Neurons that produce kisspeptin play a critical role in reproduction. However, understanding the molecular physiology of kisspeptin neurons has been limited by the lack of an in vivo marker for those cells. Here, we report the development of a Kiss1-CreGFP knockin mouse, wherein the endogenous Kiss1 promoter directs the expression of a Cre recombinase-enhanced green fluorescent protein (GFP) fusion protein. The pattern of GFP expression in the brain of the knockin recapitulates what has been described earlier for Kiss1 in the male and female mouse, with prominent expression in the arcuate nucleus (ARC) (in both sexes) and the anteroventral periventricular nucleus (in females). Single-cell RT-PCR showed that the Kiss1 transcript is expressed in 100% of GFP-labeled cells, and the CreGFP transcript was regulated by estradiol in the same manner as the Kiss1 gene (i.e. inhibited in the ARC and induced in the anteroventral periventricular nucleus). We used this mouse to evaluate the biophysical properties of kisspeptin (Kiss1) neurons in the ARC of the female mouse. GFP-expressing Kiss1 neurons were identified in hypothalamic slice preparations of the ARC and patch clamped. Whole-cell (and loose attached) recordings revealed that Kiss1 neurons exhibit spontaneous activity and expressed both h- (pacemaker) and T-type calcium currents, and hyperpolarization-activated cyclic nucleotide-regulated 1-4 and CaV3.1 channel subtypes (measured by single cell RT-PCR), respectively. N-methyl-D-aspartate induced bursting activity, characterized by depolarizing/hyperpolarizing oscillations. Therefore, Kiss1 neurons in the ARC share molecular and electrophysiological properties of other CNS pacemaker neurons.

Burst firing of neurons optimizes neurotransmitter release. GnRH neurons exhibit burst firing activity and T-type calcium channels, which are vital for burst firing activity, are regulated by 17β-estradiol (E2) in GnRH neurons. To further elucidate ion channel expression and E2 regulation during positive and negative feedback on GnRH neurosecretion, we used single cell RT-PCR and real-time qPCR to quantify channel mRNA expression in GnRH neurons. GFP-GnRH neurons expressed numerous ion channels important for burst firing activity. E2-treatment sufficient to induce an LH surge increased mRNA expression of HCN1 channels, which underlie the pacemaker current, the calcium-permeable CaV1.3, CaV2.2, CaV2.3 channels, and TRPC4 channels, which mediate the kisspeptin excitatory response. E2 also decreased mRNA expression of SK3 channels underlying the medium AHP current. Therefore, E2 exerts fundamental changes in ion channel expression in GnRH neurons, to prime them to respond to incoming stimuli with increased excitability at the time of the surge.

Trigeminal afferents convey nociceptive information from the corneal surface of the eye to trigeminal subnucleus caudalis (Vc). Trigeminal afferents, like other nociceptors, are thought to use glutamate and neuropeptides as neurotransmitters. The current studies examined whether corneal afferents contain both neuropeptides and vesicular glutamate transporters. Corneal afferents to Vc were identified using Cholera Toxin B (CTb). Corneal afferents project in two clusters to the rostral and caudal borders of Vc; regions that contain functionally distinct nociceptive neurons. Thus, corneal afferents projecting to these two regions were examined separately. Dual immunocytochemical studies combined CTb with either calcitonin gene-related peptide (CGRP), substance P (SP), vesicular glutamate transporter 1 (VGluT1) or VGluT2. Corneal afferents were more likely to contain CGRP than SP, and corneal afferents projecting to the rostral region were more likely to contain CGRP than afferents projecting caudally. Overall, corneal afferents were equally likely to contain VGluT1 or VGluT2. Together, 61% of corneal afferents contained either VGluT1 or VGluT2, suggesting that some afferents lack a vesicular glutamate transporter. Caudal corneal afferents were more likely to contain VGluT2 than VGluT1; while rostral corneal afferents were more likely to contain VGluT1 than VGluT2. Triple labeling studies combining CTb, CGRP and VGluT2 showed very few corneal afferents contain both CGRP and VGluT2; caudally (1%) and rostrally (2%). These results suggest that most corneal afferents contain a peptide or a VGluT, but rarely both. Our results are consistent with a growing literature suggesting that glutamatergic and peptidergic sensory afferents may be distinct populations.

. Molecular mechanisms that drive estradiol-dependent burst firing of Kiss1 neurons in the rostral periventricular preoptic area. Am J Physiol Endocrinol Metab 305: E1384 -E1397, 2013. First published October 8, 2013 doi:10.1152/ajpendo.00406.2013 neurons in the rostral periventricular area of the third ventricle (RP3V) provide excitatory drive to gonadotropin-releasing hormone (GnRH) neurons to control fertility. Using whole cell patch clamp recording and single-cell (sc)RT-PCR techniques targeting Kiss1-CreGFP or tyrosine hydroxylase (TH)-EGFP neurons, we characterized the biophysical properties of these neurons and identified the critical intrinsic properties required for burst firing in 17␤-estradiol (E 2)-treated, ovariectomized female mice. One-fourth of the RP3V Kiss1 neurons exhibited spontaneous burst firing. RP3V Kiss1 neurons expressed a hyperpolarization-activated h-current (I h) and a T-type calcium current (IT), which supported hyperpolarization-induced rebound burst firing. Under voltage clamp conditions, all Kiss1 neurons expressed a kinetically fast Ih that was augmented 3.4-fold by high (LH surgeproducing)-E2 treatment. scPCR analysis of Kiss1 neurons revealed abundant expression of the HCN1 channel transcripts. Kiss1 neurons also expressed a Ni 2ϩ -and TTA-P2-sensitive IT that was augmented sixfold with high-E2 treatment. CaV3.1 mRNA was also highly expressed in these cells. Current clamp analysis revealed that rebound burst firing was induced in RP3V Kiss1 neurons in high-E2-treated animals, and the majority of Kiss1 neurons had a hyperpolarization threshold of Ϫ84.7 mV, which corresponded to the V½ for IT deinactivation. Finally, Kiss1 neurons in the RP3V were hyperpolarized by -and -opioid and GABA B receptor agonists, suggesting that these pathways also contribute to rebound burst firing. Therefore, Kiss1 neurons in the RP3V express the critical channels and receptors that permit E2-dependent rebound burst firing and provide the biophysical substrate that drives the preovulatory surge of GnRH. RP3V; kisspeptin; burst firing; pacemaker current; T-type calcium channel KISSPEPTIN (KISS1) NEURONS in the the rostral periventricular area of the third ventricle (RP3V) project to GnRH neurons in the preoptic area (POA) (7) and have been identified as excitatory afferents to the gonadotropin-releasing hormone (GnRH) neurons (26). Kisspeptin is one of the most potent agonists and induces sustained firing in GnRH neurons (18,39,56). Although cell-attached recordings of firing rates of Kiss1 neurons in the RP3V have been reported (9, 11), there have been few studies characterizing the biophysical properties and molecular signature of these cells. A recent report documented the expression of an h-current (I h ) in RP3V neurons (40), but the underlying kinetic properties and channels intrinsic to these unique cells have not been characterized.Single-action potential-generated calcium influx is sufficient to spark the release of classical neurotransmitters; however, burst firing or tetanic stimulation i...

Electrophysiological devices are critical for mapping eloquent and diseased brain regions and for therapeutic neuromodulation in clinical settings and are extensively used for research in brain-machine interfaces. However, the existing clinical and experimental devices are often limited in either spatial resolution or cortical coverage. Here, we developed scalable manufacturing processes with a dense electrical connection scheme to achieve reconfigurable thin-film, multithousand-channel neurophysiological recording grids using platinum nanorods (PtNRGrids). With PtNRGrids, we have achieved a multithousand-channel array of small (30 μm) contacts with low impedance, providing high spatial and temporal resolution over a large cortical area. We demonstrated that PtNRGrids can resolve submillimeter functional organization of the barrel cortex in anesthetized rats that captured the tissue structure. In the clinical setting, PtNRGrids resolved fine, complex temporal dynamics from the cortical surface in an awake human patient performing grasping tasks. In addition, the PtNRGrids identified the spatial spread and dynamics of epileptic discharges in a patient undergoing epilepsy surgery at 1-mm spatial resolution, including activity induced by direct electrical stimulation. Collectively, these findings demonstrated the power of the PtNRGrids to transform clinical mapping and research with brain-machine interfaces.