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.

Hypothalamic magnocellular neuroendocrine cells have unique electrical properties and a remarkable capacity for morphological and synaptic plasticity. Their large somatic size, their relatively uniform and dense clustering in the supraoptic and paraventricular nuclei, and their large axon terminals in the neurohypophysis make them an attractive target for direct electrophysiological interrogation. Here, we provide a brief review of significant recent findings in the neuroplasticity and neurophysiological properties of these neurones that were presented at the symposium “Electrophysiology of Magnocellular Neurons” during the 13th World Congress on Neurohypophysial Hormones in Ein Gedi, Israel in April 2019. Magnocellular vasopressin (VP) neurones respond directly to hypertonic stimulation with membrane depolarisation, which is triggered by cell shrinkage‐induced opening of an N‐terminal‐truncated variant of transient receptor potential vanilloid type‐1 (TRPV1) channels. New findings indicate that this mechanotransduction depends on actin and microtubule cytoskeletal networks, and that direct coupling of the TRPV1 channels to microtubules is responsible for mechanical gating of the channels. Vasopressin neurones also respond to osmostimulation by activation of epithelial Na+ channels (ENaC). It was shown recently that changes in ENaC activity modulate magnocellular neurone basal firing by generating tonic changes in membrane potential. Both oxytocin and VP neurones also undergo robust excitatory synapse plasticity during chronic osmotic stimulation. Recent findings indicate that new glutamate synapses induced during chronic salt loading express highly labile Ca2+‐permeable GluA1 receptors requiring continuous dendritic protein synthesis for synapse maintenance. Finally, recordings from the uniquely tractable neurohypophysial terminals recently revealed an unexpected property of activity‐dependent neuropeptide release. A significant fraction of the voltage‐dependent neurohypophysial neurosecretion was found to be independent of Ca2+ influx through voltage‐gated Ca2+ channels. Together, these findings provide a snapshot of significant new advances in the electrophysiological signalling mechanisms and neuroplasticity of the hypothalamic‐neurohypophysial system, a system that continues to make important contributions to the field of neurophysiology.

Hypothalamic magnocellular neuroendocrine cells have unique electrical properties and a remarkable capacity for morphological and synaptic plasticity. Their large somatic size, their relatively uniform and dense clustering in the supraoptic and paraventricular nuclei, and their large axon terminals in the neurohypophysis make them an attractive target for direct electrophysiological interrogation. Here, we provide a brief review of significant recent findings in the neuroplasticity and neurophysiological properties of these neurones that were presented at the symposium "Electrophysiology of Magnocellular Neurons" during the 13th World Congress on Neurohypophysial Hormones in Ein Gedi, Israel in April 2019. Magnocellular vasopressin (VP) neurones respond directly to hypertonic stimulation with membrane depolarisation, which is triggered by cell shrinkage-induced opening of an N-terminal-truncated variant of transient receptor potential vanilloid type-1 (TRPV1) channels. New findings indicate that this mechanotransduction depends on actin and microtubule cytoskeletal networks, and that direct coupling of the TRPV1 channels to microtubules is responsible for mechanical gating of the channels. Vasopressin neurones also respond to osmostimulation by activation of epithelial Na+ channels (ENaC). It was shown recently that changes in ENaC activity modulate magnocellular neurone basal firing by generating tonic changes in membrane potential. Both oxytocin and VP neurones also undergo robust excitatory synapse plasticity during chronic osmotic stimulation. Recent findings indicate that new glutamate synapses induced during chronic salt loading express highly labile Ca2+-permeable GluA1 receptors requiring continuous dendritic protein synthesis for synapse maintenance. Finally, recordings from the uniquely tractable neurohypophysial terminals recently revealed an unexpected property of activity-dependent neuropeptide release. A significant fraction of the voltage-dependent neurohypophysial neurosecretion was found to be independent of Ca2+ influx through voltage-gated Ca2+ channels. Together, these findings provide a snapshot of significant new advances in the electrophysiological signalling mechanisms and neuroplasticity of the hypothalamic-neurohypophysial system, a system that continues to make important contributions to the field of neurophysiology.

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

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

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

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 Ca2+ 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 (ImAHP). In slices from virgin and two groups of pregnant rats [embryonic days (E18-19, or E20-21], ImAHPs were evoked with 3-, 10-, and 17-spike trains (20 Hz). With 3-spike trains, TBB increased the ImAHP 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 ImAHPs were similar among groups with 17-spike trains. In contrast, OA, while consistently reducing the ImAHP 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 (Ca2+) sensor for ImAHP, contributing to the enhanced ImAHP. 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 Ca2+-activated potassium channels (SK)/calmodulin binding protein casein kinase 2, which phosphorylates the SK channel complex and reduces its Ca2+ sensitivity.

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

In mammals, oxytocin (OT) release from neurohypophysial terminals during pregnancy and lactation is pulsatile, reflecting a coordinated burst firing from parent magnocellular OT neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (Poulain & Wakerley, 1982). Synchronized electrical bursting insures that OT receptors on uterine smooth muscle (for labour contractions) and the mammary gland myoepithelium (for milk ejection) avoid desensitization, maximizing OT's effects. Investigators have long demonstrated a significant plasticity in many aspects of this system that commence in late pregnancy and persist to lactation – changes suspected to play roles in developing these coordinated bursts (Theodosis et al. 2008; Armstrong, 2015). Electron microscopic investigations revealed that astrocyte processes withdraw from between OT neurons and dendrites during lactation, allowing an increase in close neuronal membrane appositions and a change in the number of excitatory and inhibitory synapses. These changes first appear in late pregnancy, and are thought to be programmed by steroid hormone changes, namely the precipitous drop in progesterone in late pregnancy. This morphological plasticity correlates with alterations in the synaptic and presynaptic actions of both glutamate and GABA on OT neurons. Other significant changes during late pregnancy and lactation that are specific to OT neurons include calcium-dependent afterhyperpolarizations that would modulate bursting patterns, and a significant upregulation in mitogen-activated protein kinase/extracellular signal-related kinase activity. Some of the GABAergic and glutamatergic inputs to magnocellular nuclei come from nearby neurons. In the SON, for example, the surrounding perinuclear zone (PZ) provides both types of inputs, and is an area thought to mediate the selective inhibition of vasopressin (VP) neurons to hypertension. This region has diverse morphological cell types, and receives inputs from regions known to affect magnocellular function, such as the diagonal band of Broca and the septum, as well as from within the hypothalamus itself. However, a specific role of this region in modulating OT neuronal activity has not previously been demonstrated. In this issue of The Journal of Physiology, Seymour et al. (2017) describe hypothalamic periventricular kisspeptin neurons that show significant plasticity during late pregnancy. Kisspeptin, a product of the KiSS-1 gene, is a relatively newly discovered peptide, previously implicated in the control of gonadotropin releasing hormone, but also with significant tumour suppressive actions (Murphy, 2005). In the brain, the kisspeptin periventricular hypothalamic neurons have widespread connections, including to and around the SON and paraventricular nuclei, areas controlling the peripheral release of OT and VP. In the present study, a striking increase in the density of axons within the PZ of the SON (but not in the SON itself) containing kisspeptin-like immunoreactivity was found during late pregnancy, and this was correlated with an upregulation of kisspeptin expression in the rostral periventricular hypothalamic neurons that project to the PZ. Furthermore, this increase appears specific to that cell group, as arcuate nucleus kisspeptin neurons are dramatically downregulated in the same period. These results were coincident with a significant ability of centrally administered kisspeptin to excite OT, but not VP neurons, in late pregnancy. Although mRNA for a kisspeptin receptor, Kiss1R, was found weakly localized to the PZ–SON area, its expression was not increased during late pregnancy, suggesting an upregulation of receptor numbers does not mediate this increased excitation. Indeed, the precise target of the centrally administered kisspeptin was not determined in this study, and as Seymour et al. (2017) suggest, it remains possible that its actions on OT neurons were not mediated by the Kiss1R, but possibly by related peptide receptors, or, by amplification of the Kiss1R signaling pathway. There are other questions that await further research for answers. Is kisspeptin critical for the activation of OT neurons during late pregnancy and lactation, and particularly, do its actions influence the synchronous bursting of these neurons and the consequent pulsatile OT release? Does stimulation of these neurons in the rostral periventricular area reproduce the state-dependent effects of centrally applied kisspeptin on OT neurons, and if so, is this mediated through the PZ? Are PZ neurons directly sensitive to kisspeptin? If so, what is their neurotransmitter phenotype, and are they among the PZ neurons that directly innervate SON neurons? Despite the above caveats, the novel results of this paper demonstrate for the first time a reproductive state-related plasticity in a defined system impacting on OT neurons, the periventricular-to-PZ kisspeptin projection. Whereas previous studies over the past four decades have demonstrated multiple types of neuronal and glial plasticity related to OT neurons during pregnancy and lactation, none have indicated this type of plasticity in a specifically located population of neurons thought to regulate OT neuron activity. Seymour et al. (2017) once again train focus on the PZ as an area potentially critical for integrating signals destined to impact VP and OT release through its locally connecting neurons, whether they be GABAergic, glutamatergic, or other cell types providing neuroactive inputs to the SON.

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

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.

Electrophysiological properties of genetically-identified subtypes of layer 5 1 neocortical pyramidal neurons: Ca2+-dependence and differential modulation 2 by norepinephrine. 3 4 Running Title: Subtypes of Layer 5 Pyramidal Cells 5 6 Dongxu Guan, William E. Armstrong, and Robert C. Foehring 7 Department of Anatomy and Neurobiology 8 University of Tennessee Health Science Center 9 855 Monroe Avenue 10 Memphis, TN 38163 11 Corresponding author: 12 Robert C. Foehring 13 Department of Anatomy and Neurobiology 14 University of Tennessee Health Science Center 15 855 Monroe Avenue 16 Memphis, TN 38163 17 e-mail: rfoehrin@uthsc.edu 18 Telephone: 901-448-7450 19 Articles in PresS. J Neurophysiol (January 7, 2015). doi:10.1152/jn.00524.2014

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.

One of the first neuropeptides characterized, the hypothalamo-neurohypophysial hormone oxytocin is essential for milk ejection from the mammary gland during lactation. In response to suckling, oxytocin release is pulsatile, maximizing its ability to contract the myoepithelium, and forming the classic milk ejection reflex. Neurophysiological investigations reveal that this periodicity is underlain by the brief, synchronous bursts of oxytocin neurons in the supraoptic and paraventricular nucleus. This activity is prominent during lactation, is dependent upon the sensory stimulation of the young, is regulated by the somatodendritic release of oxytocin within these two nuclei, and is further dependent upon the neurotransmitters glutamate and noradrenaline. Extensive bilateral, intrahypothalamic communication is essential for full expression of the reflex. There is remarkable neuroplasticity at many levels of this system that begins in late pregnancy and extends during lactation to shape the system for maximal periodic release. This chapter explores the neuronal pathways essential to carry and modulate the reflex, and the neurophysiological mechanisms underlying oxytocin neuronal activation.

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.