Voltage-gated sodium (Na V 1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na V 1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na V 1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na V 1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na V 1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na V 1.7 channels, inhibiting Na V 1.7 with an IC 50 value of 0.3 nM, compared with IC 50 values of 30 to 150 nM for other heterologously expressed Na V 1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on A␤-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ( 125 I-ProTx-II) binds with high affinity (K d ϭ 0.3 nM) to recombinant hNa V 1.7 channels. Binding of 125 IProTx-II is insensitive to the presence of other well characterized Na V 1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125 I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na V 1.7-selective blockers.Pain relief remains an important, currently unmet, medical need. Voltage-gated sodium channels play a critical role in modulating the excitability of most neurons, including nociceptive sensory neurons signaling pain. Despite the clinical use of systemically administered lidocaine to treat chronic pain since the 1950s (Kugelberg and Lindblom, 1959) and the approval of the weak sodium channel blocker carbamazepine for the treatment of trigeminal neuralgia (Campbell et al., 1966), several oral sodium channel blockers have failed to show efficacy in large clinical trials (Wallace et al., 2002;Vinik et al., 2007). The failure of drugs in the clinic may at least partially be attributed to the narrow therapeutic window of non-subtype-selective sodium channel blockers.Recently, three publications have shown that loss-of-function mutations in the sodium channel subtype Na V 1.7 are the cause for channelopathy-associated insensitivity to pain (CIP) (Cox et al., 2006;Ahmad et al., 2007;Goldberg et al., 2007). Furthermore, genetic linkage analysis has identified gain-of-function mutations in Na V 1.7 as the cause of inherited erythromelalgia (Yang et al., 2004;Han et al., ...

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The nucleus tractus solitarius (NTS) receives dense terminations from cranial visceral afferents

The hypothalamus coordinates autonomic responses in part through arginine vasopressin (AVP) released in medial nucleus tractus solitarius (NTS).

Cranial visceral afferents activate central pathways that mediate systemic homeostatic processes. Afferent information arrives in the brainstem nucleus of the solitary tract (NTS) and is relayed to other CNS sites for integration into autonomic responses and complex behaviors. Little is known about the organization or nature of processing within NTS. We injected fluorescent retrograde tracers into two nuclei to identify neurons that project to sites involved in autonomic regulation: the caudal ventrolateral medulla (CVLM) or paraventricular nucleus of the hypothalamus (PVN). We found distinct differences in synaptic connections and performance in the afferent path through NTS to these neurons. Anatomical studies using confocal and electron microscopy found prominent, primary afferent synapses directly on somata and dendrites of CVLM-projecting NTS neurons identifying them as second-order neurons. In brainstem slices, afferent activation evoked large, constant latency EPSCs in CVLM-projecting NTS neurons that were consistent with the precise timing and rare failures of monosynaptic contacts on second-order neurons. In contrast, most PVN-projecting NTS neurons lacked direct afferent input and responded to afferent stimuli with highly variable, intermittently failing synaptic responses, indicating polysynaptic pathways to higher-order neurons. The afferent-evoked EPSCs in most PVN-projecting NTS neurons were smaller and unreliable but also often included multiple, convergent polysynaptic responses not observed in CVLM-projecting neurons. A few PVN-projecting NTS neurons had monosynaptic EPSC characteristics. Together, we found that cranial visceral afferent pathways are structured distinctly within NTS depending on the projection target. Such, intra-NTS pathway architecture will substantially impact performance of autonomic or neuroendocrine reflex arcs.

Although the central terminals of cranial visceral afferents express vanilloid receptor 1 (VR1), little is known about their functional properties at this first synapse within the nucleus tractus solitarius (NTS). Here, we examined whether VR1 modulates afferent synaptic transmission. In horizontal brainstem slices, solitary tract (ST) activation evoked EPSCs. Monosynaptic EPSCs had low synaptic jitter (SD of latency to successive shocks) averaging 84.03 +/- 3.74 microsec (n = 72) and were completely blocked by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX). Sustained exposure to the VR1 agonist capsaicin (CAP; 100 nm) blocked ST EPSCs (CAP-sensitive) in some neurons but not others (CAP-resistant). CAP-sensitive EPSCs had longer latencies than CAP-resistant EPSCs (4.65 +/- 0.27 msec, n = 48 vs 3.53 +/- 0.28 msec, n = 24, respectively; p = 0.011), but they had similar jitter. CAP evoked two transient responses in CAP-sensitive neurons: a rapidly developing inward current (I(cap)) (108.1 +/- 22.9 pA; n = 21) and an increase in spontaneous synaptic activity. After 3-5 min in CAP, I(cap) subsided and ST EPSCs disappeared. NBQX completely blocked I(cap). The VR1 antagonist capsazepine (10-20 microm) attenuated CAP responses. Anatomically, second-order NTS neurons were identified by 4-(4-dihexadecylamino)styryl)-N-methylpyridinium iodide transported from the cervical aortic depressor nerve (ADN) to stain central terminals. Neurons with fluorescent ADN contacts had CAP-sensitive EPSCs (n = 5) with latencies and jitter similar to those of unlabeled monosynaptic neurons. Thus, consistent with presynaptic VR1 localization, CAP selectively activates a subset of ST axons to release glutamate that acts on non-NMDA receptors. Because the CAP sensitivity of cranial afferents is exclusively associated with unmyelinated axons, VR1 identifies C-fiber afferent pathways within the brainstem.

. Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS). GABAergic neurons are scattered throughout the NTS, but their relation to solitary tract (ST) afferent pathways is imprecisely known. We hypothesized that most GABAergic NTS neurons would be connected only indirectly to the ST. We identified GABAergic neurons in brain stem horizontal slices using transgenic mice in which enhanced green fluorescent protein (EGFP) expression was linked to glutamic acid decarboxylase expression (GAD ϩ ). Finely graded electrical shocks to ST recruit STsynchronized synaptic events with all-or-none thresholds and individual waveforms did not change with greater suprathreshold intensities-evidence consistent with initiation by single afferent axons. Most (ϳ70%) GAD ϩ neurons received ST-evoked excitatory postsynaptic currents (EPSCs) that had minimally variant latencies (jitter, SD of latency Ͻ200 s) and waveforms consistent with single, direct ST connections (i.e., monosynaptic). Increasing stimulus intensity evoked additional ST-synchronized synaptic responses with jitters Ͼ200 s including inhibitory postsynaptic currents (IPSCs), indicating indirect connections (polysynaptic). Shocks of suprathreshold intensity delivered adjacent (50 -300 m) to the ST failed to excite non-ST inputs to second-order neurons, suggesting a paucity of axons passing near to ST that connected to these neurons. Despite expectations, we found similar ST synaptic patterns in GAD ϩ and unlabeled neurons. Generally, ST information that arrived indirectly had small amplitudes (EPSCs and IPSCs) and frequency-dependent failures that reached Ͼ50% for IPSCs to bursts of stimuli. This ST afferent pathway organization is strongly use-dependent-a property that may tune signal propagation within and beyond NTS.

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