A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner.

One of the largest sex differences in brain neurochemistry is the expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate brain, with males having more AVP cells in the bed nucleus of the stria terminalis (BNST) than females. Despite the long-standing implication of AVP in social and anxiety-like behaviors, the circuitry underlying AVP's control of these behaviors is still not well defined. Using optogenetic approaches, we show that inhibiting AVP BNST cells reduces social investigation in males, but not in females, whereas stimulating these cells increases social investigation in both sexes, but more so in males. These cells may facilitate male social investigation through their projections to the lateral septum (LS), an area with the highest density of sexually differentiated AVP innervation in the brain, as optogenetic stimulation of BNST AVP → LS increased social investigation and anxiety-like behavior in males but not in females; the same stimulation also caused a biphasic response of LS cells ex vivo. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated all these responses. Together, these findings establish a sexually differentiated role for BNST AVP cells in the control of social investigation and anxiety-like behavior, likely mediated by their projections to the LS.

Impaired oxytocin signaling in the central amygdala in rats with chronic heart failure.

Aims: Heart failure (HF) patients often suffer from cognitive decline, depression, and mood impairments, but the molecular signals and brain circuits underlying these effects remain elusive. The hypothalamic neuropeptide oxytocin (OT) is critically involved in the regulation of mood, and OTergic signaling in the central amygdala (CeA) is a key mechanism controlling emotional responses including anxiety-like behaviors. Based on this, we used in this study a well-established ischemic rat HF model and aimed to study alterations in the hypothalamus-to-CeA OTergic circuit. Methods and Results: To study potential HF-induced changes in the hypothalamus-to-CeA OTertic circuit, we combined patch-clamp electrophysiology, immunohistochemical analysis, RNAScope assessment of OTR mRNA, brain region-specific stereotaxic injections of viral vectors and retrograde tracing, optogenetic stimulation and OT biosensors in the ischemic HF model. We found that most of OTergic innervation of the central amygdala (CeA) originated from the hypothalamic supraoptic nucleus (SON). While no differences in the numbers of SON→CeA OTertic neurons (or their OT content) was observed between sham and HF rats, we did observe a blunted content and release of OT from axonal terminals within the CeA. Moreover, we report downregulation of neuronal and astrocytic OT receptors, and impaired OTR-driven GABAergic synaptic activity within the CeA microcircuit of rats with HF. Conclusions: Our study provides first evidence that HF rats display various perturbations in the hypothalamus-to-amygdala OTergic circuit, and lays the foundation for future translational studies targeting either the OT system or GABAergic amygdala GABA microcircuit to ameliorate depression or mood impairments in rats or patients with chronic HF.

Changes in neuropeptide large dense core vesicle trafficking dynamics contribute to adaptive responses to a systemic homeostatic challenge.

Neuropeptides are packed into large dense core vesicles (LDCVs) that are transported from the soma out into their processes. Limited information exists regarding mechanisms regulating LDCV trafficking, particularly during challenges to bodily homeostasis. Addressing this gap, we used 2-photon imaging in an ex vivo preparation to study LDCVs trafficking dynamics in vasopressin (VP) neurons, which traffic and release neuropeptide from their dendrites and axons. We report a dynamic bidirectional trafficking of VP-LDCVs with important differences in speed and directionality between axons and dendrites. Acute, short-lasting stimuli known to alter VP firing activity and axonal/dendritic release caused modest changes in VP-LDCVs trafficking dynamics. Conversely, chronic/sustained systemic osmotic challenges upregulated VP-LDCVs trafficking dynamic, with a larger effect in dendrites. These results support differential regulation of dendritic and axonal LDCV trafficking, and that changes in trafficking dynamics constitute a novel mechanism by which peptidergic neurons can efficiently adapt to conditions of increased hormonal demand.

A vasopressin circuit that modulates sex-specific social interest and anxiety-like behavior in mice.

One of the largest sex differences in brain neurochemistry is the male-biased expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate social brain. Despite the long-standing implication of AVP in social and anxiety-like behavior, the precise circuitry and anatomical substrate underlying its control are still poorly understood. By employing optogenetic manipulation of AVP cells within the bed nucleus of the stria terminalis (BNST), we have unveiled a central role for these cells in promoting social investigation, with a more pronounced role in males relative to females. These cells facilitate male social investigation and anxiety-like behavior through their projections to the lateral septum (LS), an area with the highest density of sexually-dimorphic AVP fibers. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated stimulation-mediated increases in these behaviors. Together, these findings establish a distinct BNST AVP → LS V1aR circuit that modulates sex-specific social interest and anxiety-like behavior.

Lmx1a is a master regulator of the cortical hem.

Development of the nervous system depends on signaling centers - specialized cellular populations that produce secreted molecules to regulate neurogenesis in the neighboring neuroepithelium. In some cases, signaling center cells also differentiate to produce key types of neurons. The formation of a signaling center involves its induction, the maintenance of expression of its secreted molecules, and cell differentiation and migration events. How these distinct processes are coordinated during signaling center development remains unknown. By performing studies in mice, we show that Lmx1a acts as a master regulator to orchestrate the formation and function of the cortical hem (CH), a critical signaling center that controls hippocampus development. Lmx1a co-regulates CH induction, its Wnt signaling, and the differentiation and migration of CH-derived Cajal-Retzius neurons. Combining RNAseq, genetic, and rescue experiments, we identified major downstream genes that mediate distinct Lmx1a-dependent processes. Our work revealed that signaling centers in the mammalian brain employ master regulatory genes and established a framework for analyzing signaling center development.

Angiotensin II-Mediated Neuroinflammation in the Hippocampus Contributes to Neuronal Deficits and Cognitive Impairment in Heart Failure Rats.

BACKGROUND: Heart failure (HF) is a debilitating disease affecting >64 million people worldwide. In addition to impaired cardiovascular performance and associated systemic complications, most patients with HF suffer from depression and substantial cognitive decline. Although neuroinflammation and brain hypoperfusion occur in humans and rodents with HF, the underlying neuronal substrates, mechanisms, and their relative contribution to cognitive deficits in HF remains unknown. METHODS: To address this critical gap in our knowledge, we used a well-established HF rat model that mimics clinical outcomes observed in the human population, along with a multidisciplinary approach combining behavioral, electrophysiological, neuroanatomical, molecular and systemic physiological approaches. RESULTS: Our studies support neuroinflammation, hypoperfusion/hypoxia, and neuronal deficits in the hippocampus of HF rats, which correlated with the progression and severity of the disease. An increased expression of AT1aRs (Ang II [angiotensin II] receptor type 1a) in hippocampal microglia preceded the onset of neuroinflammation. Importantly, blockade of AT1Rs with a clinically used therapeutic drug (Losartan), and delivered in a clinically relevant manner, efficiently reversed neuroinflammatory end points (but not hypoxia ones), resulting in turn in improved cognitive performance in HF rats. Finally, we show than circulating Ang II can leak and access the hippocampal parenchyma in HF rats, constituting a possible source of Ang II initiating the neuroinflammatory signaling cascade in HF. CONCLUSIONS: In this study, we identified a neuronal substrate (hippocampus), a mechanism (Ang II-driven neuroinflammation) and a potential neuroprotective therapeutic target (AT1aRs) for the treatment of cognitive deficits in HF.

An analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray in rats.

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

Angiotensin-II drives changes in microglia-vascular interactions in rats with heart failure.

Activation of microglia, the resident immune cells of the central nervous system, leading to the subsequent release of pro-inflammatory cytokines, has been linked to cardiac remodeling, autonomic disbalance, and cognitive deficits in heart failure (HF). While previous studies emphasized the role of hippocampal Angiotensin II (AngII) signaling in HF-induced microglial activation, unanswered mechanistic questions persist. Evidence suggests significant interactions between microglia and local microvasculature, potentially affecting blood-brain barrier integrity and cerebral blood flow regulation. Still, whether the microglial-vascular interface is affected in the brain during HF remains unknow. Using a well-established ischemic HF rat model, we demonstrate increased vessel-associated microglia (VAM) in HF rat hippocampi, which showed heightened expression of AngII AT1a receptors. Acute AngII administration to sham rats induced microglia recruitment to the perivascular space, along with increased expression of TNFa. Conversely, administering an AT1aR blocker to HF rats prevented the recruitment of microglia to the perivascular space, normalizing their levels to those in healthy rats. These results highlight the critical importance of a rather understudied phenomenon (i.e., microglia-vascular interactions in the brain) in the context of the pathophysiology of a highly prevalent cardiovascular disease, and unveil novel potential therapeutic avenues aimed at mitigating neuroinflammation in cardiovascular diseases.

Analysis of the hypothalamic oxytocin system and oxytocin receptor-expressing astrocytes in a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hyperphagia, obesity, developmental delay and intellectual disability. Studies suggest dysfunctional signaling of the neuropeptide oxytocin as one of the key mechanisms in PWS, and administration of oxytocin via intranasal or systemic routes yielded promising results in both humans and mouse models. However, a detailed assessment of the oxytocin system in mouse models of PWS such as the Magel2-deficient Magel2tm1.Stw mouse, is lacking. In the present study, we performed an automated counting of oxytocin cells in the entire paraventricular nucleus of the hypothalamus of Magel2tm1.Stw and wild-type control mice and found a significant reduction in the caudal part, which represents the parvocellular subdivision. In addition, based on the recent discovery that some astrocytes express the oxytocin receptor (OTR), we performed detailed analysis of astrocyte numbers and morphology in various brain regions, and assessed expression levels of the astrocyte marker glial fibrillary acidic protein, which was significantly decreased in the hypothalamus, but not other brain regions in Magel2tm1.Stw mice. Finally, we analyzed the number of OTR-expressing astrocytes in various brain regions and found a significant reduction in the nucleus accumbens of Magel2tm1.Stw mice, as well as a sex-specific difference in the lateral septum. This study suggests a role for caudal paraventricular nucleus oxytocin neurons as well as OTR-expressing astrocytes in a mouse model of PWS, provides novel information about sex-specific expression of astrocytic OTRs, and presents several new brain regions containing OTR-expressing astrocytes in the mouse brain.

Hypothalamic astrocytes control systemic glucose metabolism and energy balance.

The hypothalamus is key in the control of energy balance. However, strategies targeting hypothalamic neurons have failed to provide viable options to treat most metabolic diseases. Conversely, the role of astrocytes in systemic metabolic control has remained largely unexplored. Here, we show that obesity promotes anatomically restricted remodeling of hypothalamic astrocyte activity. In the paraventricular nucleus (PVN) of the hypothalamus, chemogenetic manipulation of astrocytes results in bidirectional control of neighboring neuron activity, autonomic outflow, glucose metabolism, and energy balance. This process recruits a mechanism involving the astrocytic control of ambient glutamate levels, which becomes defective in obesity. Positive or negative chemogenetic manipulation of PVN astrocyte Ca2+ signals, respectively, worsens or improves metabolic status of diet-induced obese mice. Collectively, these findings highlight a yet unappreciated role for astrocytes in the direct control of systemic metabolism and suggest potential targets for anti-obesity strategy.

α-Melanocyte-stimulating hormone inhibition of oxytocin neurons switches to excitation in late pregnancy and lactation.

Oxytocin is secreted into the periphery by magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine contraction during birth and milk ejection during suckling. Peripheral oxytocin secretion is triggered by action potential firing, which is regulated by afferent input activity and by feedback from oxytocin secreted into the extracellular space from magnocellular neuron somata and dendrites. A prominent input to oxytocin neurons arises from proopiomelanocortin neurons of the hypothalamic arcuate nucleus that secrete an alpha-melanocyte-stimulating hormone (α-MSH), which inhibits oxytocin neuron firing in non-pregnant rats by increasing somato-dendritic oxytocin secretion. However, α-MSH inhibition of oxytocin neuron firing is attenuated in mid-pregnancy and somato-dendritic oxytocin becomes auto-excitatory in late-pregnancy and lactation. Therefore, we hypothesized that attenuated α-MSH inhibition of oxytocin neuron firing marks the beginning of a transition from inhibition to excitation to facilitate peripheral oxytocin secretion for parturition and lactation. Intra-SON microdialysis administration of α-MSH inhibited oxytocin neuron firing rate by 33 ± 9% in non-pregnant rats but increased oxytocin neuron firing rate by 37 ± 12% in late-pregnant rats and by 28 ± 10% in lactating rats. α-MSH-induced somato-dendritic oxytocin secretion measured ex vivo with oxytocin receptor-expressing "sniffer" cells, was of similar amplitude in PVN slices from non-pregnant and lactating rats but longer-lasting in slices from lactating rats. Hence, α-MSH inhibition of oxytocin neuron activity switches to excitation over pregnancy while somato-dendritic oxytocin secretion is maintained, which might enhance oxytocin neuron excitability to facilitate the increased peripheral secretion that is required for normal parturition and milk ejection.

Identification of Novel Cross-Talk between the Neuroendocrine and Autonomic Stress Axes Controlling Blood Pressure.

The hypothalamic paraventricular nucleus (PVN) controls neuroendocrine axes and the autonomic nervous system to mount responses that cope with the energetic burdens of psychological or physiological stress. Neurons in the PVN that express the angiotensin Type 1a receptor (PVNAgtr1a) are implicated in neuroendocrine and autonomic stress responses; however, the mechanism by which these neurons coordinate activation of neuroendocrine axes with sympathetic outflow remains unknown. Here, we use a multidisciplinary approach to investigate intra-PVN signaling mechanisms that couple the activity of neurons synthesizing corticotropin-releasing-hormone (CRH) to blood pressure. We used the Cre-Lox system in male mice with in vivo optogenetics and cardiovascular recordings to demonstrate that excitation of PVNAgtr1a promotes elevated blood pressure that is dependent on the sympathetic nervous system. Next, neuroanatomical experiments found that PVNAgtr1a synthesize CRH, and intriguingly, fibers originating from PVNAgtr1a make appositions onto neighboring neurons that send projections to the rostral ventrolateral medulla and express CRH type 1 receptor (CRHR1) mRNA. We then used an ex vivo preparation that combined optogenetics, patch-clamp electrophysiology, and Ca2+ imaging to discover that excitation of PVNAgtr1a drives the local, intra-PVN release of CRH, which activates rostral ventrolateral medulla-projecting neurons via stimulation of CRHR1(s). Finally, we returned to our in vivo preparation and found that CRH receptor antagonism specifically within the PVN lowered blood pressure basally and during optogenetic activation of PVNAgtr1a Collectively, these results demonstrate that angiotensin II acts on PVNAgtr1a to conjoin hypothalamic-pituitary-adrenal axis activity with sympathetically mediated vasoconstriction in male mice.SIGNIFICANCE STATEMENT The survival of an organism is dependent on meeting the energetic demands imposed by stressors. This critical function is accomplished by the CNS's ability to orchestrate simultaneous activities of neurosecretory and autonomic axes. Here, we unveil a novel signaling mechanism within the paraventricular nucleus of the hypothalamus that links excitation of neurons producing corticotropin-releasing-hormone with excitation of neurons controlling sympathetic nervous system activity and blood pressure. The implication is that chronic stress exposure may promote cardiometabolic disease by dysregulating the interneuronal cross-talk revealed by our experiments.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.

PIP2 alters of Ca2+ currents in acutely dissociated supraoptic oxytocin neurons.

Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca2+ channels that provide Ca2+ for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP2 ) is a phospholipid membrane component that has been previously shown to modulate Ca2+ channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP2 modulates these channels, and for the first time show how PIP2 modulates CaV channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP2 depletion via wortmannin (0.5 µmol/L) inhibits Ca2+ channel currents in OT but not VP neurons. Additionally, it hyperpolarizes voltage-dependent activation of the channels by ~5 mV while leaving the slope of activation unchanged, properties unaffected in VP neurons. We also identified key differences in baseline currents between the cell types, wherein VP whole cell Ca2+ currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca2+ channels in OT neurons, which we show to be mostly an effect on N-type Ca2+ channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca2+ channel currents. We conclude that PIP2 is a modulator that enhances current through N-type channels. This has implications for the afterhyperpolarization (AHP) of OT neurons, as previous work from our laboratory demonstrated the AHP is inhibited by wortmannin, and that its primary activation is from intracellular Ca2+ contributed by N-type channels.

Electrophysiological properties of identified oxytocin and vasopressin neurones.

To understand the contribution of intrinsic membrane properties to the different in vivo firing patterns of oxytocin (OT) and vasopressin (VP) neurones, in vitro studies are needed, where stable intracellular recordings can be made. Combining immunochemistry for OT and VP and intracellular dye injections allows characterisation of identified OT and VP neurones, and several differences between the two cell types have emerged. These include a greater transient K+ current that delays spiking to stimulus onset, and a higher Na+ current density leading to greater spike amplitude and a more stable spike threshold, in VP neurones. VP neurones also show a greater incidence of both fast and slow Ca2+ -dependent depolarising afterpotentials, the latter of which summate to plateau potentials and contribute to phasic bursting. By contrast, OT neurones exhibit a sustained outwardly rectifying potential (SOR), as well as a consequent depolarising rebound potential, not found in VP neurones. The SOR makes OT neurones more susceptible to spontaneous inhibitory synaptic inputs and correlates with a longer period of spike frequency adaptation in these neurones. Although both types exhibit prominent Ca2+ -dependent afterhyperpolarising potentials (AHPs) that limit firing rate and contribute to bursting patterns, Ca2+ -dependent AHPs in OT neurones selectively show significant increases during pregnancy and lactation. In OT neurones, but not VP neurones, AHPs are highly dependent on the constitutive presence of the second messenger, phosphatidylinositol 4,5-bisphosphate, which permissively gates N-type channels that contribute the Ca2+ during spike trains that activates the AHP. By contrast to the intrinsic properties supporting phasic bursting in VP neurones, the synchronous bursting of OT neurones has only been demonstrated in vitro in cultured hypothalamic explants and is completely dependent on synaptic transmission. Additional differences in Ca2+ channel expression between the two neurosecretory terminal types suggests these channels are also critical players in the differential release of OT and VP during repetitive spiking, in addition to their importance to the potentials controlling firing patterns.

Specificity in the interaction of high-voltage-activated Ca2+ channel types with Ca2+-dependent afterhyperpolarizations in magnocellular supraoptic neurons.

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 µM Cd2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 µM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

Phosphatidylinositol 4,5-bisphosphate (PIP2 ) modulates afterhyperpolarizations in oxytocin neurons of the supraoptic nucleus.

KEY POINTS: Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2 's control of these AHPs. We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+ ]i , and whole cell Ca2+ currents. Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3 -triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2 . PIP2 depletion reduced spike-evoked Ca2+ entry and voltage-gated Ca2+ currents. PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking. ABSTRACT: Oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium-dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP2 analogue (diC8 -PIP2 ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5-trisphosphate (IP3 )-triggered Ca2+ -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+ ]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+ . Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage-gated Ca2+ channels opened during spike trains.