The role of the actin cytoskeleton in oxytocin and vasopressin release from rat supraoptic nucleus neurons

Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K⁺, 50 m m ) or TG (200 n m ) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 μ m ) or depolymerizing agents (latrunculin B, 5 μ m ) to treat SON and neural lobe (NL) explants in vitro and measure high K⁺-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K⁺-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K⁺-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.     

Milk ejection burst-like electrical activity evoked in supraoptic oxytocin neurons in slices from lactating rats

To examine the mechanisms underlying milk-ejection bursts of oxytocin (OT) neurons during suckling, both in vivo and in vitro studies were performed on supraoptic OT neurons from lactating rats. The bursts were first recorded extracellularly in anesthetized rats. Burst-related electrical parameters were essentially the same as previous reports except for a trend toward transient decreases in basal firing rates immediately preceding the burst. From putative OT neurons in slices with extracellular recordings, bursts that closely mimicked the in vivo bursts were elicited by phenylephrine, an alpha1-adrenoceptor agonist, in a low-Ca(2+) medium. Moreover, in whole cell patch-clamp recordings, the in vitro bursts were recorded from immunocytochemically identified OT neurons. After a transient decrease in the basal firing rate, the in vitro bursts started with a sudden increase in the firing rate, quickly reaching a peak level, then gradually decaying, and ended with a postburst inhibition. A brief depolarization of the membrane potential and an increase in membrane conductance appeared after the onset of the burst. Spikes during a burst were characterized by a significant increase in the duration and decrease in the amplitude around the peak rate firing. These bursts were significantly different from short-lasting burst firing of vasopressin neurons in membrane potential changes, time to reach peak firing rate, spike amplitude and duration during peak rate firing. Our extensive analysis of these results suggests that the in vitro burst is a useful model for further study of mechanisms underlying milk-ejection bursts of OT neurons in vivo.     

Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats

Summary To investigate the hypothesis that oxytocin may be released within the magnocellular nuclei in vivo, push-pull cannula perfusions were performed in anaesthetized lactating rats in one supraoptic nucleus of the hypothalamus while recording the intramammary pressure and/or the electrical activity of oxytocin cells in the contralateral supraoptic nucleus. Oxytocin content was measured in samples collected over 15 min, under various conditions: 1) with no stimulation; 2) during suckling and suckling-induced reflex milk ejections; 3) during electrical stimulation of the neurohypophysis by trains of pulses that mimicked oxytocin cell bursts; 4) under osmotic stimulation by i.p. injection of 2 ml of 1.5 M NaCl to evoke a tonic and sustained oxytocin release from the neurohypophysis. Oxytocin release within the supraoptic nucleus increased significantly during the milk ejection reflex and, to a lesser extent, during burst-like electrical stimulation of the neurohypophysis. In suckled rats, the increase started before the first reflex milk ejection occurred. There was no apparent correlation between the amount of oxytocin in the perfusates and the number of milk ejections and oxytocin cell bursts occurring during each perfusion period. The amount of oxytocin in the perfusates further increased during facilitation of the milk ejection reflex by intraventricular injections of oxytocin or its analogue, isotocin. When suckling failed to evoke the milk ejection reflex, there was no change in intra-supraoptic oxytocin release. There was also no change after osmotic stimulation. When the push-pull cannula was positioned outside the supraoptic nucleus, there was no increase in the amount of oxytocin during the three types of stimulation tested. These results provide evidence for an endogenous release of oxytocin within the magnocellular nuclei in lactating rats. It is suggested that the increase in such a release induced by suckling is likely to be a pre-requisite for the onset and the maintenance of the characteristic intermittent bursting electrical activity of oxytocin cells leading to milk ejections.     

Electrophysiological evidence for the activation of supraoptic neurones during the release of oxytocin

1. Antidromically identified supraoptic (SO) units were recorded in lactating rats anaesthetized with urethane (1.1 g/kg), and their activity was studied during milk ejection evoked by the suckling of the young. Fifty-eight SO units were recorded through 174 milk ejections. Each milk ejection was the result of a neurohypophysial release of an oxytocin pulse of 0.5-1.5 m-u.2. Fifty-five of the SO units displayed background activity and three were silent. The firing rates ranged from 0 to 15.4 spikes/sec, the distribution was exponential with 26% of the units firing at < 1 spike/sec. Sixteen (28%) of the SO units displayed a phasic pattern of activity characterized by periods of activity (6-132 sec) and silence (4-71 sec).3. Twenty-five of the non-phasic units displayed a large and stereotyped acceleration in spike activity some 10.5-17.4 sec before the rise in intramammary pressure at milk ejection. Units accelerated to rates between 9-66 spikes/sec, an increase of about thirtyfold (median) on background activity. The response was brief (0.9-4.7 sec) and was followed by a period of after-inhibition.4. It was concluded from studies of double recordings and observations of multi-unit activity that all the responsive units were synchronously activated. The mean latency of 13.3 sec between the onset of the neurosecretory response and milk ejection was similar to that observed following the experimental release of endogenous oxytocin by electrical stimulation of the neurohypophysis (50 pulses/sec for 2-4 sec).5. Four of the phasically active units were correlated with the oxytocin release for milk ejection. Three of these units displayed a burst of activity superimposed on the terminal portion of an active phase, some 10.2-14.7 sec before milk ejection. The fourth unit, recorded in conjunction with a responsive non-phasic unit, consistently switched from silence to activity coincident with the onset of the SO activation.6. The remaining SO units and a further ten units that were not antidromically activated by neurohypophysial stimulation displayed no change in activity during either the period of neurosecretory activation or the period of after-inhibition.7. This activation of the SO neurones, in the formulation of oxytocin release and milk ejection, is the same as that we have previously observed in recordings from the paraventricular (PV) region, and the proportion of neurones displaying the response is similar: 48% in the SO nuclei, 58% in the PV nuclei. We conclude, since the SO nuclei contain 80% of the neurosecretory cells that project to the neurohypophysis, that the SO nuclei are as important, if not more so, than the PV nuclei in the control of oxytocin release.     

Kainic acid inhibits cholecystokinin release from rat hippocampal slices

Antagonistic interactions between cholecystokinin (CCK) and nanomolar concentrations of kainic acid (KA) have been reported in area CA3 of the rat hippocampal slice. This study tested the possibility that kainic acid inhibits the release of CCK. Elevated K⁺ was found to release CCK from hippocampal slices in a Ca²⁺-dependent manner. KA, at concentrations as low as 100 nM, inhibited this release by about one-third. Because CCK appears to exert a net inhibitory effect on the firing of CA3 pyramidal cells, the epileptogenic action of KA may be explained, in part, by the depression of CCK release.     

Activity-dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus

Exogenous cannabinoids have been shown to significantly alter neuroendocrine output, presaging the emergence of endogenous cannabinoids as important signalling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation. We showed recently that magnocellular and parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus and supraoptic nucleus (SON) respond to glucocorticoids by releasing endocannabinoids as retrograde messengers to modulate the synaptic release of glutamate. Here we show directly for the first time that both of the main endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are released in an activity-dependent fashion from the soma/dendrites of SON magnocellular neurones and suppress synaptic glutamate release and postsynaptic spiking. Cannabinoid reuptake blockade increases activity-dependent endocannabinoid levels in the region of the SON, and results in the inhibition of synaptically driven spiking activity in magnocellular neurones. Together, these findings demonstrate an activity-dependent release of AEA and 2-AG that leads to the suppression of glutamate release and that is capable of shaping spiking activity in magnocellular neurones. This activity-dependent regulation of excitatory synaptic input by endocannabinoids may play a role in determining spiking patterns characteristic of magnocellular neurones under stimulated conditions.     

低渗刺激后大鼠视上核星形胶质细胞释放牛磺酸调节神经元的活动

目的:观察低渗刺激大鼠后,视上核(SON)内星形胶质细胞调节神经元活动的机制。方法:大鼠分为4组:(1)等渗对照组,经尾静脉注入生理盐水;(2)低渗刺激组,经尾静脉注入低渗盐水(0.83%葡萄糖+0.3%NaCl);(3)氟代柠檬酸(胶质细胞代谢抑制剂,fluorocitrate,FCA)+低渗刺激组,经侧脑室注射FCA(1 nmol/μl/只)2 h后,再经尾静脉注入低渗盐水;(4)甘珀酸(缝隙连接通道阻断剂,carbennoxolon,CBX)+低渗刺激组,经侧脑室注射CBX(50μg/只,10μg/lμl)2 h后,再经尾静脉注入低渗盐水。各组动物刺激后90 min,常规固定、取材、下丘脑连续冠状切片。应用抗牛磺酸(taurine,Tau)、抗加压素(vasopressin,VP)、抗甘氨酸受体(glycine recep-tor,GlyR)、抗缝隙连接蛋白(connexin43,Cx43)的抗体进行标记,以及Fos/胶质原纤维酸性蛋白(GFAP)双标记的免疫荧光染色方法,观察其在SON神经元和星形胶质细胞内的表达。结果:与对照组相比,低渗刺激组大鼠,SON内星形胶质细胞的胞体增大、突起变粗;Tau,Cx43和GFAP的平均荧光强度(MFI)增强,神经元上的GlyR表达增强,但VP和Fos的表达减弱。FCA,而不是CBX,明显减少星形胶质细胞的Tau,GFAP,Cx43的表达。FCA和CBX均可抑制神经元上的VP、GlyR、Fos阳性反应。结论:低渗刺激激活SON内星形胶质细胞,被激活的星形胶质细胞经Cx43半通道释放牛磺酸而抑制神经元的活性,减少VP的释放。     

Naloxone potentiates the release of oxytocin induced by systemic administration of cholecystokinin without enhancing the electrical activity of supraoptic oxytocin neurones

Summary Studies performed in conscious female rats confirmed that iv injection of cholecystokinin octapeptide (CCK; 20µ/kg) increased the circulating concentration of oxytocin but not that of vasopressin, and confirmed that the stimulation of oxytocin release was markedly facilitated after iv administration of naloxone (1mg/kg), indicating attenuation of oxytocin release by endogenous opioids. To investigate the site of action of the endogenous opioids, the electrical activity of putative oxytocin neurones in the supraoptic nucleus was recorded in urethaneanaesthetised female rats. Oxytocin neurones responded to CCK injection with an increase in firing rate lasting 5–15 min, but this response was not facilitated by prior injection of naloxone. The results suggest that the opioid influence upon CCK-induced oxytocin release operates at the level of the neurosecretory terminals in the neurohypophysis rather than centrally. Since CCK does not elevate vasopressin release, it appears unlikely that dynorphin, the opioid peptide co-existing with vasopressin, is responsible in these circumstances for the cross-inhibition of oxytocin release. It is suggested that products of proenkephalin A, the met-enkephalin precursor present in the supraoptic nucleus and in the neurohypophysis itself, may be active in the regulation of oxytocin release.     

Chronic antidepressant treatment modulates the release of somatostatin in the rat nucleus accumbens

This study investigated the in vivo neuronal release of somatostatin in the rat nucleus accumbens (NAc), and the effect of chronic administration of antidepressants. Microdialysis studies were performed on male Sprague-Dawley rats, in accordance with the EU guidelines (EEC Council 86/609). Somatostatin levels were quantified by radioimmunoassay (RIA) or enzyme linked immuno sorbent assay (ELISA). A high concentration of potassium ions (K(+), 100 mM) was used to ascertain the neuronal release of somatostatin. Antidepressant treatments involved the administration of citalopram (20 mg/2 ml/kg, i.p., once daily) or desipramine (DMI, 5 mg/2 ml/kg, i.p., twice daily) for 21 days. Control groups received saline (2 ml/kg for 21 days, i.p.) once or twice daily respective of the antidepressant treatment. Basal levels of somatostatin released were found to be 20.01+/-0.52 fmol/sample. K(+) (100 mM) increased somatostatin levels at 205% of basal. Chronic citalopram and desipramine treatments also increased the somatostatin levels by 83+/-32% and 40+/-6% of basal, respectively. These findings indicate that somatostatin is released neuronally in the NAc. Antidepressants influence its release in a positive manner, suggesting the necessity of further studies for the elucidation of the involvement of somatostatin in the putative therapeutic effects of these agents.     

GABAergic projection from the arcuate nucleus to the supraoptic nucleus in the rat

Electrical stimulation of the neurones in the hypothalamic arcuate nucleus results in a transient inhibition followed by a marked post-stimulus excitation of magnocellular neurones of the supraoptic nucleus. Microdialysis administration of the gamma-aminobutyric acid agonist (GABA(A)), muscimol, directly into the supraoptic nucleus inhibited both oxytocin and vasopressin neurones and these actions were fully reversed by the GABA(A) antagonist bicuculline. In addition, bicuculline administration blocked the inhibition induced by arcuate stimulation, but had no effect on the post-stimulus excitation. Thus, part of the inhibitory pathway arising from or passing through the arcuate nucleus to the supraoptic nucleus is mediated by the neurotransmitter GABA. However, the post-inhibitory excitation induced by arcuate stimulation is not a rebound response, but appears to involve an independent excitatory pathway.     

Muscarinic modulation of acetylcholine release from slices of guinea pig nucleus basalis magnocellularis

Spontaneous and electrically evoked endogenous acetylcholine release and [³H]-choline efflux from slices of guinea pig nucleus basalis magnocellularis (nbM) were studied. Tetrodotoxin reduced the spontaneous endogenous release by 55%, while the Ca²⁺-free medium reduced it by about 30%. Evoked [³H]-choline efflux was Na⁺ and Ca²⁺ dependent and frequency related. Physostigmine, 30 μM, nearly halved the stimulation-evoked efflux; atropine, 0.15 μM, not only antagonized, but even reversed this effect into facilitation. Pirenzepine, 1 μM, and AFDX 116, 1 μM, were less effective than atropine, and reversed the inhibitory effect of physostigmine only when applied together. 4-DAMP, 0.01 μM, was ineffective. These findings indicate that acetylcholine release in guinea pig nbM slices is inhibited by the cooperation of muscarinic autoreceptors, possibly belonging to the M₁ and M₂ subclasses.     

Electrophysiological evidence for a projection from the arcuate nucleus to the supraoptic nucleus

The electrical activity of single neurones in the hypothalamic arcuate and supraoptic nuclei was recorded in urethane-anaesthetized rats. Stimulus pulses applied to the supraoptic nucleus antidromically activated 3 out of 41 cells recorded in the ipsilateral arcuate nucleus, confirming that there is a projection from the arcuate nucleus to the region of the supraoptic nucleus. Stimulation of the arcuate nucleus inhibited 17 out of 19 continuously firing (putative oxytocin) supraoptic neurones. Inhibition was followed by a marked post-stimulus excitation in 12 cells. The responses were not abolished by i.v. injection of the opioid antagonist naloxone. Thus at least part of the input to the magnocellular oxytocin system that arises from or passes through the arcuate nucleus, is not mediated by opioid peptides.     

Phosphatidylserine increases acetylcholine release from cortical slices in aged rats

Acetylcholine release was investigated in cortical slices superfused with choline-enriched Krebs solution containing physostigmine. Slices were prepared from 3 and 24 month old rats treated with either Tris buffer or sonicated suspensions of phosphatidylserine and phosphatidylcholine in Tris buffer. Slices were electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. ACh content of the superfusate was quantified by bioassay. In the 24 month old rats treated with Tris buffer, acetylcholine release, at all frequencies tested, was approximately 50% lower than that in the 3 month old rats. On the contrary, no significant decrease in ACh release was found in the 24 month old rats treated for 30 days with phosphatidylserine (15 mg/kg IP). The same treatment did not increase acetylcholine release in 3 month old rats. Acetylcholine release in 24 month old rats receiving a single administration of phosphatidylserine (15mg/kg IP) or phosphatidylcholine (15 mg/kg IP) for 30 days was as low as in the 24 month old rats receiving the Tris buffer only. It is proposed that the chronic phosphatidylserine treatment may reduce the age-induced decrease in acetylcholine release by acting on the stimulus-secretion coupling mechanism.