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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 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.

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 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.

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.

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.

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.

1. Intracellular recordings in vitro from immunochemically identified oxytocin (OT) and vasopressin (VP) neurones in the supraoptic nucleus (SON) of virgin or lactating female rats revealed no differences between neurone types in membrane potential (Vm), input resistance and current-voltage relationships (I-V), when taken at resting membrane potentials. 2. When OT (94%), but not VP, neurones (93%) were current clamped at depolarized voltages (above -50 mV), small hyperpolarizing pulses revealed a time- and voltage-dependent outward rectification that was present above -75 mV and that decreased in amplitude as Vm approached the equilibrium potential for potassium (EK). The rectification was more pronounced when the neurones were held at a more depolarized membrane potential, and was larger the longer the neurone was held depolarized, reaching a maximum at 0.6-0.9 s. 3. A rebound depolarization followed the offset of hyperpolarizing pulses that were associated with the rectification. The peak amplitude of the rebound showed a time and a voltage dependence. It followed a bell-shaped curve as the hyperpolarizing commands were made larger, attaining a peak at -65 +/- 1.5 mV. The rebound amplitude increased with pulse duration, achieving a half-maximal amplitude at 0.5 +/- 0.1 s. 4. The expression of the sustained outward rectification and the rebound in OT neurones was similar in virgin and lactating female rats. 5. These results indicate the presence of significant differences in the intrinsic membrane properties, probably K+ currents, between OT and VP neurones in both lactating and virgin female rats.

1. Intracellular recordings were made from supraoptic neurones in vitro from hypothalamic explants prepared from adult male rats. Neurones were injected with biotinylated markers, and of thirty-nine labelled neurones, nineteen were identified immunocytochemically as containing oxytocin-neurophysin and twenty as containing vasopressin-neurophysin. 2. Vasopressin and oxytocin neurones did not differ in their resting membrane potential, input resistance, membrane time constant, action potential height from threshold, action potential width at half-amplitude, and spike hyperpolarizing after-potential amplitude. Both cell types exhibited spike broadening during brief, evoked spike trains (6-8 spikes), but the degree of broadening was slightly greater for vasopressin neurones. When hyperpolarized below -75 mV, all but one neurone exhibited a transient outward rectification to depolarizing pulses, which delayed the occurrence of the first spike. 3. Both cell types exhibited a long after-hyperpolarizing potential (AHP) following brief spike trains evoked either with a square wave pulse or using 5 ms pulses in a train. There were no significant differences between cell types in the size of the AHP evoked with nine spikes, or in the time constant of its decay. The maximal AHP evoked by a 180 ms pulse was elicited by an average of twelve to thirteen spikes, and neither the size of this maximal AHP nor its time constant of decay were different for the two cell types. 4. In most oxytocin and vasopressin neurones the AHP, and concomitantly spike frequency adaptation, were markedly reduced by the bee venom apamin and by d-tubocurarine, known blockers of a Ca(2+)-mediated K+ conductance. However, a minority of neurones, of both cell types, were relatively resistant to both agents. 5. In untreated neurones, 55% of vasopressin neurones and 32% of oxytocin neurones exhibited a depolarizing after-potential (DAP) after individual spikes or, more commonly, after brief trains of spikes evoked with current pulses. For each neurone with a DAP, bursts of spikes could be evoked if the membrane potential was sufficiently depolarized such that the DAP reached spike threshold. In four out of five vasopressin neurones a DAP became evident only after pharmacological blockade of the AHP, whereas in six oxytocin neurones tested no such masking was found. 6. The firing patterns of neurones were examined at rest and after varying the membrane potential with continuous current injection. No identifying pattern was strictly associated with either cell type, and a substantial number of neurones were silent at rest.(ABSTRACT TRUNCATED AT 400 WORDS)