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Coincidence detection and cortical rhythmicity are both greatly influenced by neurons' propensity to fire bursts of action potentials. In the neocortex, repetitive burst firing can also initiate abnormal neocortical rhythmicity (including epilepsy). Bursts are generated by inward currents that underlie a fast afterdepolarization (fADP) but less is known about outward currents that regulate bursting. We tested whether Kv2 channels regulate the fADP and burst firing in labeled layer 5 PNs from motor cortex of the Thy1-h mouse. Kv2 block with guangxitoxin-1E (GTx) converted single spike responses evoked by dendritic stimulation into multispike bursts riding on an enhanced fADP. Immunohistochemistry revealed that Thy1-h PNs expressed Kv2.1 (not Kv2.2) channels perisomatically (not in the dendrites). In somatic macropatches, GTx-sensitive current was the largest component of outward current with biophysical properties well-suited for regulating bursting. GTx drove ~40% of Thy1 PNs stimulated with noisy somatic current steps to repetitive burst firing and shifted the maximal frequency-dependent gain. A network model showed that reduction of Kv2-like conductance in a small subset of neurons resulted in repetitive bursting and entrainment of the circuit to seizure-like rhythmic activity. Kv2 channels play a dominant role in regulating onset bursts and preventing repetitive bursting in Thy1 PNs., (Published by Oxford University Press 2021.)

Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca 2+ channels that provide Ca 2+ for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP 2 ) is a phospholipid membrane component that has been previously shown to modulate Ca 2+ channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP 2 modulates these channels, and for the first time show how PIP 2 modulates Ca V channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP 2 depletion via wortmannin (0.5 μmol/L) inhibits Ca 2+ 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 Ca 2+ currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca 2+ channels in OT neurons, which we show to be mostly an effect on N-type Ca 2+ channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca 2+ channel currents. We conclude that PIP 2 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 Ca 2+ contributed by N-type channels., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

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 Ca 2+ -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 Ca 2+ -dependent afterhyperpolarising potentials (AHPs) that limit firing rate and contribute to bursting patterns, Ca 2+ -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 Ca 2+ 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 Ca 2+ 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., (© 2018 British Society for Neuroendocrinology.)

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 Ca 2+ dependent and activated by Ca 2+ influx through voltage-gated Ca 2+ channels. Across central nervous system neurons that generate Ca 2+ -dependent AHPs, the Ca 2+ 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 Ca 2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca 2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 μM Cd 2+ 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 Ca 2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca 2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca 2+ 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 Ca 2+ 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 Ca 2+ channel blocker abolished the entire AHP, suggesting that additional Ca 2+ sources are involved.

Oxytocin (OT) neurons exhibit larger afterhyperpolarizations (AHPs) following spike trains during late pregnancy and lactation, times when these neurons fire in bursts and release more OT associated with labor and lactation. Calcium-dependent AHPs mediated by SK channels show this plasticity, and are reduced when the channel complex is phosphorylated by casein kinase 2 (CK2), and increased when dephosphorylated by protein phosphatase (PP)2A, by altering Ca 2+ sensitivity. We compared AHP currents in supraoptic OT neurons after CK2 inhibition with 4,5,6,7-tetrabromobenzotriazole (TBB), or PP1-PP2A inhibition with okadaic acid (OA), to determine the roles of these enzymes in AHP plasticity, focusing on the peak current at 100 ms representing the SK-mediated, medium AHP (I mAHP ). In slices from virgin and two groups of pregnant rats [embryonic days (E18-19, or E20-21], I mAHP s were evoked with 3-, 10-, and 17-spike trains (20 Hz). With 3-spike trains, TBB increased the I mAHP to the greatest extent in virgin compared with both groups of pregnant animals. A difference between virgins and E20-21 rats was also evident with a 10-spike train but the increases in I mAHP s were similar among groups with 17-spike trains. In contrast, OA, while consistently reducing the I mAHP in all cases, showed no differential effects among groups. In Western blots, CK2α, CK2β, PP2A-A, PP2A-B, and PP2A-C were found in supraoptic lysates, and expression of CK2α and CK2β was reduced in E20-21 rats. Coimmunoprecipitation revealed that calmodulin, CK2α, and PP2A-C were associated with SK3 protein. The results suggest that a downregulation of SK3-associated CK2α during late pregnancy may increase the sensitivity of the SK calmodulin (Ca 2+ ) sensor for I mAHP , contributing to the enhanced I mAHP . NEW & NOTEWORTHY The article demonstrates for the first time that enhancement in spike afterhyperpolarizations in oxytocin neurons during pregnancy may be related to a downregulation in the small-conductance Ca 2+ -activated potassium channels (SK)/calmodulin binding protein casein kinase 2, which phosphorylates the SK channel complex and reduces its Ca 2+ sensitivity.

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 PIP 2 's control of these AHPs. We examined how changes in PIP 2 levels affected AHPs, somatic [Ca 2+ ] i , and whole cell Ca 2+ currents. Manipulations of PIP 2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP 2 levels did not modulate AHPs by influencing Ca 2+ release from IP 3 -triggered Ca 2+ stores, suggesting more direct modulation of channels by PIP 2 . PIP 2 depletion reduced spike-evoked Ca 2+ entry and voltage-gated Ca 2+ currents. PIP 2 appears to influence AHPs in OT neurons by reducing Ca 2+ 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 (PIP 2 ) 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 PIP 2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (I mAHP and I sAHP ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP 2 analogue (diC 8 -PIP 2 ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the I mAHP and I sAHP . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either I mAHP or I sAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP 3 availability, i.e. PIP 2 modulation of AHPs is not likely to involve downstream Ca 2+ release from inositol 1,4,5-trisphosphate (IP 3 )-triggered Ca 2+ -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca 2+ ] i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca 2+ . Finally, wortmannin selectively reduced whole cell Ca 2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP 2 modulates both the I mAHP and I sAHP in OT neurons, most likely by controlling Ca 2+ entry through voltage-gated Ca 2+ channels opened during spike trains., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The physiological demands of parturition and lactation lead to the increased pulsatile release of oxytocin (OT) into the circulation from the neurohypophysial axons of OT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. These states of increased OT release are accompanied by a significant plasticity in magnocellular OT neurones and their synaptic connections, and many of these changes require activation of a central OT receptor. The mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK) is assumed to be up-regulated in the PVN during lactation, and many of the effects of OT in peripheral and brain tissue are mediated through a MAPK/ERK pathway. The present study investigated whether this pathway is altered in the SON and PVN during late pregnancy [embryonic day (E)20-21], which is a critical period for OT plasticity induction, and for lactation, when plastic changes are sustained. Based on immunoreactivity for phosphorylated ERK1/2 (pERK1/2), the results suggest an enhanced activation of MAPK/ERK pathway in OT neurones specifically during late pregnancy in both the SON and PVN. Although immunoblots from the SON confirm this pregnancy-associated up-regulation in late pregnancy, they also suggest enhancement into lactation as well. Together, the results suggest an important role for the MAPK/ERK pathway during reproductive changes in the SON and PVN., (© 2016 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

We studied neocortical pyramidal neurons from two lines of bacterial artificial chromosome mice (etv1 and glt; Gene Expression Nervous System Atlas: GENSAT project), each of which expresses enhanced green fluorescent protein (EGFP) in a different subpopulation of layer 5 pyramidal neurons. In barrel cortex, etv1 and glt pyramidal cells were previously reported to differ in terms of their laminar distribution, morphology, thalamic inputs, cellular targets, and receptive field size. In this study, we measured the laminar distribution of etv1 and glt cells. On average, glt cells were located more deeply; however, the distributions of etv1 and glt cells extensively overlap in layer 5. To test whether these two cell types differed in electrophysiological properties that influence firing behavior, we prepared acute brain slices from 2-4-wk-old mice, where EGFP-positive cells in somatosensory cortex were identified under epifluorescence and then studied using whole cell current- or voltage-clamp recordings. We studied the details of action potential parameters and repetitive firing, characterized by the larger slow afterhyperpolarizations (AHPs) in etv1 neurons and larger medium AHPs (mAHPS) in glt cells, and compared currents underlying the mAHP and slow AHP (sAHP) in etv1 and glt neurons. Etv1 cells exhibited lower dV/dt for spike polarization and repolarization and reduced direct current (DC) gain (lower f-I slope) for repetitive firing than glt cells. Most importantly, we found that 1) differences in the expression of Ca(2+)-dependent K(+) conductances (small-conductance calcium-activated potassium channels and sAHP channels) determine major functional differences between etv1 and glt cells, and 2) there is differential modulation of etv1 and glt neurons by norepinephrine., (Copyright © 2015 the American Physiological Society.)

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons., (Copyright © 2015 the American Physiological Society.)

The present study investigated a subpopulation of neurons in the mouse parabrachial nucleus (PbN), a gustatory and visceral relay area in the brainstem, that project to the lateral hypothalamus (LH). We made injections of the retrograde tracer Fluorogold (FG) into LH, resulting in fluorescent labeling of neurons located in different regions of the PbN. Mice were stimulated through an intraoral cannula with one of seven different taste stimuli, and PbN sections were processed for immunohistochemical detection of the immediate early gene c-Fos, which labels activated neurons. LH projection neurons were found in all PbN subnuclei, but in greater concentration in lateral subnuclei, including the dorsal lateral subnucleus (dl). Fos-like immunoreactivity (FLI) was observed in the PbN in a stimulus-dependent pattern, with the greatest differentiation between intraoral stimulation with sweet (0.5 M sucrose) and bitter (0.003 M quinine) compounds. In particular, sweet and umami-tasting stimuli evoked robust FLI in cells in the dl, whereas quinine evoked almost no FLI in cells in this subnucleus. Double-labeled cells were also found in the greatest quantity in the dl. Overall, these results support the hypothesis that the dl contains direct a projection to the LH that is activated preferentially by appetitive compounds; this projection may be mediated by taste and/or postingestive mechanisms.

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Biophysical characteristics of tetrodotoxin-sensitive sodium (Na(+)) currents were studied in vasopressin (VP) and oxytocin (OT) supraoptic neurons acutely isolated from rat hypothalamus. Na(+) current density (pA/pF) was significantly greater in VP neurons than in OT neurons. No significant difference between VP and OT neurons was detected regarding the voltage dependence of activation and steady-state inactivation, or rate of recovery from inactivation of Na(+) currents. In both VP and OT neurons, the macroscopic inactivation of the Na(+) currents was best fitted with a double-exponential expression suggesting two rates of inactivation. Also in both types, the time course of recovery from inactivation proceeded with fast and slow time constants averaging around 8 and 350 ms, respectively, suggesting the presence of multiple pathways of recovery from inactivation. The slower time constant of recovery of inactivation may be involved in the decrease in action potential (AP) amplitude that occurs after the first spike during burst firing in both neuronal types. The larger amplitude of Na(+) currents in VP vs. OT neurons may explain the previous observations that VP neurons exhibit a lower AP threshold and greater AP amplitude than OT neurons, and may serve to differently tune the firing properties and responses to neuromodulators of the respective neuronal types.

The hypothalamus contains nuclei and cell populations that are critical in reproduction and that differ significantly between the sexes in structure and function. To examine the molecular and genetic basis for these differences, we quantified gene expression in the hypothalamus of 39 pairs of adult male and female mice belonging to the BXD strains. This experimental design enabled us to define hypothalamic gene coexpression networks and provided robust estimates of absolute expression differences. As expected, sex has the strongest effect on the expression of genes on the X and Y chromosomes (e.g., Uty, Xist, Kdm6a). Transcripts associated with the endocrine system and neuropeptide signaling also differ significantly. Sex-differentiated transcripts often have well delimited expression within specific hypothalamic nuclei that have roles in reproduction. For instance, the estrogen receptor (Esr1) and neurokinin B (Tac2) genes have intense expression in the medial preoptic and arcuate nuclei and comparatively high expression in females. Despite the strong effect of sex on single transcripts, the global pattern of covariance among transcripts is well preserved, and consequently, males and females have well matched coexpression modules. However, there are sex-specific hub genes in functionally equivalent modules. For example, only in males is the Y-linked gene, Uty, a highly connected transcript in a network that regulates chromatin modification and gene transcription. In females, the X chromosome paralog, Kdm6a, takes the place of Uty in the same network. We also find significant effect of sex on genetic regulation and the same network in males and females can be associated with markedly different regulatory loci. With the exception of a few sex-specific modules, our analysis reveals a system in which sets of functionally related transcripts are organized into stable sex-independent networks that are controlled at a higher level by sex-specific modulators.

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., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

The epithelial Na⁺ channels (ENaCs) are present in kidney and contribute to Na⁺ and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.

The neurohypophysial hormones, vasopressin (VP) and oxytocin (OT), are synthesised by magnocellular cells in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus. The release of VP into the general circulation from the neurohypophysis increases during hyperosmolality, hypotension and hypovolaemia. VP neurones increase hormone release by increasing their firing rate as a result of adopting a phasic bursting. Depolarising after potentials (DAPs) following a series of action potentials are considered to be involved in the generation of the phasic bursts by summating to plateau potentials. We recently discovered a fast DAP (fDAP) in addition to the slower DAP characterised previously. Almost all VP neurones expressed the fDAP, whereas only 16% of OT neurones had this property, which implicates the involvement of fDAP in the generation of the firing patterns in VP neurones. Our findings obtained from electrophysiological experiments suggested that the ionic current underlying the fDAP is mediated by those of two closely-related Ca(2+) -activated cation channels: the melastatin-related subfamily of transient receptor potential channels, TRPM4 and TRPM5. In the present study, double/triple immunofluorescence microscopy and reverse transcriptase-polymerase chain reaction techniques were employed to evaluate whether TRPM4 and TRPM5 are specifically located in VP neurones. Using specific antibodies against these channels, TRPM5 immunoreactivity was found almost exclusively in VP neurones, but not in OT neurones in both the SON and PVN. The most prominent TRPM5 immunoreactivity was in the dendrites of VP neurones. By contrast, most TRPM4 immunoreactivity occurred in cell bodies of both VP and OT neurones. TRPM4 and TRPM5 mRNA were both found in a cDNA library derived from SON punches. These results indictate the possible involvement of TRPM5 in the generation of the fDAP, and these channels may play an important role in determining the distinct firing properties of VP neurones in the SON., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

Neurons in the mammalian neocortex receive inputs from and communicate back to thousands of other neurons, creating complex spatiotemporal activity patterns. The experimental investigation of these parallel dynamic interactions has been limited due to the technical challenges of monitoring or manipulating neuronal activity at that level of complexity. Here we describe a new massively parallel photostimulation system that can be used to control action potential firing in in vitro brain slices with high spatial and temporal resolution while performing extracellular or intracellular electrophysiological measurements. The system uses digital light processing technology to generate 2-dimensional (2D) stimulus patterns with >780,000 independently controlled photostimulation sites that operate at high spatial (5.4 μm) and temporal (>13 kHz) resolution. Light is projected through the quartz-glass bottom of the perfusion chamber providing access to a large area (2.76 mm × 2.07 mm) of the slice preparation. This system has the unique capability to induce temporally precise action potential firing in large groups of neurons distributed over a wide area covering several cortical columns. Parallel photostimulation opens up new opportunities for the in vitro experimental investigation of spatiotemporal neuronal interactions at a broad range of anatomical scales.

Potassium channels regulate numerous aspects of neuronal excitability, and several voltage-gated K(+) channel subunits have been identified in pyramidal neurons of rat neocortex. Previous studies have either considered the development of outward current as a whole or divided currents into transient, A-type and persistent, delayed rectifier components but did not differentiate between current components defined by α-subunit type. To facilitate comparisons of studies reporting K(+) currents from animals of different ages and to understand the functional roles of specific current components, we characterized the postnatal development of identified Kv channel-mediated currents in pyramidal neurons from layers II/III from rat somatosensory cortex. Both the persistent/slowly inactivating and transient components of the total K(+) current increased in density with postnatal age. We used specific pharmacological agents to test the relative contributions of putative Kv1- and Kv2-mediated currents (100 nM α-dendrotoxin and 600 nM stromatoxin, respectively). A combination of voltage protocol, pharmacology, and curve fitting was used to isolate the rapidly inactivating A-type current. We found that the density of all identified current components increased with postnatal age, approaching a plateau at 3-5 wk. We found no significant changes in the relative proportions or kinetics of any component between postnatal weeks 1 and 5, except that the activation time constant for A-type current was longer at 1 wk. The putative Kv2-mediated component was the largest at all ages. Immunocytochemistry indicated that protein expression for Kv4.2, Kv4.3, Kv1.4, and Kv2.1 increased between 1 wk and 4-5 wk of age.

In recognition of the impact that a powerful new anatomical tool, such as the Golgi method, can have, this essay highlights the enormous influence that biocytin-filling has had on modern neuroscience. This method has allowed neurones that have been recorded intracellularly, 'whole-cell' or juxta-cellularly, to be identified anatomically, forming a vital link between functional and structural studies. It has been applied throughout the nervous system and has become a fundamental component of our technical armoury. A comprehensive survey of the applications to which the biocytin-filling approach has been put, would fill a large volume. This essay therefore focuses on one area, neocortical microcircuitry and the ways in which combining physiology and anatomy have revealed rules that help us explain its previously indecipherable variability and complexity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

When released from dendrites within the supraoptic (SON) and paraventricular (PVN) nuclei (intranuclear release) during suckling, oxytocin exerts autocrine and paracrine effects on oxytocin neurons that are necessary for the unique timing and episodic pattern of oxytocin release into the systemic circulation that is characteristic of lactation. Recent reports have shown that stimulation of central noradrenergic and histaminergic receptors are both necessary for intranuclear release of oxytocin in response to suckling. In addition, in vitro studies indicate that excitatory amino acids may also be critical for central oxytocin secretion, although in vivo experiments have not provided direct support for this hypothesis. In addition to a critical role in intranuclear oxytocin release during lactation, norepinephrine has also been shown to stimulate central oxytocin during gestation. Stimulation of central oxytocin receptors during gestation appears critical for normal systemic oxytocin secretion in responses to suckling during the subsequent period of lactation. Oxytocin receptor blockade during pregnancy alters normal timing of systemic oxytocin release during suckling and reduces milk delivery. Several adaptations occur in the central oxytocin system that are necessary for determining the unique response characteristic observed during parturition and gestation. Central oxytocin receptor stimulation during gestation has been implicated in pregnancy-related morphological changes in magnocellular oxytocin neurons, disinhibition of oxytocin neurons to GABA, and adaptations in membrane response characteristics of oxytocin neurons. In conclusion, intranuclear oxytocin release during gestation and lactation are critical for establishing, and then evoking the unique pattern of systemic oxytocin secretion in response to the suckling offspring necessary for adequate milk delivery. Furthermore, activation of central noradrenergic receptors appears to be critical for release of central oxytocin in both of these reproductive states.

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) originate from hypothalamic neurosecretory cells in the paraventricular and supraoptic (SON) nuclei. The firing rate and pattern of action potentials arising from these neurones determine the timing and quantity of peripheral hormone release. We have used immunochemical identification of biocytin-filled SON neurones in hypothalamic slices in vitro to uncover differences between OT and VP neurones in membrane and synaptic properties, firing patterns, and plasticity during pregnancy and lactation. In this review, we summarise some recent findings from this approach: (i) VP neuronal excitability is influenced by slow (sDAP) and fast (fDAP) depolarising afterpotentials that underlie phasic bursting activity. The fDAP may relate to a transient receptor potential (TRP) channel, type melastatin (TRPM4 and/or TRPM5), both of which are immunochemically localised more to VP neurones, and especially, to their dendrites. Both TRPM4 and TRPM5 mRNAs are found in the SON, but single cell reverse transcriptase-polymerisation suggests that TRPM4 might be the more prominent channel. Phasic bursting in VP neurones is little influenced by spontaneous synaptic activity in slices, being shaped largely by intrinsic currents. (ii) The firing pattern of OT neurones ranges from irregular to continuous, with the coefficient of variation determined by randomly distributed, spontaneous GABAergic, inhibitory synaptic currents (sIPSCs). These sIPSCs are four- to five-fold more frequent in OT versus VP neurones, and much more frequent than spontaneous excitatory synaptic currents. (iii) Both cell types express Ca(2+)-dependent afterhyperpolarisations (AHPs), including an apamin-sensitive, medium duration AHP and a slower, apamin-insensitive AHP (sAHP). In OT neurones, both AHPs are enhanced during pregnancy and lactation. During pregnancy, the plasticity of the sAHP is blocked by antagonism of central OT receptors. AHP enhancement is mimicked by exposing slices from day 19 pregnant rats to OT and oestradiol, suggesting that central OT and sex steroids programme this plasticity during pregnancy by direct hypothalamic actions. In conclusion, the differences in VP and OT neuronal function are underlain by differences in both membrane and synaptic properties, and differentially modulated by reproductive state.

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.

The neurohypophysial hormone oxytocin (OT), synthesized in magnocellular paraventricular (PVN) and supraoptic (SON) nuclei, is well known for its effects in lactation. Our previous studies showed that central OT receptor (OTR) binding is increased during gestation and that blockade of central OTRs, specifically during mid-late gestation, causes a delay in OT release during suckling and reduces weight gain in pups, suggesting decreased milk delivery. In the present study, we tested whether central OTR blockade during late gestation disrupts the gestation-related plasticity in intrinsic membrane properties. Whole cell current-clamp recordings were performed in OT neurons from pregnant rats (19-22 days in gestation) that were infused with an OTR antagonist (OTA) or artificial cerebrospinal fluid (aCSF) and from virgin rats infused with aCSF into the third ventricle via an osmotic minipump beginning on days 12-14 of gestation. The amplitudes of both Ca(2+)-dependent afterhyperpolarizations (AHPs), an apamin-sensitive medium AHP (mAHP) and an apamin-insensitive slow AHP (sAHP), were significantly increased during late gestation in control pregnant animals. However, the amplitude of the sAHP from pregnant rats treated with the OTA was significantly smaller than that of pregnant control rats and similar to that of virgins. These results indicate that the diminished efficiency in lactation due to OTR blockade may be partly a result of an altered sAHP that would shape OT bursting. These findings suggest that central actions of OT during late gestation are necessary for programming the plasticity of at least some of the intrinsic membrane properties in OT neurons during lactation.

Oxytocin (OT) and vasopressin (VP) synthesizing magnocellular cells (MNCs) in the supraoptic nucleus (SON) display distinct firing patterns during the physiological demands for these hormones. Depolarizing afterpotentials (DAPs) in these neurons are involved in controlling phasic bursting in VP neurons. Our whole cell recordings demonstrated a Cs(+)-resistant fast DAP (fDAP; decay tau = approximately 200 ms), which has not been previously reported, in addition to the well-known Cs(+)-sensitive slower DAP (sDAP; decay tau = approximately 2 s). Immunoidentification of recorded neurons revealed that all VP neurons, but only 20% of OT neurons, expressed the fDAP. The activation of the fDAP required influx of Ca(2+) through voltage-gated Ca(2+) channels as it was strongly suppressed in Ca(2+)-free extracellular solution or by bath application of Cd(2+). Additionally, the current underlying the fDAP (I(fDAP)) is a Ca(2+)-activated current rather than a Ca(2+) current per se as it was abolished by strongly buffering intracellular Ca(2+) with BAPTA. The I-V relationship of the I(fDAP) was linear at potentials less than -60 mV but showed pronounced outward rectification near -50 mV. I(fDAP) is sensitive to changes in extracellular Na(+) and K(+) but not Cl(-). A blocker of Ca(2+)-activated nonselective cation (CAN) currents, flufenamic acid, blocked the fDAP, suggesting the involvement of a CAN current in the generation of fDAP in VP neurons. We speculate that the two DAPs have different roles in generating after burst discharges and could play important roles in determining the distinct firing properties of VP neurons in the SON neurons.

We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K+ currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and approximately 80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K+ current by 25-50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 nM) also inhibited approximately 40% of the slowly inactivating K+ current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody- and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential.

The firing pattern of magnocellular neurosecretory neurons is intimately related to hormone release, but the relative contribution of synaptic versus intrinsic factors to the temporal dispersion of spikes is unknown. In the present study, we examined the firing patterns of vasopressin (VP) and oxytocin (OT) supraoptic neurons in coronal slices from virgin female rats, with and without blockade of inhibitory and excitatory synaptic currents. Inhibitory postsynaptic currents (IPSCs) were twice as prevalent as their excitatory counterparts (EPSCs), and both were more prevalent in OT compared with VP neurons. Oxytocin neurons fired more slowly and irregularly than VP neurons near threshold. Blockade of Cl- currents (including tonic and synaptic currents) with picrotoxin reduced interspike interval (ISI) variability of continuously firing OT and VP neurons without altering input resistance or firing rate. Blockade of EPSCs did not affect firing pattern. Phasic bursting neurons (putative VP neurons) were inconsistently affected by broad synaptic blockade, suggesting that intrinsic factors may dominate the ISI distribution during this mode in the slice. Specific blockade of synaptic IPSCs with gabazine also reduced ISI variability, but only in OT neurons. In all cases, the effect of inhibitory blockade on firing pattern was independent of any consistent change in input resistance or firing rate. Since the great majority of IPSCs are randomly distributed, miniature events (mIPSCs) in the coronal slice, these findings imply that even mIPSCs can impart irregularity to the firing pattern of OT neurons in particular, and could be important in regulating spike patterning in vivo. For example, the increased firing variability that precedes bursting in OT neurons during lactation could be related to significant changes in synaptic activity.

Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-cell RT-PCR, immunocytochemistry, and whole cell recordings with specific peptide toxins revealed that individual pyramidal cells express multiple Kv1 alpha-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as axonal cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these cells. The alpha-dendrotoxin (alpha-DTX)-sensitive current activated more rapidly and at more negative potentials than the alpha-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The alpha-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from -70 mV. From -50 mV, this percentage increased to approximately 20%. All cells expressed an alpha-DTX-sensitive current with slow inactivation kinetics. In some cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the alpha-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.

The central and systemic release of oxytocin (OT) has been well documented during parturition and lactation. In preparation for the demands of these events, the magnocellular hypothalamic neurons of the central OT system undergo a variety of biochemical, molecular, electrophysiological, and anatomical adaptations during gestation. However, the mechanisms responsible for these changes have not been well established. A number of neurochemical mediators have been implicated in contributing to the plasticity in the OT magnocellular system during gestation, including ovarian hormones, as well as central neurotransmitters, such as glutamate, gamma-amino butyric acid (GABA), and central neurosteroids, e.g., allopregnanolone. In addition, several lines of evidence suggest that central OT release and subsequent OT receptor stimulation may contribute to adaptations of the OT system during gestation, and may be necessary for its subsequent functioning during lactation. Here, we review evidence for involvement of the neurochemical systems implicated in contributing to adaptations that occur in the OT system during the course of gestation.

Supraoptic nucleus (SON) neurons possess a prominent afterhyperpolarization (AHP) that contributes to spike patterning. This AHP is probably underlain by a small-conductance, CA2+-dependent, K+ type 3 (SK3) channel. To determine the distribution of SK3 channels within the SON, we used immunocytochemistry in rats and in transgenic mice with a regulatory cassette on the SK3 gene, allowing regulated expression with dietary doxycycline (DOX). In rats and wild-type mice, SK3 immunostaining revealed an intense lacy network surrounding SON neurons, with weak staining in neuronal somata and dendrites. In untreated, conditional SK3 knockout mice, SK3 was overexpressed, but the pericellular pattern in the SON was similar to that of rats. DOX-treated transgenic mice exhibited no SK3 staining in the SON. Double staining for oxytocin or vasopressin neurons revealed weak co-localization with SK3 but strong staining surrounding each neuron type. Electron microscopy showed that SK3-like immunoreactivity was intense between neuronal somata and dendrites, in apparent glial processes, but weak in neurons. This was confirmed by using confocal microscopy and double staining for glial fibrillary acidic protein (GFAP) and SK3: many GFAP-positive processes in the SON, and in the ventral dendritic/glial lamina, were shown to contain SK3-like immunoreactivity. These studies suggest a prominent role of SK3 channels in astrocytes. Given the marked plasticity in glial/neuronal relationships, as well as studies suggesting that astrocytes in the central nervous system can generate prominent CA2+ transients to various stimuli, a CA2+-dependent K+ channel may help SON astrocytes with K+ buffering whenever astrocyte intracellular CA2+ is increased., ((c) 2005 Wiley-Liss, Inc.)

The firing pattern of oxytocin (OT) hormone synthesizing neurons changes dramatically immediately before each milk ejection, when a brief burst of action potentials is discharged. OT neurons possess intrinsic currents that would modulate this burst. Our previous studies showed the amplitude of the Ca2+ -dependent afterhyperpolarization (AHP) following spike trains is significantly larger during lactation. In the present study we sought to determine which component of the AHP is enhanced, and whether the enhancement could be related to changes in whole-cell Ca2+ current or the Ca2+ transient in identified OT or vasopressin (VP) neurons during lactation. We confirmed, with whole-cell current-clamp recordings, our previous finding from sharp electrodes that the size of the AHP following spike trains increased in OT, but not VP neurons during lactation. We then determined that an apamin-sensitive medium-duration AHP (mAHP) and an apamin-insensitive slow AHP (sAHP) were specifically increased in OT neurons. Simultaneous Ca2+ imaging revealed that the peak change in somatic [Ca2+]i was not altered in either cell type, but the slow decay of the Ca2+ transient was faster in both cell types during lactation. In voltage clamp, the whole-cell, Ca2+ current was slightly larger during lactation in OT cells only, but current density was unchanged when corrected for somatic hypertrophy. The currents, ImAHP and IsAHP, also were increased in OT neurons only, but only the apamin-sensitive ImAHP showed an increase in current density after adjusting for somatic hypertrophy. These findings suggest a specific modulation (e.g. increased number) of the small-conductance Ca2+ -dependent K+ (SK) channels, or their interaction with Ca2+, underlies the increased mAHP/ImAHP during lactation. This larger mAHP may be necessary to limit the explosive bursts during milk ejection.

We identified Kv3-like high-threshold K+ currents in hypothalamic supraoptic neurons using whole cell recordings in hypothalamic slices and in acutely dissociated neurons. Tetraethylammonium (TEA)-sensitive currents (< 1 mM TEA) evoked from -50 mV were characterized by a large component that inactivated in 10-30 ms, and a smaller, persistent component that inactivated in 1-2 s. I/V relations in dissociated neurons revealed TEA-subtracted currents with a slope and voltage dependency consistent with the presence of Kv3-like channels. In slices, tests with 0.01-0.7 mM TEA produced an IC50 of 200-300 nM for both fast and persistent currents. The fast transient current was similar to currents associated with the expression of Kv3.4 subunits, given that it was sensitive to BDS-I (100 nM). The persistent TEA-sensitive current appeared similar to those attributed to Kv3.1/3.2 subunits. Although qualitatively similar, oxytocin (OT) and vasopressin (VP) neurons in slices differed in the stronger presence of persistent current in VP neurons. In both cell types, the IC50 for TEA-induced spike broadening was similar to that observed for current suppression in voltage clamp. However, TEA had a greater effect on the spike width of VP neurons than of OT neurons. Immunochemical studies revealed a stronger expression of the Kv3.1b alpha-subunit in VP neurons, which may be related to the greater importance of this current type in VP spike repolarization. Because OT and VP neurons are not considered fast firing, but do exhibit frequency- and calcium-dependent spike broadening, Kv3-like currents may be important for maintaining spike width and calcium influx within acceptable limits during repetitive firing.

To better understand the plasticity of intrinsic membrane properties of supraoptic magnocellular neuroendocrine cells associated with reproductive function, intracellular recordings were performed in oxytocin (OT) and vasopressin (VP) neurones from virgin, late pregnant (E19-22), and lactating (8-12 days of lactation) rats in vitro, using hypothalamic explants. OT neurones from virgin rats displayed a narrower spike width than neurones from pregnant and lactating rats, characterized by faster rise and decay times. Spike width changes in VP neurones were not as prominent as those observed in OT neurones. In OT neurones, the amplitude and the decay of the afterhyperpolarization following spike trains was significantly larger and faster, respectively, in pregnant and lactating rats compared to virgin rats. These properties did not change during pregnancy and lactation in VP neurones. The incidence of the depolarizing afterpotential following spikes significantly increased from approximately 20% in virgin rats to 40-50% during pregnancy and lactation in OT neurones, but was stable (80-90%) across states in VP neurones. Repetitive firing properties (frequency adaptation, the first interspike interval frequency and frequency-current (F-I) relationship) were altered during pregnancy and lactation in OT neurones, but not VP neurones. The increased incidence of depolarizing afterpotentials in OT neurones enhances excitability, while the increased afterhyperpolarization results in suppression of firing rate. Thus, the changes may favour the short bursting activity seen in OT neurones during lactation. These results confirmed reproductive state-dependent changes in intrinsic membrane properties of OT neurones during lactation, and suggest these changes are in place during late pregnancy. This argues that the plasticity in the electrical properties in OT neurones associated with lactation is not instigated by suckling.

In mammals, the neurohypophysial hormone oxytocin (OT) is released into the bloodstream during labor and lactation to promote uterine contraction and milk ejection, respectively. Electrophysiological studies have established that OT neurons fire in brief, synchronized bursts during this release. During pregnancy and lactation, the intrinsic membrane and synaptic properties of OT, and to a lesser extent vasopressin (VP) neurons, are altered as a part of the adaptation to these specialized states. During lactation OT neurons specifically exhibit an enhanced rebound depolarization which could assist in instigating bursts and an increased gating of firing frequency which is correlated with an enhanced Ca(2+)-dependent after hyperpolarization. Spike broadening occurs in both VP and OT neurons, but in OT neurons this and other changes are present during late pregnancy, suggesting involvement of steroidal hormones in programming neuronal adaptations. Excitatory and inhibitory synaptic activity also are altered by reproductive state. There is a doubling of glutamatergic activity specific to OT neurons which is consistent with an increase in terminal numbers, but this is accompanied by an increase in paired-pulse facilitation, suggesting an increase in the probability of glutamate release during lactation as well. Together with profound changes in both pre- and postsynaptic GABAergic synaptic activity, these data suggest that neurosecretory, and particularly OT neuronal, properties are state-dependent. These modifications may adjust the responsiveness of these neurons to afferent stimulation during periods of increased hormone demand and thereby enhance stimulus-secretion coupling., (Copyright 2002 Wiley-Liss, Inc.)

The increased release of oxytocin during lactation has been shown to be dependent upon glutamatergic transmission and is associated with an increased synaptic innervation of the supraoptic nucleus (SON). To determine whether the glutamatergic synaptic properties of oxytocin neurones are changed during lactation, we recorded excitatory postsynaptic currents (EPSCs) from identified oxytocin neurones in the SON of slices taken from adult virgin and lactating rats. The frequency of AMPA-mediated miniature EPSCs (mEPSCs) more than doubled during lactation. In addition, the decay time constant, but not the amplitude of the mEPSCs was significantly increased in both vasopressin and oxytocin neurones. Paired-pulse facilitation (PPF) was significantly reduced in oxytocin neurones during lactation, whereas no change was observed in vasopressin neurones. Elevating Ca(2+) reduced PPF in oxytocin neurones in virgin rats but did not alter PPF in oxytocin neurones from lactating rats. Collectively, our results suggest that excitatory glutamatergic transmission is strengthened in oxytocin neurones during lactation, probably by a combination of an increased number of terminals, slower decay kinetics, and an increase in the probability of release.

Oxytocin (OT) and vasopressin (VP) hormone release from neurohypophysial terminals is controlled by the firing pattern of neurosecretory cells located in the hypothalamic supraoptic (SON) and paraventricular nuclei. Although glutamate is a key modulator of the electrical activity of both OT and VP neurons, a differential contribution of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) has been proposed to mediate glutamatergic influences on these neurons. In the present study we examined the distribution and functional properties of synaptic currents mediated by AMPARs and NMDARs in immunoidentified SON neurons. Our results suggest that the properties of AMPA-mediated currents in SON neurons are controlled in a cell type-specific manner. OT neurons displayed AMPA-mediated miniature EPSCs (mEPSCs) with larger amplitude and faster decay kinetics than VP neurons. Furthermore, a peak-scaled nonstationary noise analysis of mEPSCs revealed a larger estimated single-channel conductance of AMPARs expressed in OT neurons. High-frequency summation of AMPA-mediated excitatory postsynaptic potentials was smaller in OT neurons. In both cell types, AMPA-mediated synaptic currents showed inward rectification, which was more pronounced in OT neurons, and displayed Ca2+ permeability. On the other hand, NMDA-mediated mEPSCs of both cell types had similar amplitude and kinetic properties. The cell type-specific expression of functionally different AMPARs can contribute to the adoption of different firing patterns by these neuroendocrine neurons in response to physiological stimuli.

Visceral inputs to the brain make their initial synapses within the nucleus of the solitary tract (NTS), where information is relayed to other brain regions. These inputs relate to markedly different physiological functions and provide a tool for investigating the topography of visceral processing in brainstem nuclei. Therefore, Fos immunoreactivity was used to determine whether a gastric stimulus affects neurones within different or similar parts of the NTS, ventrolateral medulla (VLM) and parabrachial nucleus (PBN), compared to a baroreceptive stimulus. The contribution of catecholaminergic neurones in these areas was studied by combining Fos and tyrosine hydroxylase (TH) immunoreactivity. Conscious male rats received either cholecystokinin (CCK) intraperitoneally to activate gastrointestinal afferents, or were made hypertensive by intravenous infusion of phenylephrine (PE) to activate baroreceptors. Tissue sections were processed immunocytochemically for Fos and/or TH. Phenylephrine infusion and CCK injection elicited Fos expression in distinct and in overlapping regions of the NTS and the VLM. Cholecystokinin injections increased the number of Fos-immunoreactive neurones in the area postrema (AP) and throughout the rostral-caudal extent of the NTS, including commissural neurones and the medial subnuclei. Some reactive neurones in NTS were also positive for TH, but most were not, and most of the TH-positive NTS neurones were not Fos-positive. In contrast, PE infusion produced a more restricted distribution of Fos-positive neurones in the NTS, with most neurones confined to a dorsolateral strip containing few TH-positive neurones. The medial NTS at the level of the AP and the AP itself were largely unresponsive, but rostral to the AP the medial NTS was labelled, including some TH-positive neurones. Both treatments produced labelling in the caudal and mid-VLM, but PE infusion had a stronger effect in the rostral VLM. In the PBN, CCK elevated Fos expression in several subregions, whereas PE infusion failed to specifically alter any subdivision. The results suggest that stimulation of baroreceptor and gastric afferents evoke both overlapping and cytoarchitectonically distinct pathways in the brainstem.

Oxytocin (OT) and vasopressin (VP) release from the neurohypophysis are correlated with the electrical activity of magnocellular cells (MNCs) in the supraoptic (SON) and paraventricular nuclei. Synaptic inputs to MNCs influence their electrical activity and, hence, hormone release. During lactation OT neurons display a synchronized high-frequency bursting activity preceding each milk ejection. In parallel to the adoption of this pattern of electrical activity, an ultrastructural reorganization of the SON has been observed during lactation. In the present study we performed a light microscopic, morphometric analysis of identified OT and VP neurons in the SON to determine whether the dendrites of these neurons participate in the plasticity observed during lactation. The dendritic trees of OT neurons shrunk during lactation ( approximately 41% decrease in the total dendritic length) because of a decreased dendritic branching concentrated at a distance of 100-200 microm from the soma. No changes in the maximal distal extension were observed. The distribution pattern of dendritic length into branch orders also was affected. Strikingly, opposite effects were observed in VP neurons. The dendritic trees during lactation elongated ( approximately 48% increase in the total dendritic length) because of an increased branching close to the soma. No changes in the maximal distal extension were observed. These results indicate that the length and geometry of the dendritic trees of OT and VP neurons are altered in opposite ways during lactation. These changes would influence the availability of postsynaptic space and alter the electrotonic properties of the neurons, affecting the efficacy of synaptic inputs.

Oxytocin and vasopressin neurons can be differentiated from one another, and from neurons in the immediately adjacent perinuclear zone, by their electrophysiological properties. In both sexes, oxytocin and vasopressin neurons are characterized by a prominent transient outward rectification which is conspicuously lacking in most perinuclear neurons. In addition, perinuclear neurons, some of which project to the supraoptic nucleus, exhibit a transient depolarization which underlies short bursts of spikes. Oxytocin neurons are characterized by: 1) the presence of a sustained outward rectifier above -50 mV, active below spike threshold; 2) a rebound depolarization following deactivation of the sustained rectification which can sustain short spike trains; and 3) a smaller transient outward rectification, probably associated with the potassium current, Ia. Vasopressin neurons show little of the sustained outward rectification and rebound depolarization, but have a stronger transient outward rectification. Although both cell types exhibit depolarizing afterpotentials, in vasopressin neurons these lead to plateau potentials underlying prolonged discharges. In oxytocin neurons, the depolarizing potential usually sustains a short spike discharge, but less often leads to prolonged bursts. These data suggest that the intrinsic properties of oxytocin and vasopressin neurons lead to quantitatively different forms of burst discharges, both of which may facilitate hormone release.

Oxytocin and vasopressin secreting neurones of the hypothalamic supraoptic nucleus share many membrane characteristics and a roughly similar morphology. However, these two neurone types differ in the relative expression of some intrinsic and synaptic currents, and in the extent of their respective dendritic arbors. Spike depolarizing afterpotentials are present in both types, but more frequently give rise to prolonged burst discharges in vasopressin neurones. Oxytocin, but not vasopressin neurones, are characterized by a depolarization-activated, sustained outward rectifier which turns on near spike threshold, and which can produce prolonged spike frequency adaptation. When this sustained current is deactivated by small hyperpolarizing pulses, a rebound depolarization sufficient to evoke short spike trains follows the offset of these pulses. Both oxytocin and vasopressin neurones exhibit a transient outward rectification underlain by an Ia-type current. This transient rectifier delays spiking to depolarizing stimuli from a relatively hyperpolarized baseline, and is more prominent in vasopressin neurones. As a result, oxytocin neurones may be more reactive to depolarizing inputs. Both cell types receive glutamatergic, excitatory synaptic inputs and both possess R,S- alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subtypes. The AMPA receptor channel on both cell types is characterized by a relatively high calcium permeability and voltage-dependent rectification, characteristic of a diminished presence of the GluR2 AMPA subunit. However, AMPA-mediated synaptic transients are larger, and decay faster, in oxytocin compared with vasopressin neurones, suggesting a potential difference for synaptic integration. The characteristics of NMDA-mediated synaptic transients are similar in oxytocin and vasopressin neurones, but some data suggest NMDA receptors may be less involved in the glutamatergic activation of oxytocin neurones. In both cell types, synaptic release of glutamate often coactivates AMPA and NMDA receptors. The dendritic morphology of oxytocin and vasopressin neurones in female rats differs from one another and exhibits considerable plasticity as a function of endocrine state. In virgin rats, oxytocin neurones have more dendritic branches and a greater total dendritic length compared with lactation, when the arbor is much less extensive. A complementary change occurs in vasopressin dendrites, which are more extensive during lactation. This reorganization suggests that oxytocin neurones may be more electronically compact during lactation. In addition, such dramatic shifts in overall dendritic length imply that significant gains and losses in either the total number of synapses, or in synaptic density, are incurred by both cell types as a function of reproductive state.

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

1. Intracellular recordings were obtained in vitro from oxytocin and vasopressin neurones from dioestrous and lactating female rats. Oxytocin neurones were characterized under current clamp by the expression of a depolarization-activated, sustained outward rectification (SOR) and a rebound depolarization (RD). 2. An increment in extracellular K+ shifted the expression of the SOR and RD towards a more depolarized membrane potential, indicating that the mechanisms underlying these events are dependent on extracellular potassium. 3. The SOR and RD were blocked by external tetraethylammonium (10 mM) and Ba2+ (0.1-0.5 mM). Cs+ (2 mM) blocked the hyperpolarization-activated inward rectification without affecting the expression of the SOR and RD. 4. The SOR was not affected by 4-aminopyridine (6 mM). However, the rebound amplitude was significantly enhanced, indicating that the activation of a transient outward current interacts with the expression of the rebound. 5. Iberiotoxin (100 nM) and apamin (50 nM), toxins known to block some calcium-dependent potassium conductances, did not affect the expression of the SOR and RD. 6. The SOR and RD were significantly reduced by Cd2+ (0.5 mM) but not by Ni2+ (0.25 mM). 7. Muscarine (10 microM) did not affect the SOR or the RD. 8. These results indicate that the SOR and RD depend upon a depolarization-activated, sustained outward potassium current, which might be calcium dependent. A current with these characteristics has never been described before in the magnocellular system. Voltage-clamp experiments are needed to completely characterize this potassium conductance selectively expressed by oxytocin neurones.

The median preoptic nucleus (MnPo) is critical for normal fluid balance, mediating osmotically evoked drinking and neurohypophysial hormone secretion. The influence of the MnPo on vasopressin and oxytocin release is in part through direct connections to the supraoptic and paraventricular nucleus. In the present investigation the synaptic contacts between the MnPo and supraoptic neurons were investigated in rats by ultrastructural examination of terminals labeled anterogradely with the tracers Phaseolus vulgaris-leucoagglutinin or biotinylated dextran. At the light microscopic level, labeled fibers within the supraoptic nucleus branched frequently, were punctuated by varicosities, and were distributed throughout the nucleus without preference for the known distributions of oxytocin and vasopressin neurons. At the ultrastructural level, synapses were associated with many of these varicosities. The ratio of labeled axodendritic to axosomatic synapses encountered was roughly consistent with a uniform innervation of dendrites and somata. The great majority of synapses were characterized by symmetrical contacts. Similar results were found for a few injections made in the organum vasculosum of the lamina terminalis, just rostral to the MnPo, and in the immediately adjacent periventricular preoptic area. Coupled with other recent anatomical and electrophysiological evidence, these results suggest there is a strong monosynaptic pathway from structures along the ventral lamina terminalis to the supraoptic nucleus.

1. The ionic basis of the histamine-induced depolarization of immunohistochemically identified neurones in the supraoptic nucleus (SON) was investigated in the hypothalamo-neurohypophysial explant of male rats. Histamine (0.1-100 microM) caused an H1 receptor-mediated, dose-dependent depolarization of fifty of sixty-two vasopressin neurones in the SON. In contrast, twenty-three oxytocin neurones were either depolarized (n = 6), hyperpolarized (n = 4), or unaffected (n = 13) by histamine. Due to the low percentage of responding cells, oxytocin neurones were not further investigated. 2. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA; 100-500 mM) blocked the depolarization, whereas blocking Ca2+ influx and synaptic transmission with equimolar Co2+ or elevated (5-20 mM) Mg2+ in nominally Ca(2+)-free solutions was without effect. 3. The amplitude of the histamine-induced depolarization was relatively independent of membrane potential. The input resistance was unaltered by histamine in nine neurones, but in nine other neurones it was decreased and in two neurones it was increased by more than 5%. Neither elevating extracellular K+ nor addition of the K+ channel blockers, apamin, d-tubocurarine, tetraethylammonium (TEA), or intracellular Cs+ decreased the histamine effect. Indeed, broadly blocking K+ currents with TEA and Cs+ significantly increased the depolarization to histamine. 4. Tetrodotoxin (2-3 microM) did not inhibit the histamine-induced depolarization. However, equimolar replacement of approximately 50% of extracellular Na+ with Tris+ or N-methyl-D-glucamine reduced or eliminated the response. 5. The depolarization of vasopressin neurones by histamine thus requires extracellular Na+ and intracellular Ca2+. Activation of a Ca(2+)-activated non-specific cation current or a Ca(2+)-Na+ pump are possible mechanisms for this effect.

The electrophysiological and morphological properties of layer I neurons were studied in visual cortex slices from 7- to 19-d-old rats using whole-cell recording and biocytin labeling. A heterogeneous population of small, nonpyramidal neurons was found. Approximately one third of the cells we recorded were neurogliaform cells; another third were multipolar neurons with axons descending out of layer I. The remaining cells were heterogeneous and were not classified. In slices from 7- to 10-d-old animals only, we identified Cajal-Retzius cells. Neurogliaform neurons had a very dense local axonal field, which was largely contained within layer I. Cells with descending axons had a relatively sparse local axonal arbor and projected at least to layer II and sometimes deeper. Spiking in neurogliaform neurons was followed by an afterdepolarizing potential, whereas spiking in cells with descending axons was followed by a slow after-hyperpolarizing potential (AHP). In addition, neurogliaform cells exhibited less spike broadening and a larger fast AHP after single spikes than did cells with descending axons. Generally, cells in layer I received synaptic inputs characterized as either GABA- or glutamate-mediated, suggesting the presence of excitatory and inhibitory inputs. With their output largely limited to layer I, neurogliaform cells could synapse with other layer I neurons, the most distal dendritic branches of pyramidal cells, or the dendrites of layer II/III interneurons, which invade layer I. Cells with descending axons could contact a wide variety of cortical cells throughout their vertical projection.