L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons.

In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca(2+)) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca(2+)]o, suggesting a critical role for both intracellular Ca(2+) release and Ca(2+) influx. TRH-induced inward current was unchanged by T-type Ca(2+) channel blockade, but was decreased by blockade of high-voltage-activated Ca(2+) channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca(2+) influx via L-type Ca(2+) channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca(2+)-induced Ca(2+) release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca(2+) influx via opening of L-type Ca(2+) channels, a sensitivity of a TRPC-like component to intracellular Ca(2+) release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component.

Novel coupling between TRPC-like and KNa channels modulates low threshold spike-induced afterpotentials in rat thalamic midline neurons.

Neurons in thalamic midline and paraventricular nuclei (PVT) display a unique slow afterhyperpolarizing potential (sAHP) following the low threshold spike (LTS) generated by activation of their low voltage Ca(2+) channels. We evaluated the conductances underlying this sAHP using whole-cell patch-clamp recordings in rat brain slice preparations. Initial observations recorded in the presence of TTX revealed a marked dependency of the LTS-induced sAHP on extracellular Na(+): replacing Na(+) with TRIS(+) in the external medium eliminated the LTS-induced sAHP; substitution of Na(+) with either Li(+) or choline(+) in the external medium resulted in a gradual loss of the sAHP and its replacement with a prolonged slow afterdepolarizing potential (sADP). The LTS-induced sAHP was reduced by quinidine and potentiated by loxapine, suggesting involvement of KNa-like channels. Canonical transient receptor potential (TRPC) channels were considered the source for Na(+) based on observations that the sAHP was suppressed by nonselective TRPC channel blockers (2-APB, flufenamic acid and ML204) but unchanged in the presence of TRPV1 channel blocker (SB-366791). In addition, after replacement of Na(+) with Li(+), the isolated LTS-induced sADP was significantly suppressed in the presence of 2-APB or ML204, after replacement of extracellular Ca(2+) with Sr(2+), and by intracellular Ca(2+) chelation with EGTA, data that collectively suggest involvement of Ca(2+)-activated TRPC-like conductances containing TRPC4/5 subunits. The isolated LTS-induced sADP also exhibited a strong voltage dependency, decreasing at hyperpolarizing potentials, further support for involvement of TRPC4/5 subunits. This sADP exhibited neurotransmitter receptor sensitivity, with suppression by 5-CT, a 5-HT7 receptor agonist, and enhancement by the neuropeptide orexin A. These data suggest that LTS-induced slow afterpotentials reflect a simultaneous interplay between KNa and TRPC-like conductances, novel for midline thalamic neurons.

Intrinsic properties and neuropharmacology of midline paraventricular thalamic nucleus neurons.

Neurons in the midline and intralaminar thalamic nuclei are components of an interconnected brainstem, limbic and prefrontal cortex neural network that is engaged during arousal, vigilance, motivated and addictive behaviors, and stress. To better understand the cellular mechanisms underlying these functions, here we review some of the recently characterized electrophysiological and neuropharmacological properties of neurons in the paraventricular thalamic nucleus (PVT), derived from whole cell patch clamp recordings in acute rat brain slice preparations. PVT neurons display firing patterns and ionic conductances (IT and IH) that exhibit significant diurnal change. Their resting membrane potential (RMP) is maintained by various ionic conductances that include inward rectifier (Kir), hyperpolarization-activated nonselective cation (HCN) and TWIK-related acid sensitive (TASK) K(+) channels. Firing patterns are regulated by high voltage-activated (HVA) and low voltage-activated (LVA) Ca(2+) conductances. Moreover, transient receptor potential (TRP)-like nonselective cation channels together with Ca(2+)- and Na(+)-activated K(+) conductances (KCa; KNa) contribute to unique slow afterhyperpolarizing potentials (sAHPs) that are generally not detectable in lateral thalamic or reticular thalamic nucleus neurons. The excitability of PVT neurons is also modulated by activation of neurotransmitter receptors associated with afferent pathways to PVT and other thalamic midline nuclei. We report on receptor-mediated actions of GABA, glutamate, monoamines and several neuropeptides: arginine vasopressin, gastrin-releasing peptide, thyrotropin releasing hormone and the orexins (hypocretins). This review represents an initial survey of intrinsic and transmitter-sensitive ionic conductances that are deemed to be unique to this population of midline thalamic neurons, information that is fundamental to an appreciation of the role these thalamic neurons may play in normal central nervous system (CNS) physiology and in CNS disorders that involve the dorsomedial thalamus.

GIRK-like and TRPC-like conductances mediate thyrotropin-releasing hormone-induced increases in excitability in thalamic paraventricular nucleus neurons.

The thalamic paraventricular nucleus (PVT), reported to participate in arousal and motivated behaviors, contains abundant receptors for thyrotropin-releasing hormone (TRH), a neuropeptide also known to modulate arousal and mood. To test the hypothesis that TRH could influence the excitability of PVT neurons, whole cell patch-clamp recordings obtained in rat brain slice preparations were evaluated during bath applied TRH. In the majority of neurons tested, TRH induced reversible TTX-resistant membrane depolarization. Under voltage-clamp, TRH induced a concentration-dependent G protein- mediated inward current. The mean net TRH-induced current exhibited a decrease in membrane conductance. Further analyses identified two concurrent conductances contributing to the TRH-induced response. One conductance featured a Na(+)-independent and K(+)-dependent net current that displayed rectification and was suppressed by micromolar concentrations of Ba(2+) and two GIRK antagonists, tertiapin Q and SCH 23390. The second conductance featured a Na(+)-dependent net inward current with an I-V relationship that exhibited double rectification with a negative slope conductance below -40 mV. This conductance was suppressed by nonselective TRPC channel blockers 2-APB, flufenamic acid and ML204, enhanced by La(3+) in a subpopulation of cells, and unchanged by the TRPV1 antagonist capsazepine or a Na(+)/Ca(2+) exchanger blocker KB-R7943. TRH also enhanced hyperpolarization-activated low threshold spikes, a feature that was sensitive to pretreatment with either 2-APB or ML204. Collectively, the data imply that TRH enhances excitability in PVT neurons via concurrently decreasing a G-protein-gated inwardly rectifying K(+) conductance and activating a cationic conductance with characteristics reminiscent of TRPC-like channels, possibly involving TRPC4/C5 subunits.

Midline thalamic paraventricular nucleus neurons display diurnal variation in resting membrane potentials, conductances, and firing patterns in vitro.

Neurons in the rodent midline thalamic paraventricular nucleus (PVT) receive inputs from brain stem and hypothalamic sites known to participate in sleep-wake and circadian rhythms. To evaluate possible diurnal changes in their excitability, we used patch-clamp techniques to record and examine the properties of neurons in anterior PVT (aPVT) in coronal rat brain slices prepared at zeitgeber time (ZT) 2-6 vs. ZT 14-18 and recorded at ZT 8.4 ± 0.2 (day) vs. ZT 21.2 ± 0.2 (night), the subjective quiet vs. aroused states, respectively. Compared with neurons recorded during the day, neurons from the night period were significantly more depolarized and exhibited a lower membrane conductance that in part reflected loss of a potassium-mediated conductance. Furthermore, these neurons were also significantly more active, with tonic and burst firing patterns. Neurons from each ZT period were assessed for amplitudes of two conductances known to contribute to bursting behavior, i.e., low-threshold-activated Ca(2+) currents (I(T)) and hyperpolarization-activated cation currents (I(h)). Data revealed that amplitudes of both I(T) and I(h) were significantly larger during the night period. In addition, biopsy samples from the night period revealed a significant increase in mRNA for Ca(v)3.1 and Ca(v)3.3 low-threshold Ca(2+) channel subtypes. Neurons recorded from the night period also displayed a comparative enhancement in spontaneous bursting at membrane potentials of approximately -60 mV and in burst firing consequent to hyperpolarization-induced low-threshold currents and depolarization-induced current pulses. These novel in vitro observations reveal that midline thalamic neurons undergo diurnal changes in their I(T), I(h), and undefined potassium conductances. The underlying mechanisms remain to be characterized.

Ca2+-dependent and Na+-dependent K+ conductances contribute to a slow AHP in thalamic paraventricular nucleus neurons: a novel target for orexin receptors.

Thalamic paraventricular nucleus (PVT) neurons exhibit a postburst apamin-resistant slow afterhyperpolarization (sAHP) that is unique to midline thalamus, displays activity dependence, and is abolished in tetrodotoxin. Analysis of the underlying sI(AHP) confirmed a requirement for Ca(2+) influx with contributions from P/Q-, N-, L-, and R subtype channels, a reversal potential near E(K)(+) and a significant reduction by UCL-2077, barium or TEA, consistent with a role for K(Ca) channels. sI(AHP) was significantly reduced by activation of either the cAMP or the protein kinase C (PKC) signaling pathway. Further analysis of the sAHP revealed an activity-dependent but Ca(2+)-independent component that was reduced in high [K(+)](o) and blockable after Na(+) substitution with Li(+) or in the presence of quinidine, suggesting a role for K(Na) channels. The Ca(2+)-independent sAHP component was selectively reduced by activation of the PKC signaling pathway. The sAHP contributed to spike frequency adaptation, which was sensitive to activation of either cAMP or PKC signaling pathways and, near the peak of membrane hyperpolarization, was sufficient to cause de-inactivation of low threshold T-Type Ca(2+) channels, thus promoting burst firing. PVT neurons are densely innervated by orexin-immunoreactive fibers, and depolarized by exogenously applied orexins. We now report that orexin A significantly reduced both Ca(2+)-dependent and -independent sI(AHP), and spike frequency adaptation. Furthermore orexin A-induced sI(AHP) inhibition was mediated through activation of PKC but not PKA. Collectively, these observations suggest that K(Ca) and K(Na) channels have a role in a sAHP that contributes to spike frequency adaptation and neuronal excitability in PVT neurons and that the sAHP is a novel target for modulation by the arousal- and feeding-promoting orexin neuropeptides.

Metabotropic glutamate receptors in median preoptic neurons modulate neuronal excitability and glutamatergic and GABAergic inputs from the subfornical organ.

Cardiovascular and behavioral responses to circulating angiotensin require intact connectivity along the upper lamina terminalis joining the subfornical organ (SFO) with the median preoptic nucleus (MnPO). In the present study on MnPO neurons, we used whole cell patch-clamp recording techniques in brain slice preparations to evaluate the influence of metabotropic glutamate receptor (mGluR) agonists on modulating their intrinsic excitability and SFO-evoked glutamatergic and GABAergic postsynaptic currents. In 22/36 cells, bath application of a mGluR group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced a TTX-resistant inward current coupled with decrease in a membrane K(+) conductance but also a possible increase in a nonselective cationic conductance. By contrast, 27/49 cells responded to a mGluR group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) with a TTX-resistant outward current and increase in membrane conductance that reversed around -95 mV, suggesting opening of K(+) channels. None of 19 cells responded to the mGluR group III agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4). Agonists for all mGluR groups suppressed SFO-evoked excitatory postsynaptic currents and significantly increased paired-pulse ratios, implying a presynaptic mechanism. Only the mGluR group II agonist significantly reduced SFO-evoked inhibitory postsynaptic currents and caused an increase in paired-pulse ratios. These results suggest a complexity of pre- and postsynaptic mGluRs are available to modulate rapid neurotransmission along the upper lamina terminalis from SFO to MnPO.

Properties of a T-type Ca2+channel-activated slow afterhyperpolarization in thalamic paraventricular nucleus and other thalamic midline neurons.

Burst firing mediated by a low-threshold spike (LTS) is the hallmark of many thalamic neurons. However, postburst afterhyperpolarizations (AHPs) are relatively uncommon in thalamus. We now report data from patch-clamp recordings in rat brain slice preparations that reveal an LTS-induced slow AHP (sAHP) in thalamic paraventricular (PVT) and other midline neurons, but not in ventrobasal or reticular thalamic neurons. The LTS-induced sAHP lasts 8.9 +/- 0.4 s and has a novel pharmacology, with resistance to tetrodotoxin and cadmium and reduction by Ni(2+) or nominally zero extracellular calcium concentration, which also attenuate both the LTS and sAHP. The sAHP is inhibited by 10 mM intracellular EGTA or by equimolar replacement of extracellular Ca(2+) with Sr(2+), consistent with select activation of LVA T-type Ca(2+) channels and subsequent Ca(2+) influx. In control media, the sAHP reverses near E(K(+)), shifting to -78 mV in 10.1 mM [K(+)](o) and is reduced by Ba(2+) or tetraethylammonium. Although these data are consistent with opening of Ca(2+)-activated K(+) channels, this sAHP lacks sensitivity to specific Ca(2+)-activated K(+) channel blockers apamin, iberiotoxin, charybdotoxin, and UCL-2077. The LTS-induced sAHP is suppressed by a beta-adrenoceptor agonist isoproterenol, a serotonin 5-HT(7) receptor agonist 5-CT, a neuropeptide orexin-A, and by stimulation of the cAMP/protein kinase A pathway with 8-Br-cAMP and forskolin. The data suggest that PVT and certain midline thalamic neurons possess an LTS-induced sAHP that is pharmacologically distinct and may be important for information transfer in thalamic-limbic circuitry during states of attentiveness and motivation.

Enhanced LTP of primary afferent neurotransmission in AMPA receptor GluR2-deficient mice.

Ca(2+)-permeable-AMPA receptors (AMPARs) are expressed in the superficial dorsal horn (SDH, laminae I/II) of the spinal cord, the area involved in transmission and modulation of sensory information, including nociception. A possible role of Ca(2+)-permeable-AMPARs in synaptic strengthening has been suggested in postnatal DH cultures, but their role in the long-lasting activity-dependent synaptic plasticity of primary afferent neurotransmission in the adult mouse SDH has not been investigated. In the present study the role of Ca(2+)-permeable-AMPARs in the regulation of long-lasting synaptic plasticity, specifically long-term potentiation (LTP) and long-term depression (LTD) in the SDH, was investigated using mice deficient in AMPAR GluR2 subunit. We show here that the GluR2 mutants exhibited no changes in passive membrane properties, but a significant increase in rectification of excitatory postsynaptic currents, the finding suggesting increased expression of Ca(2+)-permeable-AMPARs. In the absence of GluR2, high-frequency stimulation (HFS) of small-diameter primary afferent fibers induced LTP that is enhanced and non-saturating in the SDH at both primary afferent Adelta- and/or C-fibers monosynaptic and polysynaptic pathways, whereas neuronal excitability and paired-pulse depression were normal. The LTP could be induced in the presence of the NMDA receptor antagonist d-AP5, and L-type Ca(2+) channel blockers, suggesting that Ca(2+)-permeable-AMPARs are sufficient to induce LTP in the SDH neurons of adult mouse spinal cord. In contrast, the induction of HFS-LTD is reduced in the SDH of GluR2 mutants. These results suggest an important role for AMPAR GluR2 subunit in regulating synaptic plasticity with potential relevance for long-lasting hypersensitivity in pathological states.

Presynaptic alpha-adrenoceptors in median preoptic nucleus modulate inhibitory neurotransmission from subfornical organ and organum vasculosum lamina terminalis.

The median preoptic nucleus (MnPO) in the lamina terminalis receives a prominent catecholaminergic innervation from the dorsomedial and ventrolateral medulla. The present investigation used whole cell patch-clamp recordings in rat brain slice preparations to evaluate the hypothesis that presynaptic adrenoceptors could modulate GABAergic inputs to MnPO neurons. Bath applications of norepinephrine (NE; 20-50 microM) induced a prolonged and reversible suppression of inhibitory postsynaptic currents (IPSCs) and reduced paired-pulse depression evoked by stimulation in the subfornical organ and organum vasculosum lamina terminalis. These events were not correlated with any observed changes in membrane conductance arising from NE activity at postsynaptic alpha(1)- or alpha(2)-adrenoceptors. Consistent with a role for presynaptic alpha(2)-adrenoceptors, responses were selectively mimicked by an alpha(2)-adrenoceptor agonist (UK-14304) and blockable with an alpha(2)-adrenoceptor antagonist (idazoxan). Although the alpha(1)-adrenoceptor agonist cirazoline and the alpha(1)-adrenoceptor antagonist prazosin were without effect on these evoked IPSCs, NE was noted to increase (via alpha(1)-adrenoceptors) or decrease (via alpha(2)-adrenoceptors) the frequency of spontaneous and tetrodotoxin-resistant miniature IPSCs. Collectively, these observations imply that both presynaptic and postsynaptic alpha(1)- and alpha(2)-adrenoceptors in MnPO are capable of selective modulation of rapid GABA(A) receptor-mediated inhibitory synaptic transmission along the lamina terminalis and therefore likely to exert a prominent influence in regulating cell excitability within the MnPO.

Heterogeneity in low voltage-activated Ca2+ channel-evoked Ca2+ responses within neurons of the thalamic paraventricular nucleus.

Low voltage-activated Ca2+ channels (LVA or T-type Ca2+ channels) are crucial to burst firing and oscillations in thalamocortical relay cells and are exhibited by neurons in the paraventricular nucleus of thalamus (PVT), a dorsal midline nucleus deemed important in the neural representation of motivational behaviours. We used a functional approach (whole-cell patch-clamp electrophysiology combined with confocal laser scanning microscopy) to analyse the spatial distribution of LVA Ca2+ channel-evoked Ca2+ transients in PVT neurons. We observed that the magnitude of LVA Ca2+ channel-evoked Ca2+ transients was significantly greater in proximal dendrites (located up to 20 microm from the soma) than in the soma. In addition, the magnitudes of these Ca2+ transients varied significantly not only among different dendrites of the same cell but also within individual dendrites. These findings suggest that LVA Ca2+ channels are expressed (i) predominantly on the proximal dendrites and (ii) heterogeneously within individual dendrites of PVT neurons. The spatial characteristics of dendritic LVA Ca2+ channels in PVT neurons suggest that these channels may regulate burst firing by modulating dendritic afferent inputs.

Low voltage-activated Ca2+ channels are coupled to Ca2+-induced Ca2+ release in rat thalamic midline neurons.

High voltage-activated Ca2+ channels are coupled to the release of Ca2+ from intracellular stores. Here we present evidence that, in the paraventricular thalamic nucleus and other midline thalamic nuclei, activation of low voltage-activated (LVA) Ca2+ channels stimulates Ca2+-induced Ca2+ release (CICR) from intracellular stores. Voltage-clamp activation of LVA Ca2+ channels in fluo-4 AM-loaded neurons induced an initial transient increase in intracellular Ca2+ concentrations ([Ca2+]i) (mean increase, 19.4%; decay time constant, 71 ms) that reflected the entry of extracellular Ca2+. This was followed by a sustained secondary elevation in [Ca2+]i (mean increase, 4.7%; decay time constant, 7310 ms) that was attributable to CICR. Repeated activation of LVA Ca2+ channels to evoke CICR caused a progressive buildup of baseline [Ca2+]i (mean increase, 13.12 +/- 3.41%) that was reduced by depletion of intracellular Ca2+ stores with thapsigargin or caffeine. In contrast, LVA Ca2+ channel-evoked CICR was absent from ventrolateral thalamocortical relay neurons, suggesting that LVA Ca2+ channel coupling to Ca2+-dependent intracellular signaling may be a property that is unique to nonspecific and midline thalamocortical neurons.

GABAB receptor modulation of rapid inhibitory and excitatory neurotransmission from subfornical organ and other afferents to median preoptic nucleus neurons.

Cardiovascular and behavioral responses to circulating angiotensin require intact connectivity along the upper lamina terminalis joining the subfornical organ (SFO) with the median preoptic nucleus (MnPO). Whole cell patch-clamp recordings in sagittal rat brain slice preparations revealed that 28/40 MnPO neurons responded to electrical stimulation of SFO efferents with bicuculline-sensitive GABA(A) receptor-mediated inhibition and glutamate-mediated postsynaptic excitation involving AMPA and N-methyl-d-aspartate (NMDA) receptor subtypes, blockable with 2,3-dioxo-6nitro-1, 2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfoamide disodium (NBQX) and d-2-amino-4-phosphonovaleric acid (d-APV), respectively. Bath applications of baclofen induced a concentration-dependent (0.3-10 microM) reduction in these SFO-evoked postsynaptic currents, attenuation of SFO-evoked paired-pulse depression, and reduction in frequency (but not amplitude) of miniature postsynaptic currents, consistent with an action at presynaptic GABA(B) receptors. Baclofen's effects on miniature currents lacked sensitivity to barium, omega-conotoxin GVIA, and cadmium. Acting at postsynaptic GABA(B) receptors, baclofen hyperpolarized a majority of MnPO neurons by increasing a G protein-coupled inwardly rectifying potassium conductance and suppressing an N-type high-voltage-activated calcium conductance. The latter contributed to reduction in action potential afterhyperpolarization and enhanced cell firing and spike frequency adaptation when tested with a depolarizing stimulus. All baclofen-induced effects were blockable with CGP52432. CGP52432 alone had no significant effect on SFO-evoked postsynaptic current amplitudes or paired-pulse ratios, but did induce an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in 2/4 cells tested, indicating that ambient levels of GABA could activate presynaptic GABA(B) receptors on undefined inputs. These observations indicate that MnPO neurons receive both a GABAergic and glutamatergic innervation from SFO. Both forms of rapid neurotransmission are subject to modulation via pre- and postsynaptic GABA(B) receptors.