Magnocellular Neurons Of Supraoptic Nucleus Research Articles

Rescue of Vasopressin Synthesis in Magnocellular Neurons of the Supraoptic Nucleus Normalises Acute Stress-Induced Adrenocorticotropin Secretion and Unmasks an Effect on Social Behaviour in Male Vasopressin-Deficient Brattleboro Rats.

The relevance of vasopressin (AVP) of magnocellular origin to the regulation of the endocrine stress axis and related behaviour is still under discussion. We aimed to obtain deeper insight into this process. To rescue magnocellular AVP synthesis, a vasopressin-containing adeno-associated virus vector (AVP-AAV) was injected into the supraoptic nucleus (SON) of AVP-deficient Brattleboro rats (di/di). We compared +/+, di/di, and AVP-AAV treated di/di male rats. The AVP-AAV treatment rescued the AVP synthesis in the SON both morphologically and functionally. It also rescued the peak of adrenocorticotropin release triggered by immune and metabolic challenges without affecting corticosterone levels. The elevated corticotropin-releasing hormone receptor 1 mRNA levels in the anterior pituitary of di/di-rats were diminished by the AVP-AAV-treatment. The altered c-Fos synthesis in di/di-rats in response to a metabolic stressor was normalised by AVP-AAV in both the SON and medial amygdala (MeA), but not in the central and basolateral amygdala or lateral hypothalamus. In vitro electrophysiological recordings showed an AVP-induced inhibition of MeA neurons that was prevented by picrotoxin administration, supporting the possible regulatory role of AVP originating in the SON. A memory deficit in the novel object recognition test seen in di/di animals remained unaffected by AVP-AAV treatment. Interestingly, although di/di rats show intact social investigation and aggression, the SON AVP-AAV treatment resulted in an alteration of these social behaviours. AVP released from the magnocellular SON neurons may stimulate adrenocorticotropin secretion in response to defined stressors and might participate in the fine-tuning of social behaviour with a possible contribution from the MeA.

Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.

Furosemide, which is used worldwide as a diuretic agent, inhibits sodium reabsorption in the Henle's loop, resulting in diuresis and natriuresis. Arginine vasopressin (AVP) is synthesized in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) of the hypothalamus. The synthesis AVP in the magnocellular neurons of SON and PVN physiologically regulated by plasma osmolality and blood volume and contributed water homeostasis by increasing water reabsorption in the collecting duct. Central AVP dynamics after peripheral administration of furosemide remain unclear. Here, we studied the effects of intraperitoneal (i.p.) administration of furosemide (20 mg/kg) on hypothalamic AVP by using transgenic rats expressing AVP-enhanced green fluorescent protein (eGFP) under the AVP promoter. The i.p. administration of furosemide did not affect plasma osmolality in the present study; however, eGFP in the SON and magnocellular divisions of the PVN (mPVN) were significantly increased after furosemide administration compared to the control. Immunohistochemical analysis revealed Fos-like immunoreactivity (IR) in eGFP-positive neurons in the SON and mPVN 90 min after i.p. administration of furosemide, and AVP heteronuclear (hn) RNA and eGFP mRNA levels were significantly increased. These furosemide-induced changes were not observed in the suprachiasmatic AVP neurons. Furthermore, furosemide induced a remarkable increase in Fos-IR in the organum vasculosum laminae terminals (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), locus coeruleus (LC), nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVLM) after i.p. administration of furosemide. In conclusion, we were able to visualize and quantitatively evaluate AVP-eGFP synthesis and neuronal activations after peripheral administration of furosemide, using the AVP-eGFP transgenic rats. The results of this study may provide new insights into the elucidation of physiological mechanisms underlying body fluid homeostasis induced by furosemide. This article is protected by copyright. All rights reserved.

Effect of interaction between acute administration of morphine and cannabinoid compounds on spontaneous excitatory and inhibitory postsynaptic currents of magnocellular neurons of supraoptic nucleus.

Opioids and cannabinoids are two important compounds that have been shown to influence the activity of magnocellular neurons (MCNs) of supraoptic nucleus (SON). The interaction between opioidergic and cannabinoidergic systems in various structures of the brain and spinal cord is now well established, but not in the MCNs of SON. In this study, whole cell patch clamp recording of neurons in rat brain slice was used to investigate the effect of acute morphine and cannabinoid administration on spontaneous inhibitory and excitatory spostsynaptic currents (sIPSCs and sEPSCs) in MCNs. Bath application of morphine produced an increase in sEPSCs frequency and a decrease in sIPSCs frequency. In contrast, bath application of URB597 (fatty acid amide hydrolase (FAAH) inhibitor) produced a decrease in sEPSCs frequency but an increase in sIPSCs frequency. WIN55212-2 (cannabinoid receptor agonist) decreased both sIPSCs and sEPSCs frequencies of MCNs. Co-application of morphine and URB597 attenuated the effect of morphine on MCNs. Taken together, these data indicated that at the cellular level, pharmacological augmentation of endocannabinoids could attenuate morphine effects on MCNs.

Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

The comparison of the effects of acute and repeated morphine administration on fast synaptic transmission in magnocellular neurons of supraoptic nucleus, plasma vasopressin levels, and urine volume of male rats.

The activity of the magnocellular neurons (MCNs) of supraoptic nucleus (SON) is regulated by a variety of excitatory and inhibitory inputs. Opioids are one of the important compounds that affect these inputs at SON synapses. In this study, whole-cell patch clamp recording of SON neurons was used to investigate the effect of acute and repeated morphine administration on spontaneous inhibitory and excitatory post synaptic currents (sIPSCs and sEPSCs) in MCNs. While acute bath application of morphine to brain slice of intact rat produced an increase in sEPSCs frequency and a decrease in sIPSCs frequency, repeated in-vivo administration of morphine produced opposite effect. Moreover, repetitive i.c.v. administration of morphine for three consecutive days caused significant increase in urine volume, but had no significant alteration in water consumption compared to control group. The increase in urine volume was consistent with a significant decrease in plasma arginine vasopressin (AVP) levels after repetitive i.p. morphine administration. The results suggest that acute administration of morphine stimulates whereas repeated administration of morphine inhibits the MCNs. Morphine-induced MCN inhibition could result in diminished plasma AVP levels and eventually an increase in urine volume of rats.

Galanin Modulates Neuronal and Synaptic Properties in the Rat Supraoptic Nucleus in a Use and State Dependent Manner

The magnocellular neurons of the hypothalamic supraoptic nucleus (SON) synthesize and secrete oxytocin (OXT) and vasopressin (AVP) from their dendrites. These peptides, and several other neurotransmitters, have been shown to modulate afferent glutamatergic neurotransmission in the SON. The neuropeptide, galanin (GAL) is also localized in SON magnocellular neurons and in afferent fibers in the nucleus. We show that GAL dose-dependently reduces evoked excitatory postsynaptic currents (eEPSCs), alters paired pulse ratio and decreases mEPSC frequency, but not amplitude or decay kinetics in both OXT and AVP neurons. GAL therefore modulates excitatory neurotransmission at a likely presynaptic receptor. Neither OXT/AVP, GABA(B) nor cannabinoid antagonists blocked this effect. A GAL2/3 agonist mimicked GAL's action while GAL1 antagonist did not block GAL's effect, suggesting that GAL2/3 receptors mediate the presynaptic effect. In nondehydrated rats GAL causes a small postsynaptic response, as assessed by input resistance measurements. When the rats were water deprived for 2 days the presynaptic response to GAL was unaltered; however, the postsynaptic decrease in input resistance and hyperpolarization was increased, an effect consistent with a previously described increase in GAL1 receptor expression in dehydration. A GAL1 receptor antagonist blocked the postsynaptic effects. Last, when a train of eEPSCs was elicited, GAL was found to inhibit the earlier events in a train but not the latter. This indicates that GAL may modulate a single synaptic event more effectively than trains of synaptic inputs, thereby acting as a high-pass filter.

Noradrenergic regulation of galanin expression in the supraoptic nucleus in the rat hypothalamus. An ex vivo study

Galanin is coexpressed with vasopressin and oxytocin in magnocellular neurons of the rat neuroendocrine hypothalamus. Various physiological stimuli, such as osmotic stimulation or lactation, that affect vasopressin and oxytocin expression and release also modulate galanin expression. Magnocellular neurons are highly innervated by noradrenergic inputs from the brainstem. The noradrenergic system plays a critical excitatory role in the activation of vasopressin-expressing and oxytocin-expressing neurons. Here, we have evaluated the possible regulation of Gal expression by noradrenaline in the magnocellular neurons of supraoptic nucleus in an ex vivo acute model of rat hypothalamic slices. The slices containing the supraoptic nucleus were incubated with 10(-4) M noradrenaline for 1 or 4 hr. The levels of galanin and galanin mRNA were estimated by semiquantitative immunohistochemistry and in situ hybridization, respectively. Our results show that the amount of galanin-immunopositive material in the cell bodies of the magnocellular neurons increased significantly after incubation with noradrenaline compared with control slices at the same time point and that this effect was more pronounced after 4 hr than after 1 hr. In situ hybridization showed that radiolabeling of the supraoptic nucleus with a radioactive galanin probe increased slightly after 1 hr of incubation and increased considerably after 4 hr of incubation with noradrenaline. Our study shows that galanin may be a target in the regulation of the hypothalamic magnocellular-neurohypophysial system by noradrenaline.

Activity-Dependent Modulation of Neurotransmitter Innervation to Vasopressin Neurons of the Supraoptic Nucleus

Confocal microscopy was used to assess activity-dependent neuroplasticity in neurotransmitter innervation of vasopressin immunoreactive magnocellular neurons in the supraoptic nucleus (SON). Vesicular glutamate transporter 2, glutamic acid decarboxylase, and dopamine beta-hydroxylase (DBH) synaptic boutons were visualized in apposition to vasopressin neurons in the SON. A decrease in DBH synaptic boutons per cell was seen upon salt loading, indicating diminished noradrenergic/adrenergic innervation. Loss of DBH appositions to vasopressin neurons was associated with a general loss of DBH immunoreactivity in the SON. In contrast, the number of vesicular glutamate transporter 2 synaptic boutons per neuron increased with salt loading, consistent with increased glutamatergic drive of magnocellular SON neurons. Salt loading also caused an increase in the total number of glutamic acid decarboxylase synaptic boutons on vasopressinergic neurons, suggesting enhanced inhibitory innervation as well. These studies indicate that synaptic plasticity compensates for increased secretory demand and may indeed underlie increased secretion, perhaps via neurotransmitter-specific, activity-related changes in synaptic contacts on vasopressinergic magnocellular neurons in the SON.

Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release.

Apelin, a recently isolated neuropeptide that is expressed in the supraoptic and the paraventricular nuclei, acts on specific receptors located on vasopressinergic neurons. The increased phasic pattern of these neurons facilitates sustained antidiuresis during dehydration or lactation. Here, we investigated whether apelin interacts with arginine vasopressin (AVP) to maintain body fluid homeostasis. We first characterized the predominant molecular forms of endogenous hypothalamic and plasma apelin as corresponding to apelin 13 and, to a lesser extent, to apelin 17. We then demonstrated that, in lactating rats, apelin was colocalized with AVP in supraoptic nucleus magnocellular neurons and given intracerebroventricularly inhibited the phasic electrical activity of AVP neurons. In lactating mice, intracerebroventricular administration of apelin 17 reduced plasma AVP levels and increased diuresis. Moreover, water deprivation, which increases systemic AVP release and causes depletion of hypothalamic AVP stores, decreased plasma apelin concentrations and induced hypothalamic accumulation of the peptide, indicating that AVP and apelin are conversely regulated to facilitate systemic AVP release and suppress diuresis. Opposite effects of AVP and apelin are likely to occur at the hypothalamic level through autocrine modulation of the phasic electrical activity of AVP neurons. Altogether, these data demonstrate that apelin acts as a potent diuretic neuropeptide counteracting AVP actions through inhibition of AVP neuron activity and AVP release. The coexistence of apelin and AVP in magnocellular neurons, their opposite biological effects, and regulation are likely to play a key role for maintaining body fluid homeostasis.

Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus.

Oxytocin (OT) and vasopressin (VP) are peptide hormones that are derived from genes predominantly expressed in distinct magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Recent evidence suggests that some magnocellular neurons coexpress both peptides. Our qualitative RT-PCR experiments on single cells show that the majority of magnocellular neurons coexpress both peptide messenger RNAs (mRNAs) in varying amounts. Using a competitive RT-PCR method combined with a standard calibration curve, we quantitatively determined OT and VP mRNA in single magnocellular neurons from the normal female rat SON, with a detection sensitivity of less than 30 mRNA molecules/cell. We defined the phenotypes of the single magnocellular neurons according to their ratios of these two peptide mRNAs. Using this approach, we identified three major phenotypes: oxytocin neurons, where the average OT to VP mRNA ratio is about 256; vasopressin neurons, where the average VP to OT mRNA ratio is about 182; and one oxytocin/vasopressin coexisting neuron, where the OT/VP mRNA ratio is 2. Thus, there is some OT and VP mRNA coexpression in virtually all of the magnocellular neurons in supraoptic nuclei of hypothalamus. However, clear phenotypes are identifiable by considering quantitative as opposed to qualitative differences.

Activation of an Inwardly Rectifying K+ Conductance by Orphanin-FQ/Nociceptin in Vasopressin-Containing Neurons

The orphanin-FQ/nociceptin (OFQ/N) receptor (previously, ORL₁, LC132) has been shown to be coupled to an inwardly rectifying K⁺ conductance in several neuronal populations. Although OFQ/N receptor mRNA is densely expressed in the supraoptic nucleus (SON), little is known about its coupling to effector system(s). The present study examined the effects of OFQ/N on guinea pig magnocellular neurons within the SON using intracellular recording from hypothalamic slices. In the presence of tetrodotoxin, OFQ/N hyperpolarized 48 of 48 SON magnocellular neurons, 24 of which were subsequently identified by immunocytochemistry as arginine vasopressin positive (AVP⁺). Nineteen of the 48 SON neurons, including 7 which were AVP⁺, responded to OFQ/N with an outward current that reversed at the K⁺ equilibrium potential (E_K+) and a decrease in slope resistance consistent with the activation of an inwardly rectifying K⁺ channel. In 4 of these neurons, BaCl₂ significantly attenuated both the hyperpolarization and the decrease in slope resistance induced by OFQ/N. Twenty-one SON neurons, 13 of which were AVP⁺, responded to OFQ/N with an increase in slope resistance which did not reverse at E_K+. An additional 5 neurons (2 were AVP⁺) were treated with the gap junction blocking agent carbenoxolone (CARB). CARB induced a small hyperpolarization, increased slope resistance and significantly reduced the subsequent OFQ/N-induced hyperpolarization. However, when the CARB and CARB plus OFQ/N hyperpolarizations were summed in these 5 cells, they were no different than the OFQ/N hyperpolarization alone. The effect of two putative OFQ/N receptor antagonists was also evaluated. The ĸ₃-opioid antagonist naloxone benzoylhydrazone was without effect (n = 3), and the 13-amino-acid [Phe¹Ψ(CH₂-NH)Gly²]OFQ/N(1–13)NH₂ OFQ/N analog produced a small hyperpolarization on its own in addition to partially antagonizing the effects of OFQ/N (n = 3). Taken together, these results suggest that OFQ/N acts upon SON neurons through two mechanisms, one which hyperpolarizes the neuron by activating an inwardly rectifying K⁺ conductance, and another which may increase membrane resistance by closing the low-resistance gap junctions.

Electrophysiological studies of neurohypophysial neurons and peptides

We have used hypothalamic slices of the supraoptic nucleus (SON) to investigate synaptic control of magnocellular vasopressinergic and oxytocinergic neurons. With the use of perforated patch recording techniques we identified and isolated excitatory or inhibitory postsynaptic currents elicited by electrical stimulation of afferent fibers. Both inhibitory and excitatory afferent fibers displayed presynaptic GABAB receptors; the GABAB agonist, baclofen caused a dose-dependent suppression of the evoked potentials in the absence of any effects on postsynaptic input resistance. Further evidence for a presynaptic locus included an increase in paired pulse ratio and a lack of effect on currents elicited by exogenously applied muscimol (a GABAA receptor agonist) or AMPA (a glutamate agonist). With the use of an GABAB receptor antagonist we demonstrated an action of endogenously released GABA, acting at GABAB receptors on excitatory terminals, to reduce excitatory transmission. In addition to presynaptic modulation by GABA of afferent inputs, we also observed actions of vasopressin and oxytocin, released from dendrites of magnocellular SON neurons, to gate afferent, excitatory transmission in the SON. Exogenously applied vasopressin and oxytocin, or these peptides when released by depolarizing stimuli of magnocellular neurons, reduced the size of evoked excitatory postsynaptic potentials at a presynaptic locus. We have also observed actions of arginine vasopressin to modulate the action of glutamate in slices of the ventral septal area and to attenuate a glutamate-mediated excitatory postsynaptic current in slices of the parabrachial nucleus.

Acute osmotic/stress stimuli induce a transient decrease of transcriptional activity in the neurosecretory neurons of supraoptic nuclei.

Administration of hypertonic NaCl solutions by intraperitoneal injection evokes a transient expression of immediate-early genes in the hypothalamic magnocellular neurons of supraoptic nuclei (SON), which is followed by an upregulation of arginine vasopressin synthesis and a general increase in cellular metabolic activity. Here we have analysed the changes that occur in the nucleus of SON neurons during the period of transient Fos expression after injection of hypertonic saline. Within the first 30 minutes after injection, the nuclei become significantly smaller, contain more condensed chromatin and incorporate less 3H-uridine than the controls. By 12 hours these effects are reverting and at 24 hours the nuclei are already more active than the controls. Additionally, we observe an initial decrease in the number of coiled bodies per nucleus within the first 2 hours, followed by a 3-fold increase at 24 hours after injection. As coiled bodies are transcription-dependent subnuclear 'organelles', these results further support the view that injection of hypertonic saline causes a transient inhibition of nuclear activity. Our data show that SON neurons respond to acute osmotic/stress stimuli first with inhibition and then with activation of gene expression. Importantly, inhibition of transcriptional activity occurs simultaneously with maximal accumulation of Fos protein in the nucleus, raising the possibility that activation of c-fos expression may cause repression of target genes.

Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus.

Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus. J. Neurophysiol. 78: 2427-2437, 1997. Neurons in the perinuclear zone (PZ) of the supraoptic nucleus (SON) are thought to serve as interneurons and may mediate changes in neurohypophysial hormone release in response to physiological changes in blood pressure. However, the morphology and electrophysiological characteristics of PZ neurons are unknown. In the present study, PZ neurons from male and female rats were recorded intracellularly to determine some membrane properties, then filled with biocytin or biotinamide for morphological analysis. In general, PZ neurons had faster spikes than magnocellular SON neurons, and the great majority were characterized by a subthreshold depolarizing hump when depolarized from a hyperpolarized (less than -80 mV) membrane potential. In most neurons, this hump was similar to low-threshold spikes described in other CNS regions. Near-threshold, fast action potentials were clustered near the onset of these depolarizations. Conspicuously absent in all PZ neurons was the strong transient and subthreshold outward rectification characteristic of vasopressin and oxytocin neurons of the SON. These results suggest that PZ neurons are electrophysiologically distinct from neurosecretory neurons of the SON. No differences were found between male and female rats in any of the basic properties examined, including input resistance, membrane time constant, spike height, spike width, spike threshold, and the size of the spike afterhyperpolarization. Morphologically, PZ neurons were diverse but were divided into spiny and aspiny groups. Three spiny neurons and one aspiny neuron contributed an axonal projection to the SON characterized by varicosities suggestive of terminals. In the case of the three spiny neurons, the SON projection was clearly a minor collateral projection. The axon arborized in the PZ, but one or more branches were cut at the edge of the explant, indicating a longer projection. In the remaining neurons, no axonal projection to the SON was detected and several had axons leaving the explant. Some portion of the dendritic tree penetrated the SON in several neurons. The morphology of PZ neurons was thus heterogeneous and suggests that, for some cells at least, the projection to the SON may be a minor collateral component of a much wider axonal projection.

Inhibition of spontaneous EPSCs and IPSCs by presynaptic GABAB receptors on rat supraoptic magnocellular neurons.

1. The function of presynaptic GABA receptors in the regulation of transmitter release in supraoptic nucleus (SON) magnocellular neurons was investigated by recording spontaneous postsynaptic currents from rat magnocellular SON neurons in a slice preparation (150 microns thick, 1.8 mm in diameter) using the whole-cell patch-clamp technique. 2. Both the spontaneous EPSCs and IPSCs were TTX resistant. The EPSCs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the IPSCs were abolished by picrotoxin, suggesting that the EPSCs and IPSCs are synaptic inputs from glutamatergic and GABAergic neurons, respectively. 3. The selective GABAB agonist, baclofen, reduced the frequency of both the EPSCs and IPSCs without affecting the amplitude. The time constant of the decay phase of both the EPSCs and IPSCs remained unchanged after baclofen application. 4. The reduction of the frequency of the synaptic currents by baclofen was dose dependent (10 nM to 100 microM) and the EC50 values were 5.8 and 8.5 microM for the EPSCs and IPSCs, respectively. 5. The effect of baclofen (10 microM) was antagonized by the selective GABAB antagonist, 2-hydroxy-saclofen (2OH-saclofen), at 300 microM. 6. When given alone, 2OH-saclofen (100 microM) increased the frequency of both the EPSCs and IPSCs without affecting their amplitude, suggesting that endogenously released GABA in the slice acts on presynaptic GABAB receptors. 7. The GABAA agonist, muscimol, reduced the frequency of EPSCs, and picrotoxin increased the frequency of the EPSCs, suggesting that GABAA receptors also participate in the presynaptic inhibition of glutamate release. 8. Taken together, these data suggest that GABAB receptors are present on the presynaptic terminals of both GABA and glutamate neurons in the SON, and that these presynaptic GABAB receptors play an important role in the regulation of the neuronal activity in SON magnocellular neurons.

Presynaptic modulation by metabotropic glutamate receptors of excitatory and inhibitory synaptic inputs to hypothalamic magnocellular neurons.

The effects of activation of metabotropic glutamate receptors (mGluRs) on synaptic inputs to magnocellular neurons of the hypothalamic supraoptic nucleus (SON) were studied with the use of whole cell patch-clamp and microelectrode recordings in acute hypothalamic slices. Application of the mGluR agonist trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 100 microM) elicited an increase in the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs) in 20% of the cells, and of spontaneous inhibitory postsynaptic potentials (IPSPs) and inhibitory postsynaptic currents (IPSCs) in 50% of the cells tested in normal medium. The increased frequency of spontaneous EPSPs/EPSCs and IPSPs/IPSCs was blocked by tetrodotoxin (TTX), indicating that mGluRs act to excite the somata/dendrites of presynaptic glutamatergic and GABAergic neurons. (RS)-3,5-dihydroxyphenylglycine (50 microM), a selective group I receptor agonist, mimicked the presynaptic somatic/dendritic effects of trans-ACPD, suggesting that the presynaptic somatic/dendritic receptors responsible for increased spike-dependent glutamate and gamma-aminobutyric acid (GABA) release belong to the group I mGluRs. In the presence of TTX, trans-ACPD caused a decrease in the frequency of miniature EPSCs (up to 90%) in 13 of 16 cells, and a decrease in the frequency of miniature IPSCs (up to 80%) in 10 of 16 cells tested. Miniature EPSC and IPSC amplitudes usually did not change in trans-ACPD, suggesting that activation of metabotropic receptors located at presynaptic glutamatergic and GABAergic terminals led to a reduction in transmitter release onto SON magnocellular neurons. L(+)-2-amino-4-phosphonobutyric acid (100-250 microM), a selective group III receptor agonist, mimicked the effects of trans-ACPD at presynaptic terminals, decreasing the frequency of miniature EPSCs and IPSCs by up to 85% without affecting their amplitude. Thus the metabotropic receptors at presynaptic glutamate and GABA terminals in the SON belong to group III mGluRs. EPSCs evoked by electrical stimulation were enhanced by the group III receptor antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid, suggesting that presynaptic metabotropic receptors are activated by the release of endogenous glutamate. These data indicate that mGluRs in the hypothalamus have opposing actions at presynaptic somata/dendrites and at presynaptic terminals. Activation of group I receptors (mGluR1 and/or mGluR5) on presynaptic somata/dendrites led to an increase in spike-dependent transmitter release, whereas activation of the group III receptors (mGluR4, 7, and/or 8) on presynaptic terminals suppressed glutamate and GABA release onto SON neurons. No differences were seen in the effects of mGluR activation between immunohistochemically identified oxytocin and vasopressin neurons of the SON.

Cellular Localization of the Prohormone Convertases in the Hypothalamic Paraventricular and Supraoptic Nuclei: Selective Regulation of PC1 in Corticotrophin-Releasing Hormone Parvocellular Neurons Mediated by Glucocorticoids

The prohormone convertases (PCs) are processing enzymes that activate proproteins via cleavage at specific single or pairs of basic residues. The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) are primary sites of biosynthesis of several neuroendocrine hormone precursors, including provasopressin (pro-AVP), pro-oxytocin (pro-OT), and procorticotrophin-releasing hormone (pro-CRH), which require post-translational processing to yield active products. Using in situ hybridization, we observed PC1 and PC5 mRNAs in PVN and SON magnocellular neurons, while PC2 mRNA was observed in both magnocellular and parvocellular PVN neurons as well as magnocellular SON neurons. Similar to furin, PC7 mRNA was expressed throughout the PVN and SON, whereas PACE4 mRNA levels were undetectable. Both immunohistochemical and Western blot studies were performed to demonstrate the presence of PC proteins and forms in the PVN and SON. Using double-labeling in situ hybridization, we examined the cellular colocalization of each PC mRNA with pro-AVP, pro-OT, and pro-CRH mRNAs in PVN and SON. PC1 mRNA was colocalized with both AVP and OT mRNA in PVN and SON magnocellular neurons. All AVP, OT, and CRH neurons expressed PC2. In contrast, PC5 mRNA was colocalized only with OT mRNA. We examined the effects of adrenalectomy (ADX) on PVN PC mRNA levels. PC1 mRNA levels were increased selectively within CRH/AVP parvocellular neurons but were unchanged in PVN magnocellular AVP or OT neurons. These results established the anatomical organization of each convertase and proneuropeptide substrates in the PVN and SON and suggested potential roles for each enzyme under resting and stimulated conditions.

Depolarizing Action of Secretory Granule Protein 7B2 on Rat Supraoptic Neurosecretory Neurons

A novel precursor neuropeptide termed 7B2 is present within specific brain areas, including the hypothalamic magnocellular neurosecretory neurons, and appears to be processed to smaller fragments. In order to determine whether specific C-terminal fragments of 7B2 might exert local effects on neurosecretory cells, we used intracellular current-clamp recordings in supraoptic neurons maintained in superfused hypothalamic explants to evaluate membrane potential and resistance changes in 25 supraoptic nucleus neurons during bolus applications of 7B2 174-186 and two other C-terminal peptide fragments 7B2 156-173 and 7B2 141-150. In 15 supraoptic neurons, only the 7B2 174-186 fragment induced a gradual 2-8 mV membrane depolarization that lasted for 4 to 30 min and was accompanied by 15+/-8% reduction in input resistance. Immunocytochemical identification of the recorded cells revealed that both vasopressin (VP)- and oxytocin (OT)-containing neurons were depolarized by 7B2 174-186. These data suggest that 7B2 174-186 is a biologically active fragment of 7B2 and may regulate the excitability of magnocellular supraoptic nucleus neurons.

Activation of supraoptic magnocellular neurons by gamma 2-melanocyte stimulating hormone ( γ2-MSH)

The effects of intracarotid infusions of the peptide gamma 2-melanocyte stimulating hormone ( γ 2-MSH) on electrophysiologically and immunohistochemically identified supraoptic nucleus (SON) units were investigated Over a wide dose range this agent always excited SON units, while control infusions of vehicle had no effect. Because neural responses invariably preceded blood pressure elevation, it appears that γ 2-MSH excitation of the magnocellular system was due to a direct effect on the central nervous system and was not a result of systemic cardiovascular responses. These results suggest a forebrain γ 2-MSH sensitive site in the activation of SON magnocellular neurons.

Two possible actions for circulating angiotensin II in the control of vasopressin release

The supraoptic-hypophyseal tract is a primary system for the synthesis and release of vasopressin. Angiotensin II (AII) has been shown to release vasopressin when injected into the cerebral ventricles (IVT). However, intravenous (IV) AII injections have not produced consistent results. The present studies were conducted to examine the effects of AII delivered by either route on the unit activity of supraoptic nucleus (SON) magnocellular neurons. Rats were prepared with intracranial cannulas to insure delivery of drugs to the left lateral ventricle and with polyethylene catheters in the left jugular vein, femoral vein, and femoral artery for systemic injections and arterial pressure recordings. A ventral approach permitted recording from the SON without violating the ventricular-SON partition. Magnocellular neurons were electrophysiologically identified. In the majority of identified cells, IVT AII increased activity. In others pressor doses of AII IV inhibited firing while blood pressure was elevated. After sino-aortic denervation, AII IV excited SON neurons. Based on latency, and the fact that lesioning the anteroventral third ventricle blocked the action of AII IVT, the results indicate that AII IVT acts on a periventricular site to influence SON magnocellular neurons. Furthermore, systemic AII may have two effects on SON neurons: a central excitatory action, and an inhibition due to a baroreceptor reflex.