The adaptive brain: Glenn Hatton and the supraoptic nucleus.

In December 2009, Glenn Hatton died, and neuroendocrinology lost a pioneer who had done much to forge our present understanding of the hypothalamus and whose productivity had not faded with the passing years. Glenn, an expert in both functional morphology and electrophysiology, was driven by a will to understand the significance of his observations in the context of the living, behaving organism. He also had the wit to generate bold and challenging hypotheses, the wherewithal to expose them to critical and elegant experimental testing, and a way with words that gave his papers and lectures clarity and eloquence. The hypothalamo-neurohypophysial system offered a host of opportunities for understanding how physiological functions are fulfilled by the electrical activity of neurones, how neuronal behaviour changes with changing physiological states, and how morphological changes contribute to the physiological response. In the vision that Glenn developed over 35 years, the neuroendocrine brain is as dynamic in structure as it is adaptable in function. Its adaptability is reflected not only by mere synaptic plasticity, but also by changes in neuronal morphology and in the morphology of the glial cells. Astrocytes, in Glenn's view, were intimate partners of the neurones, partners with an essential role in adaptation to changing physiological demands.

Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine.

Osmotic regulation of supraoptic nucleus (SON) neuron activity depends in part on activation of neuronal glycine receptors (GlyRs), most probably by taurine released from adjacent astrocytes. In the neurohypophysis in which the axons of SON neurons terminate, taurine is also concentrated in and osmo-dependently released by pituicytes, the specialized glial cells ensheathing nerve terminals. We now show that taurine release from isolated neurohypophyses is enhanced by hypo-osmotic and decreased by hyper-osmotic stimulation. The high osmosensitivity is shown by the significant increase on only 3.3% reduction in osmolarity. Inhibition of taurine release by 5-nitro-2-(3-phenylpropylamino)benzoic acid, niflumic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid suggests the involvement of volume-sensitive anion channels. On purified neurohypophysial nerve endings, activation of strychnine-sensitive GlyRs by taurine or glycine primarily inhibits the high K(+)-induced rise in [Ca(2+)](i) and subsequent release of vasopressin. Expression of GlyRs in vasopressin and oxytocin terminals is confirmed by immunohistochemistry. Their implication in the osmoregulation of neurohormone secretion was assessed on isolated whole neurohypophyses. A 6.6% hypo-osmotic stimulus reduces by half the depolarization-evoked vasopressin secretion, an inhibition totally prevented by strychnine. Most importantly, depletion of taurine by a taurine transport inhibitor also abolishes the osmo-dependent inhibition of vasopressin release. Therefore, in the neurohypophysis, an osmoregulatory system involving pituicytes, taurine, and GlyRs is operating to control Ca(2+) influx in and neurohormone release from nerve terminals. This elucidates the functional role of glial taurine in the neurohypophysis, reveals the expression of GlyRs on axon terminals, and further defines the role of glial cells in the regulation of neuroendocrine function.

High voltage-activated Ca2+ currents in rat supraoptic neurones: biophysical properties and expression of the various channel alpha1 subunits.

The diversity of Ca2+ currents was studied in voltage-clamped acutely dissociated neurones from the rat supraoptic nucleus (SON), and the expression of the various corresponding pore-forming alpha1 subunits determined by immunohistochemistry. We observed the presence of all high voltage-activated L-, N-, P/Q- and R-type currents. We did not observe low-voltage-activated T-type current. The multimodal current/voltage relationships of L- and R-type currents indicated further heterogeneity within these current types, each exhibiting two components that differed by a high (-20 mV) and a lower (-40 mV) threshold potential of activation. L- and R-type currents were fast activating and showed time-dependent inactivation, conversely to N- and P/Q-type currents, which activated more slowly and did not inactivate. The immunocytochemical staining indicated that the soma and proximal dendrites of SON neurones were immunoreactive for Cav1.2, Cav1.3 (forming L-type channels), Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 subunits (R-type). Each subunit exhibited further specificity in its distribution throughout the nucleus, and we particularly observed strong immunostaining of Cav1.3 and Cav2.3 subunits within the dendritic zone of the SON. These data show a high heterogeneity of Ca2+ channels in SON. neurones, both in their functional properties and cellular distribution. The lower threshold and rapidly activating L- and R-type currents should underlie major Ca2+ entry during action potentials, while the slower and higher threshold N- and P/Q-type currents should be preferentially recruited during burst activity. It will be of key interest to determine their respective role in the numerous Ca2+-dependent events that control the activity and physiology of SON neurones

Correlation between electrophysiological and morphological characteristics during maturation of rat supraoptic neurons.

The neurohypophysial peptides oxytocin (OT) and vasopressin (AVP) are well known for their role in reproductive functions and fluid balance regulation, respectively. During development, these peptides are thought to act as trophic factors on both peripheral and central structures. However, despite this early developmental function, the maturation of their secreting neurons remains poorly investigated. In this study, we have characterized the electrical and morphological characteristics displayed by OT and AVP supraoptic (SO) neurons between embryonic day 21 and postnatal day 20. Transient changes in passive membrane properties, correlated with a transient increase in the dendritic arborization, were observed at the beginning of the second postnatal week (PW2). The action potential matured mostly during PW1 and its threshold progressively hyperpolarized in parallel with the resting membrane potential. During PW1, SO neurons displayed unique characteristics with a low-threshold Ca(2+)-dependent depolarizing potential and a prominent hyperpolarization-activated current (I(h) ). This latter is involved in a depolarizing sag during hyperpolarization and an after hyperpolarizing potential following a depolarization. During this period, maintaining E(Cl) unchanged by the use of gramicidin-perforated patch recordings revealed excitatory GABAergic potentials, that became inhibitory during PW2, whilst glutamatergic potential appeared. The electrical activity was very erratic in young neurons and progressively differentiated in the typical firing observed in mature neurons (tonic and phasic for OT and AVP neurons, respectively) during PW2--3. These results show that the development of electrical properties of SO neurons is correlated with the maturation of their dendritic arborization.

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.

To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins.

Developmental regulation of a local positive autocontrol of supraoptic neurons.

Mature oxytocin (OT) and vasopressin (AVP) magnocellular neurons of the hypothalamic supraoptic nuclei (SON) autocontrol their electrical activity via somatodendritic release of their respective peptides. Because OT and AVP are synthesized early in development and could play an important role in the maturation of these neurons, we checked whether the peptides are released within the SON and act on their secreting neurons during 3 weeks of postnatal development. We used patch-clamp recordings from SON neurons in rat hypothalamic horizontal slices to show that the spontaneous electrical activity of immature SON neurons is blocked by OT or AVP receptor antagonists, demonstrating a basal somatodendritic release of the peptides. Application of OT or AVP depolarizes SON neurons and stimulates activity typical of the corresponding mature neurons. This effect is directly on SON neurons because it is recorded in dissociated neurons. Radioimmunoassays from isolated SON were used to show that each peptide facilitates its own release at a somatodendritic level, exhibiting a self-sustaining positive feedback loop. This autocontrol is not uniform during development because the proportion of neurons depolarized by the peptides, the amplitude of the depolarization, and the propensity of the peptides to facilitate their own release are maximal during the second postnatal week and decrease thereafter. These data are consistent with a role of autocontrol in the maturation of SON neurons because it is maximal during the delimited period of postnatal development that corresponds to the period of major synapse formation.

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure.

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.

The variability of basal action potential firing is positively correlated with bursting in hypothalamic oxytocin neurones.

Magnocellular oxytocin neurones are proposed as a suitable system for studying the mechanisms involved in the regulation of neuronal bursting activity. They display high frequency (50 sp./s) bursts of spikes (approximately every 300 s), in response to specific stimuli, which are superimposed on a variable level of basal activity and are tightly co-ordinated as a result of network interactions. The relationship between the strength of the bursting activity (as quantified by burst amplitude and interburst interval) and the characteristics of the interburst basal activity were assessed. During control conditions, mean basal activity and variability of firing increased just before bursts. During experimental conditions leading to burst facilitation, burst amplitude increased and interburst interval decreased while a sustained increase in mean firing rate occurred. Variability of firing (measured by both the standard deviation of firing rate, and the index of dispersion which corrected this standard deviation for differences in mean firing rate), increased demonstrating an increase in spike clustering greater than expected as a result of increased basal activity. When bursting was restrained (i.e. interburst interval increased), mean basal activity increased substantially, but index of dispersion decreased. A narrowing of the interspike interval distribution occurred, indicating increased regularity of firing. The aspect of basal activity most strongly correlated with bursting was variability of firing rate. The strongest correlate of burst amplitude was the standard deviation of mean firing rate, whereas the strongest and most consistent correlate of interburst interval was the index of dispersion. In conclusion, bursting behaviour is most strongly related to the irregularity rather than the level of basal activity.

Expression of the genes encoding the vasopressin-activated calcium-mobilizing receptor and the dual angiotensin II/vasopressin receptor in the rat central nervous system.

The distributions of two newly discovered receptors, the vasopressin-activated calcium-mobilizing receptor (VACM-1) and the dual angiotensin II/vasopressin receptor (AII/AVP), in the central nervous system (CNS) of the rat were determined using reverse transcriptase-polymerase chain reaction and in situ hybridization. The sequence of the rat VACM-1 cDNA was determined and found very homologous to the rabbit and human sequences. Both VACM-1 and AII/AVP receptor genes were widely expressed in the brain, but differed according to the cell type studied. Glial cells were very faintly labelled. The epithelial cells of the choroid plexuses, the ependymal cells and the pia mater were all labelled. Both genes were most active in neurones throughout the CNS. VACM-1 and AII/AVP receptors were detected in neurones previously shown to possess V1a and V1b vasopressin receptors, and/or the AT1 and AT2 angiotensin II receptors in many brain areas. This was the case for the magnocellular neurones of the supraoptic and paraventricular nuclei of the hypothalamus. We suggest that the VACM-1 and AII/AVP receptors may account for the V2-like responses to vasopressin by these neurones which lack a genuine V2 vasopressin receptor.

Tyrosine phosphorylation modulates the osmosensitivity of volume-dependent taurine efflux from glial cells in the rat supraoptic nucleus.

1. In the supraoptic nucleus, taurine, selectively released in an osmodependent manner by glial cells through volume-sensitive anion channels, is likely to inhibit neuronal activity as part of the osmoregulation of vasopressin release. We investigated the involvement of various kinases in the activation of taurine efflux by measuring [3H]taurine release from rat acutely isolated supraoptic nuclei. 2. The protein tyrosine kinase inhibitors genistein and tyrphostin B44 specifically reduced, but did not suppress, both the basal release of taurine and that evoked by a hypotonic stimulus. Inhibition of tyrosine phosphatase by orthovanadate had the opposite effect. 3. The tyrosine kinase and phosphatase inhibitors shifted the relationship between taurine release and medium osmolarity in opposite directions, suggesting that tyrosine phosphorylation modulates the osmosensitivity of taurine release, but is not necessary for its activation. 4. Genistein also increased the amplitude of the decay of the release observed during prolonged hypotonic stimulation. Potentiation of taurine release by tyrosine kinases could serve to maintain a high level of taurine release in spite of cell volume regulation. 5. Taurine release was unaffected by inhibitors and/or activators of PKA, PKC, MEK and Rho kinase. 6. Our results demonstrate a unique regulation by protein tyrosine kinase of the osmosensitivity of taurine efflux in supraoptic astrocytes. This points to the presence of specific volume-dependent anion channels in these cells, or to a specific activation mechanism or regulatory properties. This may relate to the particular role of the osmodependent release of taurine in this structure in the osmoregulation of neuronal activity.

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.

V1a- and V2-type vasopressin receptors mediate vasopressin-induced Ca2+ responses in isolated rat supraoptic neurones.

1. The pharmacological profile of receptors activated by vasopressin (AVP) in freshly dissociated supraoptic magnocellular neurones was investigated using specific V1a- and V2-type AVP receptor agonists and antagonists. 2. In 97 % of AVP-responding neurones (1-3000 nM) V1a or V2 receptor type agonists (F-180 and dDAVP, respectively) elicited dose-dependent [Ca2+]i transients that were suppressed by removal of external Ca2+. 3. The [Ca2+]i response induced by 1 microM F-180 or dDAVP was selectively blocked by 10 nM of V1a and V2 antagonists (SR 49059 and SR 121463A, respectively). The response to V1a agonist was maintained in the presence of the V2 antagonist, and the V2 agonist-induced response persisted in the presence of the V1a antagonist. 4. The [Ca2+]i response induced by 1 microM AVP was partially (61 %) blocked by 10 nM SR 121463A. This blockade was increased by a further 31 % with the addition of 10 nM SR 49059. Similarly, the AVP-induced response was partially (47 %) decreased by SR 49059, and a further inhibition of 33 % was achieved in the presence of SR 121463A. 5. We demonstrate that AVP acts on the magnocellular neurones via two distinct types of AVP receptors that exhibit the pharmacological profiles of V1a and V2 types. However, since V2 receptor mRNA is not expressed in the supraoptic nucleus (SON), and since V1b receptor transcripts are observed in the SON, we propose that the V2 receptor agonist and antagonist act on a 'V2-like' receptor or a new type of AVP receptor that remains to be elucidated. The possibility that V2 ligands act on the V1b receptor cannot be excluded.

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Vasopressin regularizes the phasic firing pattern of rat hypothalamic magnocellular vasopressin neurons.

Vasopressin (AVP) magnocellular neurons of hypothalamic nuclei express specific phasic firing (successive periods of activity and silence), which conditions the mode of neurohypophyseal vasopression release. In situations favoring plasmatic secretion of AVP, the hormone is also released at the somatodendritic level, at which it is believed to modulate the activity of AVP neurons. We investigated the nature of this autocontrol by testing the effects of juxtamembrane applications of AVP on the extracellular activity of presumed AVP neurons in paraventricular and supraoptic nuclei of anesthetized rats. AVP had three effects depending on the initial firing pattern: (1) excitation of faintly active neurons (periods of activity of <10 sec), which acquired or reinforced their phasic pattern; (2) inhibition of quasi-continuously active neurons (periods of silences of <10 sec), which became clearly phasic; and (3) no effect on neurons already showing an intermediate phasic pattern (active and silent periods of 10-30 sec). Consequently, AVP application resulted in a narrower range of activity patterns of the population of AVP neurons, with a Gaussian distribution centered around a mode of 57% of time in activity, indicating a homogenization of the firing pattern. The resulting phasic pattern had characteristics close to those established previously for optimal release of AVP from neurohypophyseal endings. These results suggest a new role for AVP as an optimizing factor that would foster the population of AVP neurons to discharge with a phasic pattern known to be most efficient for hormone release.

Activation of N-methyl-D-aspartate receptors regulates basal electrical activity of oxytocin and vasopressin neurons in lactating rats.

The control of suckling-induced bursting activity of oxytocin neurons and of phasic activity of vasopressin neurons by N-methyl-D-aspartate receptors was investigated in anaesthetized lactating rats. Receptor antagonist or agonist was applied in the vicinity of supraoptic neurons recorded extracellularly. The basal activity of oxytocin neurons was tonically decreased and increased by sustained application of the antagonist and agonist respectively. These effects occurred independently of the effectiveness of suckling to trigger the bursting pattern. When drugs were applied during an ongoing series of milk-ejection-related bursts, these changes were accompanied by parallel modifications in burst amplitude, but burst periodicity was unaffected. In rats failing to milk-eject, antagonist or agonist application did not facilitate the occurrence of bursts. Simultaneous recordings from oxytocin neurons in the contralateral supraoptic nucleus showed that neither their basal nor their bursting activity were affected, indicating the absence of cross-talk between nuclei during such application. The excitatory effect of N-methyl-D-aspartate differed from that induced in the same neurons by i.c.v. injection of oxytocin, which enhanced basal level of activity and burst amplitude, but also increased burst frequency. Furthermore, the distribution of interspike intervals indicated that N-methyl-D-aspartate, but not oxytocin, induced a regularization of the spike pattern. For vasopressin neurons, application of the receptor antagonist inhibited phasic activity by decreasing burst duration and increasing silences. Conversely, N-methyl-D-aspartate enhanced phasic activity, increasing both the duration of the active phases and the frequency of spikes during active phases. When applied to silent vasopressin neurons, N-methyl-D-aspartate induced a regular phasic activity. These results provide evidence that functional N-methyl-D-aspartate receptors regulate the excitability of both oxytocin and vasopressin neurons in lactating rats. These receptors play a paramount role in maintaining a certain level of basal activity which will favour appropriate discharge patterns, tonic for oxytocin neurons and phasic for vasopressin neurons. For oxytocin neurons, this sustained control by ambient glutamate influences the amplitude of bursts, but N-methyl-D-aspartate receptors are probably not involved in the generation of the bursting pattern.

GABA-induced facilitation of the periodic bursting activity of oxytocin neurones in suckled rats.

1. GABAergic innervation of oxytocin neurones is particularly abundant during lactation, but little is known about its functional role. In this study, the role of GABAA receptors in the suckling-induced bursting activity of oxytocin neurones was investigated in lactating rats. GABAA agonists or antagonists were applied by pressure injection into the immediate neighbourhood of recorded neurones while simultaneous recordings were made from oxytocin neurones in the contralateral supraoptic nucleus. 2. GABA and the GABA agonist isoguvacine decreased the basal electrical activity while application of GABAA antagonists (picrotoxin and gabazine) increased the basal electrical activity. However, in marked and unexpected contrast, application of GABA and isoguvacine facilitated or triggered milk-ejection reflex bursting activity whereas GABAA antagonists interrupted this reflex activity. 3. Systemic injection of hypertonic saline is known to increase the firing rate of neurones in the supraoptic nucleus and temporarily to interrupt suckling-induced bursting activity. Application of GABA into one supraoptic nucleus counteracted this inhibitory effect on milk ejection. 4. These observations can be explained if the role of the important GABAergic innervation of oxytocin neurones during lactation is to favour the expression of the stereotyped suckling-induced bursting activity. It might do this by attenuating inputs unrelated to suckling which are incompatible with bursts.

Electrical recordings of magnocellular supraoptic and paraventricular neurons displaying both oxytocin- and vasopressin-related activity.

In suckled rats, some magnocellular neurons displayed both vasopressin-related phasic activity and oxytocin-related milk ejection bursts. Characteristics of basal activity and interspike intervals resembled those of vasopressin neurons. Bursts were coincident with those of oxytocin neurons and were facilitated by centrally injected oxytocin, but had lower maximum instantaneous frequency and often no after-inhibition. These data provide evidence of magnocellular neurones of mixed electrophysiological phenotype and complement reports of peptide coexistence.

A rise in the intracellular Ca2+ concentration of isolated rat supraoptic cells in response to oxytocin.

1. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in single cells isolated from adult rat supraoptic (SO) nuclei. The great majority of cells (85%) were neurones and most were immunoreactive to oxytocin or to vasopressin (AVP). 2. The resting [Ca2+]i of the majority (80%) of the neurones remained stable while 20% of the neurones displayed spontaneous [Ca2+]i oscillations which disappeared in low-Ca2+ (100 nM) EGTA buffer. 3. Addition of 100 nM oxytocin increased the [Ca2+]i in both stable and oscillating cells. Two types of responses were observed: (i) a sustained response with [Ca2+]i being maintained at an elevated level and (ii) a brief response with [Ca2+]i quickly returning to a near-resting level. Responses were reproducible, dose dependent and blocked with a specific oxytocin antagonist. 4. Removal of extracellular Ca2+ did not block the oxytocin response. In EGTA buffer, application of thapsigargin (200 nM) onto oxytocin-sensitive cells induced an increase in [Ca2+]i and inhibited the oxytocin response. These effects were not induced by other intracellular Ca2+ mobilizers such as tBuBHQ (see Methods) or caffeine. 5. In conclusion, half of the SO cells respond to oxytocin with a rise in [Ca2+]i. The effect is mediated by oxytocin receptors and results from release of Ca2+ from thapsigargin-sensitive stores.