5-HT3R-sourced calcium enhances glutamate release from a distinct vesicle pool.

The serotonin 3 receptor (5-HT3R) is a calcium-permeant channel heterogeneously expressed in solitary tract (ST) afferents. ST afferents synapse in the nucleus of the solitary tract (NTS) and rely on a mix of voltage-dependent calcium channels (CaVs) to control synchronous glutamate release (ST-EPSCs). CaV activation triggers additional, delayed release of glutamate (asynchronous EPSCs) that trails after the ST-EPSCs but only from afferents expressing the calcium-permeable, transient receptor potential vanilloid type 1 receptor (TRPV1). Most afferents express TRPV1 and have high rates of spontaneous glutamate release (sEPSCs) that is independent of CaVs. Here, we tested whether 5-HT3R-sourced calcium contributes to these different forms of glutamate release in horizontal NTS slices from rats. The 5-HT3R selective agonist, m-chlorophenyl biguanide hydrochloride (PBG), enhanced sEPSCs and/or delayed the arrival times of ST-EPSCs (i.e. increased latency). The specific 5-HT3R antagonist, ondansetron, attenuated these effects consistent with direct activation of 5-HT3Rs. PBG did not alter ST-EPSC amplitude or asynchronous EPSCs. These independent actions suggest two distinct 5-HT3R locations; axonal expression that impedes conduction and terminal expression that mobilizes a spontaneous vesicle pool. Calcium chelation with EGTA-AM attenuated the frequency of 5-HT3R-activated sEPSCs by half. The mixture of chelation-sensitive and resistant sEPSCs suggests that 5-HT3R-activated vesicles span calcium diffusion distances that are both distal (micro-) and proximal (nanodomains) to the channel. Our results demonstrate that the calcium domains of 5-HT3Rs do not overlap other calcium sources or their respective vesicle pools. 5-HT3Rs add a unique calcium source on ST afferents as part of multiple independent synaptic signaling mechanisms.

Vasopressin inhibits glutamate release via two distinct modes in the brainstem.

The hypothalamus coordinates autonomic responses in part through arginine vasopressin (AVP) released in medial nucleus tractus solitarius (NTS). However, the mechanisms and sites of AVP action within NTS pathways are uncertain. In brainstem slices, we activated solitary tract (ST) primary afferents to release glutamate and tested whether AVP modulated synaptic transmission to second-order neurons. NTS neurons were classified as second order by ST synaptic characteristics or the presence of anterograde tracers from peripheral baroreceptor afferents. Stimulus recruitment curves indicated ST-EPSCs on individual neurons were evoked by stimulation of single ST axons. Variance-mean (V-M) analysis of ST-EPSCs in individual neurons revealed uniformly high release probability (p approximately 0.9) from an average of 19 release sites (N) and a quantal size (q) of 34.0 +/- 4.7 pA. In 26 of 49 neurons, AVP inhibited afferent synaptic transmission. In most neurons, AVP reduced ST-EPSC amplitudes (n = 20) by decreasing p to 0.65, whereas q, N, and conduction times were unaffected. The V1a antagonist SR49059 alone decreased ST-EPSC V and increased M, suggesting tonic AVP actions, and blocked exogenous AVP action (n = 4). In other neurons with identical ST release properties, AVP induced synaptic failures and increased conduction time without altering the V-M relationship of successful ST-EPSCs (n = 6). Interestingly, frequency-depressed ST-EPSCs were not affected by AVP. AVP failed to alter holding or voltage-dependent potassium currents. Thus, AVP regulates NTS neurons by two distinct novel and state-dependent mechanisms: one, an analog, graded presynaptic inhibition of terminal glutamate release and the other, a binary, extraterminal block of conducted excitation.

Proopiomelanocortin neurons in nucleus tractus solitarius are activated by visceral afferents: regulation by cholecystokinin and opioids.

The nucleus tractus solitarius (NTS) receives dense terminations from cranial visceral afferents, including those from the gastrointestinal (GI) system. Although the NTS integrates peripheral satiety signals and relays this signal to central feeding centers, little is known about which NTS neurons are involved or what mechanisms are responsible. Proopiomelanocortin (POMC) neurons are good candidates for GI integration, because disruption of the POMC gene leads to severe obesity and hyperphagia. Here, we used POMC-enhanced green fluorescent protein (EGFP) transgenic mice to identify NTS POMC neurons. Intraperitoneal administration of cholecystokinin (CCK) induced c-fos gene expression in NTS POMC-EGFP neurons, suggesting that they are activated by afferents stimulated by the satiety hormone. We tested the synaptic relationship of these neurons to visceral afferents and their modulation by CCK and opioids using patch recordings in horizontal brain slices. Electrical activation of the solitary tract (ST) evoked EPSCs in NTS POMC-EGFP neurons. The invariant latencies, low failure rates, and substantial paired-pulse depression of the ST-evoked EPSCs indicate that NTS POMC-EGFP neurons are second-order neurons directly contacted by afferent terminals. The EPSCs were blocked by the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline. CCK increased the amplitude of the ST-stimulated EPSCs and the frequency of miniature EPSCs, effects attenuated by the CCK1 receptor antagonist lorglumide. In contrast, the orexigenic opioid agonists [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin and met-enkephalin inhibited both ST-stimulated EPSCs and the frequency of miniature EPSCs. These findings identify a potential satiety pathway in which visceral afferents directly activate NTS POMC-EGFP neurons with excitatory inputs that are appropriately modulated by appetite regulators.

Strategies for cellular identification in nucleus tractus solitarius slices.

The indistinct regional anatomy and intermixing of second order neurons with projection and interneurons make cellular studies more difficult within the nucleus tractus solitarius (NTS). Here, we outline experimental strategies to join in vitro electrophysiological with neuroanatomical protocols to discriminate specific subpopulations of NTS neurons. Horizontally cutting the brain stem produces slices in which electrical activation of the solitary tract (ST) is free of local interneuron contamination. Such ST excitatory synaptic currents (EPSCs) functionally identify second order NTS neurons by their minimal variation of latency (jitter). Sapphire blades, cold cutting temperatures and a mechanically stable microtome were critical to consistently obtain viable slices that were optimized for infrared and fluorescence microscopy. Anterogradely transported carbocyanine dye implanted on the aortic depressor nerve anatomically identified second order NTS neurons and their ST synaptic performance conformed to the minimal jitter signature of second order neurons. Retrograde tracers and green fluorescent protein labeled neurons afford two additional promising approaches for discriminating NTS neuron phenotypes in broader system contexts. Detailed methods and troubleshooting are described. Coupling tracing techniques with electrophysiology adds important new dimensions to NTS studies and such strategies provide bridging information between cellular mechanisms, neuroanatomy and systems integration.

Ketamine differentially blocks sensory afferent synaptic transmission in medial nucleus tractus solitarius (mNTS).

BACKGROUND: Ketamine increases blood pressure and heart rate by unknown mechanisms, but studies suggest that an intact central nervous system and arterial baroreceptors are required. In the brain stem, medial nucleus tractus solitarius receives afferents from nodose neurons that initiate cardiovascular autonomic reflexes. Here, the authors assessed ketamine actions on afferent medial nucleus tractus solitarius synaptic transmission. METHODS: Ketamine was applied to horizontally sliced brain stems. Solitary tract (ST) stimulation evoked excitatory postsynaptic currents (eEPSCs) in medial nucleus tractus solitarius neurons. Capsaicin (200 nm) block of ST eEPSCs sorted neurons into sensitive (n = 19) and resistant (n = 23). In nodose ganglion slices, shocks to the peripheral vagal trunk activated afferent action potentials in sensory neurons classified by conduction velocities and capsaicin. RESULTS: Ketamine potently (10-100 mciro m) blocked small, ST-evoked -methyl-d-aspartate synaptic currents found only in a subset of capsaicin-resistant neurons (6 of 12). Surprisingly, ketamine reversibly inhibited ST eEPSC amplitudes and induced synaptic failure at lower concentrations in capsaicin-sensitive than in capsaicin-resistant neurons (P < 0.005; n = 11 and 11). Spontaneous EPSCs using non- -methyl-d-aspartate receptors were insensitive even to 1-3 mm ketamine, suggesting that ST responses were blocked presynaptically. Similarly, ketamine blocked C-type action potential conduction at lower concentrations than A-type nodose sensory neurons. CONCLUSION: The authors conclude that ketamine inhibits postsynaptic -methyl-d-aspartate receptors and presynaptic afferent processes in medial nucleus tractus solitarius. Unexpectedly, capsaicin-sensitive (C-type), unmyelinated afferents are significantly more susceptible to block than capsaicin-resistant (A-type), myelinated afferents. This differentiation may be related to tetrodotoxin-resistant sodium currents. Since C-type afferents mediate powerful arterial baroreflexes effects, these differential actions may contribute to ketamine-induced cardiovascular dysfunction.

Vanilloid receptors presynaptically modulate cranial visceral afferent synaptic transmission in nucleus tractus solitarius.

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.

Vanilloid-sensitive afferents activate neurons with prominent A-type potassium currents in nucleus tractus solitarius.

Cranial visceral afferents innervate second-order nucleus tractus solitarius (NTS) neurons via myelinated (A-type) and unmyelinated (C-type) axons in the solitary tract (ST). A- and C-type afferents often evoke reflexes with distinct performance differences, especially with regard to their frequency-dependent properties. In horizontal brainstem slices, we used the vanilloid receptor 1 agonist capsaicin (CAP; 100 nm) to identify CAP-sensitive and CAP-resistant ST afferent pathways to second-order NTS neurons and tested whether these two groups of neurons had similar intrinsic potassium currents. ST stimulation evoked monosynaptic EPSCs identified by minimal synaptic jitter (<150 microsec) and divided into two groups: CAP-sensitive (n = 37) and CAP-resistant (n = 22). EPSCs in CAP-sensitive neurons had longer latencies (5.1 +/- 0.3 vs 3.6 +/- 0.3 msec; p = 0.001) but similar jitter (p = 0.57) compared with CAP-resistant neurons, respectively. Transient outward currents (TOCs) were significantly greater in CAP-sensitive than in CAP-resistant neurons. Steady-state currents were similar in both groups. 4-Aminopyridine or depolarized conditioning blocked the TOC, but tetraethylammonium had no effect. Voltage-dependent activation and inactivation of TOC were consistent with an A-type K+ current, I(KA). In current clamp, the activation of I(KA) reduced neuronal excitability and action potential responses to ST transmission. Our results suggest that the potassium-channel differences of second-order NTS neurons contribute to the differential processing of A- and C-type cranial visceral afferents beginning as early as this first central neuron. I(KA) can act as a frequency transmission filter and may represent a key target for the modulation of temporal processing of reflex responsiveness such as within the baroreflex arc.