Vasopressin(4-9) fragment activates V1a-type vasopressin receptor in rat supraoptic neurones.

The effect of vasopressin fragment 4-9 (AVP(4-9)) was investigated on freshly dissociated rat supraoptic neurones by measuring changes in intracellular calcium concentration ([Ca2+]i) using fura-2 microspectrofluorimetry. In 60% of neurones responding to vasopressin, AVP(4-9) induced a transient rise in [Ca2+]i that was dose-dependent in the concentration range 10 nM to 1 microM AVP(4-9) and strongly decreased in Ca2+-free buffer (84% inhibition). This [Ca2+]i response was completely and reversibly abolished by SR 49059 (1O nM), a specific V1a receptor antagonist, but not by SR 121463A, a specific V2 receptor antagonist. Our results demonstrate the presence of functional receptors activated by AVP(4-9) on vasopressin-sensitive neurones that possess the apparent pharmacological profile of the V1a-type vasopressin receptor.     

Intracellular calcium increase and somatodendritic vasopressin release by vasopressin receptor agonists in the rat supraoptic nucleus: involvement of multiple intracellular transduction signals

Vasopressin neurones of the supraoptic nucleus are autoregulated by vasopressin released from their soma and dendrites. Vasopressin binds to specific autoreceptors to trigger an influx of Ca(2+), and this response involves both phospholipase C (PLC) and adenylate cyclase (AC) pathways that, in the periphery, are activated by V(1) (V(1a) and V(1b))- and V(2)-type receptors. To investigate the pathways involved in the [Ca(2+)](i) response, [Ca(2+)](i) measurements were made on freshly dissociated neurones using Fura-2 microspectrofluorimetry, and vasopressin release was measured from isolated supraoptic nuclei. The [Ca(2+)](i) increase and vasopressin release induced by the V(1a) agonist were strongly inhibited by a PLC blocker, an IP(3) receptor antagonist, and a PKC blocker. An AC inhibitor did not affect the V(1a) response, while PKA inhibitors significantly reduced the V(1a)-induced [Ca(2+)](i) and release responses. The [Ca(2+)](i) increase and vasopressin release elicited by the V(2) agonist were attenuated not only by AC pathway blockers, but also by PLC inhibitors. Surprisingly, the V(1b) agonist showed no [Ca(2+)](i) or vasopressin release response. In conclusion, the V(1a) agonist activates both PLC and AC pathway, confirming the functional expression of a V(1a) vasopressin receptor on vasopressin neurones. The V(2) agonist activation of both PLC and AC pathways could result from an action on the PLC-linked unknown receptor, and/or the AC-linked dual angiotensin II-vasopressin receptor.     

Activation of κ-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca in secretory nerve terminals of the neurohypophysis

Nerve endings of the magnocellular neurohypophysial neurones possess kappa-opioid receptors. Using a preparation of isolated terminals from the neurohypophysis we studied kappa-opioid effects on secretion of oxytocin and vasopressin and on intracellular Ca2+ concentration ([Ca2+]i) measured fluorimetrically or using digital video imaging with Fura-2. The dihydropyridine Ca(2+)-channel antagonist nicardipine reduced [Ca2+]i responses to K(+)-depolarisation (30-40 mM K+) by 55-75% and inhibited evoked secretion of oxytocin and vasopressin to a similar extent. The selective kappa-receptor agonist D-Pro10 Dynorphin A 1-11 (DPDYN) substantially inhibited K+ evoked secretion of oxytocin by 40-90% and secretion of arginine vasopressin (AVP) by 20-50%. DPDYN caused only a 10% reduction in the average total population [Ca2+]i response to K+ depolarisation. No sub-population of inhibitory responses was observed when samples of individual terminal [Ca2+]i responses were examined with imaging. Although kappa-receptors are coupled to Ca(2+)-channels at neuronal somata our data suggest that alternative effector mechanisms operate in these secretory nerve endings.     

Patch-Clamp Analysis of the Mechanism of PACAP-Induced Excitation in Rat Supraoptic Neurones

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP)causes an increase in [Ca²⁺]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca²⁺]i revealed that the membrane depolarization correlated well with increases in [Ca²⁺]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of ?70 or ?80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was ?33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAPreceptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca²⁺]i and vasopressin (VP) release observed in SON neurones.     

Involvement of Prolactin‐Releasing Peptide in the Activation of Oxytocin Neurones in Response to Food Intake

Food intake activates neurones expressing prolactin‐releasing peptide (PrRP) in the medulla oblongata and oxytocin neurones in the hypothalamus. Both PrRP and oxytocin have been shown to have an anorexic action. In the present study, we investigated whether the activation of oxytocin neurones following food intake is mediated by PrRP. We first examined the expression of PrRP receptors (also known as GPR10) in rats. Immunoreactivity of PrRP receptors was observed in oxytocin neurones and in vasopressin neurones in the paraventricular and supraoptic nuclei of the hypothalamus and in the bed nucleus of the stria terminalis. Application of PrRP to isolated supraoptic nuclei facilitated the release of oxytocin and vasopressin. In mice, re‐feeding increased the expression of Fos protein in oxytocin neurones of the hypothalamus and bed nucleus of the stria terminalis. The increased expression of Fos protein in oxytocin neurones following re‐feeding or i.p. administration of cholecystokinin octapeptide (CCK), a peripheral satiety factor, was impaired in PrRP‐deficient mice. CCK‐induced oxytocin increase in plasma was also impaired in PrRP‐deficient mice. Furthermore, oxytocin receptor‐deficient mice showed an increased meal size, as reported in PrRP‐deficient mice and in CCK_A receptor‐deficient mice. These findings suggest that PrRP mediates, at least in part, the activation of oxytocin neurones in response to food intake, and that the CCK–PrRP–oxytocin pathway plays an important role in the control of the termination of each meal.     

Impaired long-term potentiation in vivo in the dentate gyrus of pituitary adenylate cyclase-activating polypeptide (PACAP) or PACAP type 1 receptor-mutant mice

The present study was conducted to clarify a role of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type 1 receptor (PAC1R) in learning and memory function. We demonstrated long-term potentiation (LTP) in vivo in the dentate gyrus of PAC1R exon 2-deficient (PAC1R-/-) mice and heterozygous PACAP-deficient (PACAP+/-) mice using extracellular recording techniques. We used two paradigms of tetanic stimulation, suprathreshold and at threshold tetanus, which both induced LTP in vivo in PAC1R-/- and PACAP+/- mice. However, the population spike of 'at threshold' but not 'suprathreshold' LTP decreased significantly in PAC1R-/- and PACAP+/- mice. At threshold LTP of PACAP+/- mice was impaired greater than the one of PAC1R-/- mice. Thus, both PACAP and PAC1R could contribute to the establishment of LTP in a gene dosage-dependent manner, although PACAP rather than PAC1R might play a pivotal role in learning and memory function.     

Involvement of N-methyl-D-aspartic acid receptor activation in oxytocin and vasopressin release after osmotic stimuli in rats

The present study aimed to examine roles of N-methyl-D-aspartic acid (NMDA) receptors in oxytocin and vasopressin release after osmotic stimuli. A noncompetitive NMDA receptor antagonist, MK-801 (0.2 mg/kg body weight, i.p.), significantly decreased plasma concentrations of oxytocin and vasopressin after hypertonic saline injection (0.3 or 0.6 M NaCl, i.p., 20 ml/kg). By contrast, oxytocin release induced by injection of cholecystokinin octapeptide (20 microg/kg, i.p.) was not significantly changed by MK-801. Hypertonic saline injection increased the number of cells expressing Fos in the supraoptic nucleus and in the regions anterior and ventral to the third ventricle (AV3V) regions [the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus]. MK-801 decreased the number of cells expressing protein in these areas after hypertonic saline injection. A microdialysis method showed that a hypertonic saline injection (0.6 M NaCl, 20 ml/kg, i.p.) facilitated glutamic acid release in and near the OVLT. The results support the view that NMDA receptor in the AV3V region modulates in a facilitative fashion the AV3V inputs to the supraoptic neurosecretory neurones.     

Comparative Examination of Inner Ear in Wild Type and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-Deficient Mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with well-known neuroprotective and neurotrophic effects. The involvement of PACAP in sensory processing has also been documented, but little is known about its effects in the auditory system. PACAP and its specific receptor (PAC1) are present in the cochlea and in brain structures involved in auditory pathways. Recently, we have shown that PACAP protects cochlear cells against oxidative stress-induced apoptosis. The endolymphatic Ca(2+) concentration controlled by Ca(2+) buffers of the hair cells is essential for the normal hearing processes. In this study we examined the localization of PAC1 receptor and Ca(2+) buffering proteins (parvalbumin, calretinin, calbindin) in the inner ear of 5-day-old PACAP-deficient mice compared with wild-type mice in order to get a closer insight into the effect of endogenous PACAP in the cochlear function. We did not find differences in the distribution pattern of PAC1 receptors between the two groups, but wild-type animals showed significantly higher PAC1 receptor expression. In contrast, inner and outer hair cells of PACAP-deficient mice showed more pronounced parvalbumin, calbindin, and calretinin immunopositivity compared with wild-type mice. Elevated endolymphatic Ca(2+) is deleterious for cochlear function, while the high concentration of Ca(2+) buffers in hair cells may offer protection. The increased immunoreactivity of Ca(2+) binding proteins in the absence of PACAP provide further evidence the important role of PACAP in the hearing processes.     

mu-Opioid receptor modulates peptide release from rat neurohypophysial terminals by inhibiting Ca(2+) influx

The activation of opioid receptors in neurones of the central nervous system leads to a variety of effects including the modulation of diuresis and parturition, processes that are directly controlled by the hypothalamic-neurohypophysial system (HNS). The effects of mu-opioid receptor activation on peptide release, voltage-gated Ca2+ currents and intracellular calcium levels ([Ca2+]i) were studied in isolated nerve terminals of the HNS. The mu-receptor agonist, DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) inhibited high K+-induced peptide release in a dose-dependent manner, with oxytocin release being more sensitive to block than vasopressin release at all concentrations tested. The addition of the mu-receptor antagonist CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide) was able to overcome the inhibitory effects of DAMGO. By contrast to previous results, voltage-gated Ca2+ currents were sensitive to blockage by DAMGO and this inhibition was also prevented by CTOP. Furthermore, [Ca2+]i measurements with Fura-2 corroborated the inhibition by DAMGO of calcium entry and its reversal by the micro -receptor antagonist in these nerve terminals. Thus, the decrease in neuropeptide release, particularly for oxytocin, induced by the activation of mu-opioid receptors in neurohypophysial terminals is mediated, at least in part, by a corresponding decrease in Ca2+ entry due to the inhibition of voltage-gated Ca2+ channels.     

Effects of membrane depolarization on intracellular calcium in single nerve terminals

The [Ca2+]i of individual neurosecretory nerve terminals loaded with the fluorescent probe fura-2 was monitored during depolarizing stimuli and in the presence of substances known to induce or block neurohormone release. Induction of membrane depolarization with elevated [K+] or veratridine led to a rapid increase in [Ca2+]i that was sensitive to block by substances which block voltage-sensitive L-type Ca2+ channels such as the dihydropyridine nicardipine and by D-888. Relaxin, cholecystokinin and enkephalin which have been reported to regulate vasopressin and oxytocin secretion at the nerve endings were without effect on basal [Ca2+]i or K(+)-stimulated increases in [Ca2+]i.     

Bradykinin and Angiotensin II-Induced [Ca2+]i Rise in Cultured Rat Pituitary Folliculo-Stellate Cells

Folliculo-stellate cells of the anterior pituitary are thought to modulate pituitary hormone secretion through a paracrine mechanism. Angiotensin II and pituitary adenylate cyclase-activating polypeptide (PACAP) have previously been shown to increase the intracellular Ca2+ concentration ([Ca2+]i) of these cells. In the present study, we examined the effects of various peptides such as bradykinin, angiotensin II, endothelin-1, PACAP, galanin and neurotensin by Ca2+-imaging of folliculo-stellate cells in primary culture. Bradykinin and angiotensin II increased [Ca2+]i in folliculo-stellate cells. Both responses were completely suppressed by thapsigargin and were significantly suppressed by the phospholipase C inhibitor, U-73122. Ryanodine did not significantly modify the responses. A B2 antagonist and angiotensin II receptor antagonist inhibited the response induced by bradykinin and angiotensin II, respectively. Endothelin-1 and PACAP increased [Ca2+]i in fewer than 50% of folliculo-stellate cells but galanin and neurotensin did not influence [Ca2+]i in any of the folliculo-stellate cells tested. These results indicate that bradykinin and angiotensin II increase [Ca2+]i in folliculo-stellate cells by activating phospholipase C through B2 receptor and AT1 receptor, respectively, and that endothelin-1 and PACAP also increase [Ca2+]i in some folliculo-stellate cells.     

Expression of exocytosis proteins in rat supraoptic nucleus neurones

In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca(2+) -dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory.     

Arg8]-vasopressin-induced increase in intracellular Ca2+ concentration in cultured rat hippocampal neurons.

Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by [Arg8]-vasopressin (AVP) were studied in cultured rat hippocampal neurons by fura-2 fluorometry. AVP (10-1,000 nM) caused a dose-dependent increase in [Ca2+]i. The selective V1 vasopressin receptor agonist [Phe2, Ile3, Orn8]-vasopressin also induced a significant increase in [Ca2+]i, whereas the selective V2 vasopressin receptor agonist [deamino Cys1, D-Arg8]-vasopressin showed no effect. The AVP-induced increase in [Ca2+]i was inhibited by the selective V1 vasopressin receptor antagonist d(CH2)5[Tyr2(Me), Arg8]-vasopressin and nonpeptide V1 antagonist OPC-21268. On the other hand, no antagonistic effects were observed with the V2 vasopressin antagonist desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin and nonpeptide V2 antagonist OPC-31260. The increase in [Ca2+]i induced by AVP was abolished after removal of extracellular Ca2+. In addition, AVP-induced [Ca2+]i elevation was not affected by treatment with verapamil, which blocked the [Ca2+]i increase induced by an isotonic high K(+)-medium (50 mM). However, omega-conotoxin GVIA completely inhibited the effect of AVP. These results suggested that the AVP-induced [Ca2+]i increase in cultured rat hippocampal neurons is due to influx of Ca2+ through V1 VP receptors coupled with N-type calcium channels.     

Interleukin-1beta release in the supraoptic nucleus area during osmotic stimulation requires neural function

Interleukin (IL)-1beta is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1beta is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0-180 min; basal), and after (180-240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1beta was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1beta+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1beta+ in the SON area was measurable and stable (pg/ml; mean +/- SEM) from 0-60 min (2.2 +/- 0.06), 60-120 min (2.32 +/- 0.05) and 120-180 min (2.33 +/- 0.06), likely originating locally from activated microglia (OX42+; IL-1beta+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1beta. In response to osmotic stimulation, IL-1beta increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1beta in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones.     

PACAP/PAC1 autocrine system promotes proliferation and astrogenesis in neural progenitor cells

The Pituitary adenylate cyclase-activating peptide (PACAP) ligand/type 1 receptor (PAC1) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC1 system induces differentiation of neural progenitor cells (NPCs) through the Gs-mediated cAMP-dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC1 were highly expressed and co-localized in NPCs of mouse cortex at embryonic day 14.5 (E14.5) and found that the PACAP/PAC1 system potentiated growth factor-induced proliferation of mouse cortical NPCs at E14.5 via Gq-, but not Gs-, mediated PLC/IP3-dependent signaling pathway in an autocrine manner. Moreover, PAC1 activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)-incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC1 signaling. These results suggest that the PACAP/PAC1 system may play a dual role in neural/glial progenitor cells not only differentiation but also proliferation in the cortical astrocyte lineage via Ca2+-dependent signaling pathways through PAC1.     

An essential role for peptidergic signalling in the control of circadian rhythms in the suprachiasmatic nuclei

Two structurally related neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP), colocalized with glutamate in neurones of the retinohypothalamic tract, and vasoactive intestinal peptide (VIP), present in light-responsive cells of the suprachiasmatic nuclei (SCN), appear to play distinct and important roles in the control of mammalian circadian rhythms. Mice deficient in the PACAP-selective PAC1 receptor exhibit altered responsiveness of the SCN clock to light-induced phase-shifts, but display robust circadian patterns of wheel-running behaviour. By contrast, our studies of mice lacking the VPAC2 receptor, which responds to both PACAP and VIP, indicate that this receptor plays a critical role in rhythm generation in the SCN. The predominant factor determining wheel-running activity in VPAC2 receptor null (Vipr2-/-) mice is "masking" by light. Mutant animals re-entrain immediately to advances or delays in the light/dark cycle and do not exhibit robust circadian rhythms of behaviour when in constant darkness. The mice do not exhibit circadian expression of core clock genes (mPer1, mPer2, mCry1), or of the clock-controlled gene arginine vasopressin (AVP), in the SCN. We propose that VIP signalling between SCN neurones provides a paracrine reinforcing signal that is essential for sustained rhythm generation. The presence of VIP signalling in the SCN may explain why SCN neurones are capable of generating long-lasting self-sustained oscillations, whereas rhythmic clock gene expression in other tissues is dependent on periodic reinforcement by neural or hormonal signals.     

Agonistic Behavior of PACAP6-38 on Sensory Nerve Terminals and Cytotrophoblast Cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) are mediated through G-protein-coupled receptors, the specific PAC1 receptor and VPAC1 and VPAC2 receptors which bind vasoactive intestinal peptide with similar affinity. Based on binding affinity studies, PACAP6-38 was discovered as a potent antagonist of PAC1 and it has been used by hundreds of studies as a PACAP antagonist. Recently, we have found that in certain cells/tissues, PACAP6-38 does not antagonize PACAP-induced effects, but surprisingly, it exerts similar actions to PACAP1-38, behaving as an agonist. In the present study, we report on the agonistic behavior of PACAP6-38 on neuropeptide release from sensory nerves of the isolated rat trachea and on the MAPK signaling pathways in cytotrophoblast cells. In isolated rat tracheae, PACAP6-38, similarly to PACAP1-38, induced significant inhibitory effects on the release of three simultaneously measured sensory neuropeptides, substance P, calcitonin gene-related peptide, and somatostatin evoked by both chemical excitation and electrical field stimulation of capsaicin-sensitive afferents. Effects of PACAP6-38 were the same as those of PACAP1-38 on MAPK signaling in human cytotrophoblast cells. Western blot analysis showed that both peptide forms stimulated ERK1/2 and JNK phosphorylation, while they both inhibited p38 MAPK phosphorylation. The most pronounced effects were observed when both peptides were present. In summary, our results show that PACAP6-38, which is a PACAP receptor antagonist in most cells/tissues, can behave as an agonist in other systems. The increasing interest in the effects of PACAP requires further studies on the pharmacological properties of the peptide and its analogues.     

Tonic regulation of GABAergic synaptic activity on vasopressin neurones by cannabinoids

Synaptic activity in magnocellular neurosecretory neurones is influenced by the retrograde (i.e. somatodendritic) release of vasopressin, oxytocin and cannabinoids (CBs). For oxytocin neurones, oxytocin exerts constitutive effects on pre-synaptic activity through its ability to release CBs post-synaptically. In the present study, we examined evoked inhibitory post-synaptic currents (eIPSCs) and spontaneous inhibitory post-synaptic currents (sIPSCs) in identified vasopressin (VP) neurones in coronal slices from virgin rats to determine: (i) the extent to which CBs may also tonically modulate VP synaptic activity; and (ii) to determine whether depolarisation-induced suppression of inhibition was present in VP neurones, and if so, whether it was mediated by VP or CBs. The CB1 antagonists AM251 (1 μm) and SR14171 (1 μm) consistently increased the frequency of sIPSCs in VP neurones without affecting their amplitude, suggesting a tonic CB presence. This effect on frequency was independent of action potential activity, and blocked by chelating intracellular calcium with 10 mm ethylene glycol tetraacetic acid (EGTA). AM251 also increased the amplitude of eIPSCs and decreased the paired-pulse ratio (PPR) in VP neurones-effects that were completely blocked with even low (1 mm EGTA) internal calcium chelation. Bouts of evoked firing of VP neurones consistently suppressed sIPSCs but had no effect on eIPSCs or the PPR. This depolarisation-induced suppression of IPSCs was reduced by AM251, and was totally blocked by 10 μm of the mixed vasopressin/oxytocin antagonist, Manning compound. We then tested the effect of vasopressin on IPSCs at the same time as blocking CB1 receptors. Vasopressin (10-100 nm) inhibited sIPSC frequency but had no effect on sIPSC or eIPSC amplitudes, or on the PPR, in the presence of AM251. Taken together, these results suggest a tonic, pre-synaptic inhibitory modulation of IPSCs in VP neurones by CBs that is largely dependent on post-synaptic calcium, and an inhibitory effect of VP on IPSCs that is independent of CB release.