in conscious male rats was blocked by intravenous pretreatment with a corticotropin-releasing hormone antagonist. This suggested an action of NPW in the parvocellular division of the PVN. Indeed, in hypothalamic slice preparations (whole cell patch recording), bath application of NPW depolarized and increased the spike frequency of the majority of electrophysiologically identified putative neuroendocrine PVN neurons. Effects on membrane potential were maintained in the presence of TTX, suggesting them to be direct postsynaptic actions on these neuroendocrine cells. Our data suggest that endogenous NPW, produced in brain, may play a physiologically relevant role in the neuroendocrine response to stress.