Jessica A. Fawley

Primary afferent activation of thermosensitive TRPV1 triggers asynchronous glutamate release at central neurons.

TRPV1 receptors feature prominently in nociception of spinal primary afferents but are also expressed in unmyelinated cranial visceral primary afferents linked to homeostatic regulation. Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS) to control the heart, lungs, and other vital organs. Here we identify a role for central TRPV1 in the activity-dependent facilitation of glutamatergic transmission from solitary tract (ST) afferents. Fast, synchronous ST-NTS transmission from capsaicin-sensitive (TRPV1+) and -insensitive (TRPV1-) afferents was similar. However, afferent activation triggered long-lasting asynchronous glutamate release only from TRPV1+ synapses. Asynchronous release was proportional to synchronous EPSC amplitude, activity, and calcium entry. TRPV1 antagonists and low temperature blocked asynchronous release, but not evoked EPSCs. At physiological afferent frequencies, asynchronous release strongly potentiated the duration of postsynaptic spiking. This activity-dependent TPRV1-mediated facilitation is a form of synaptic plasticity that brings a unique central integrative feature to the CNS and autonomic regulation.

Thermally Active TRPV1 Tonically Drives Central Spontaneous Glutamate Release

Central synapses spontaneously release neurotransmitter at low rates. In the brainstem, cranial visceral afferent terminals in caudal solitary tract nucleus (NTS) display pronounced, activity-dependent, asynchronous release of glutamate and this extra release depends on TRPV1 receptors (TRPV1+). Asynchronous release is absent for afferents lacking TRPV1 (TRPV1−) and resting EPSC frequency was greater in TRPV1+. Here, we studied this basal activity difference by assessing thermal sensitivity of spontaneous and miniature synaptic events in TRPV1+ and TRPV1− second-order NTS neurons. The spontaneous EPSC rate decreased when temperature was decreased, increased steeply between 30 and 42°C only in TRPV1+ neurons, and was calcium-dependent. TRPV1-specific antagonist SB366791, but not TTX, strongly attenuated thermal responses. Temperature changes failed to alter EPSC frequency in TRPV1− neurons. EPSC amplitudes and decay kinetics changed little with temperature. IPSCs in these second-order NTS neurons were unaltered by temperature. Such results suggest that activated, presynaptic TRPV1+ receptors trigger continuous resting release of glutamate vesicles at physiological temperatures only in capsaicin-responsive terminals. In mechanically isolated individual neurons harvested from medial NTS, increases in temperature increased the rate of glutamate release only in TRPV1+ neurons, whereas IPSC rates were unaffected. Cadmium failed to block thermal increases in glutamate release, suggesting that calcium entry through TRPV1 channels may trigger glutamate release independently of voltage-activated calcium channels. Together, our findings indicate a new form of afferent signaling in which TRPV1 channels within central terminals of peripheral afferents tonically generate glutamate release in NTS at 37°C in the absence of afferent action potentials.

Cannabinoid 1 and Transient Receptor Potential Vanilloid 1 Receptors Discretely Modulate Evoked Glutamate Separately from Spontaneous Glutamate Transmission

Action potentials trigger synaptic terminals to synchronously release vesicles, but some vesicles release spontaneously. G-protein-coupled receptors (GPCRs) can modulate both of these processes. At cranial primary afferent terminals, the GPCR cannabinoid 1 (CB1) is often coexpressed with transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel present on most afferents. Here we tested whether CB1 activation modulates synchronous, action potential-evoked (eEPSCs) and/or spontaneous (sEPSCs) EPSCs at solitary tract nucleus neurons. In rat horizontal brainstem slices, activation of solitary tract (ST) primary afferents generated ST-eEPSCs that were rapidly and reversibly inhibited from most afferents by activation of CB1 with arachidonyl-2′-chloroethylamide (ACEA) or WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate]. The CB1 antagonist/inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] blocked these responses. Despite profound depression of ST-eEPSCs during CB1 activation, sEPSCs in these same neurons were unaltered. Changes in temperature changed sEPSC frequency only from TRPV1+ afferents (i.e., thermal sEPSC responses only occurred in TRPV1+ afferents). CB1 activation failed to alter these thermal sEPSC responses. However, the endogenous arachidonate metabolite N-arachidonyldopamine (NADA) promiscuously activated both CB1 and TRPV1 receptors. NADA inhibited ST-eEPSCs while simultaneously increasing sEPSC frequency, and thermally triggered sEPSC increases in neurons with TRPV1+ afferents. We found no evidence for CB1/TRPV1 interactions suggesting independent regulation of two separate vesicle pools. Together, these data demonstrate that action potential-evoked synchronous glutamate release is modulated separately from TRPV1-mediated glutamate release despite coexistence in the same central terminations. This two-pool arrangement allows independent and opposite modulation of glutamate release by single lipid metabolites.

TRPV1 Marks Synaptic Segregation of Multiple Convergent Afferents at the Rat Medial Solitary Tract Nucleus

TRPV1 receptors are expressed on most but not all central terminals of cranial visceral afferents in the caudal solitary tract nucleus (NTS). TRPV1 is associated with unmyelinated C-fiber afferents. Both TRPV1+ and TRPV1- afferents enter NTS but their precise organization remains poorly understood. In horizontal brainstem slices, we activated solitary tract (ST) afferents and recorded ST-evoked glutamatergic excitatory synaptic currents (ST-EPSCs) under whole cell voltage clamp conditions from neurons of the medial subnucleus. Electrical shocks to the ST produced fixed latency EPSCs (jitter<200 µs) that identified direct ST afferent innervation. Graded increases in shock intensity often recruited more than one ST afferent and ST-EPSCs had consistent threshold intensity, latency to onset, and unique EPSC waveforms that characterized each unitary ST afferent contact. The TRPV1 agonist capsaicin (100 nM) blocked the evoked TRPV1+ ST-EPSCs and defined them as either TRPV1+ or TRPV1- inputs. No partial responses to capsaicin were observed so that in NTS neurons that received one or multiple (2–5) direct ST afferent inputs – all were either blocked by capsaicin or were unaltered. Since TRPV1 mediates asynchronous release following TRPV1+ ST-evoked EPSCs, we likewise found that recruiting more than one ST afferent further augmented the asynchronous response and was eliminated by capsaicin. Thus, TRPV1+ and TRPV1- afferents are completely segregated to separate NTS neurons. As a result, the TRPV1 receptor augments glutamate release only within unmyelinated afferent pathways in caudal medial NTS and our work indicates a complete separation of C-type from A-type afferent information at these first central neurons.

The unsilent majority–TRPV1 drives “spontaneous” transmission of unmyelinated primary afferents within cardiorespiratory NTS

Cranial primary afferent sensory neurons figure importantly in homeostatic control of visceral organ systems. Of the two broad classes of visceral afferents, the role of unmyelinated or C-type class remains poorly understood. This review contrasts key aspects of peripheral discharge properties of C-fiber afferents and their glutamate transmission mechanisms within the solitary tract nucleus (NTS). During normal prevailing conditions, most information arrives at the NTS through myelinated A-type nerves. However, most of visceral afferent axons (75-90%) in NTS are unmyelinated, C-type axons. Centrally, C-type solitary tract (ST) afferent terminals have presynaptic transient receptor potential vanilloid type 1 (TRPV1) receptors. Capsaicin activation of TRPV1 blocks phasic or synchronous release of glutamate but facilitates release of glutamate from a separate pool of vesicles. This TRPV1-operated pool of vesicles is active at normal temperatures and is responsible for actively driving a 10-fold higher release of glutamate at TRPV1 compared with TRPV1- terminals even in the absence of afferent action potentials. This novel TRPV1 mechanism is responsible for an additional asynchronous release of glutamate that is not present in myelinated terminals. The NTS is rich with presynaptic G protein-coupled receptors, and the implications of TRPV1-operated glutamate offer unique targets for signaling in C-type sensory afferent terminals from neuropeptides, inflammatory mediators, lipid metabolites, cytokines, and cannabinoids. From a homeostatic view, this combination could have broad implications for integration in chronic pathological disturbances in which the numeric dominance of C-type endings and TRPV1 would broadly disturb multisystem control mechanisms.

GABA B -mediated inhibition of multiple modes of glutamate release in the nucleus of the solitary tract

In the caudal portions of the solitary tract (ST) nucleus, primary sensory afferents fall into two broad classes based on the expression of transient receptor potential vanilloid type 1 (TRPV1) receptors. Both afferent classes (TRPV1+/-) have indistinguishable glutamate release mechanisms for ST-evoked excitatory postsynaptic currents (EPSCs). However, TRPV1+ terminals release additional glutamate from a unique, TRPV1-operated vesicle pool that is temperature sensitive and facilitated by ST activity to generate asynchronous EPSCs. This study tested whether presynaptic γ-aminobutyric acid (GABA)(B) receptors inhibit both the evoked and TRPV1-operated release mechanisms on second-order ST nucleus neurons. In horizontal slices, shocks activated single ST axons and evoked the time-invariant (latency jitter <200 μs), glutamatergic EPSCs, which identified second-order neurons. Gabazine eliminated GABA(A) responses in all recordings. The GABA(B) agonist baclofen inhibited the amplitude of ST-EPSCs from both TRPV1+ and TRPV1- afferents with a similar EC(50) (∼1.2 μM). In TTX, GABA(B) activation decreased miniature EPSC (mEPSC) rates but not amplitudes, suggesting presynaptic actions downstream from terminal excitability. With calcium entry through voltage-activated calcium channels blocked by cadmium, baclofen reduced mEPSC frequency, indicating that GABA(B) reduced vesicle release by TRPV1-dependent calcium entry. GABA(B) activation also reduced temperature-evoked increases in mEPSC frequency, which relies on TRPV1. Our studies indicate that GABA(B) G protein-coupled receptors are uniformly distributed across all ST primary afferent terminals and act at multiple stages of the excitation-release cascades to suppress both action potential-triggered and TRPV1-coupled glutamate transmission pathways. Moreover, the segregated release cascades within TRPV1+ ST primary afferents represent independent, potential targets for differential modulation.

Peptide and Lipid Modulation of Glutamatergic Afferent Synaptic Transmission in the Solitary Tract Nucleus

The brainstem nucleus of the solitary tract (NTS) holds the first central neurons in major homeostatic reflex pathways. These homeostatic reflexes regulate and coordinate multiple organ systems from gastrointestinal to cardiopulmonary functions. The core of many of these pathways arise from cranial visceral afferent neurons that enter the brain as the solitary tract (ST) with more than two-thirds arising from the gastrointestinal system. About one quarter of ST afferents have myelinated axons but the majority are classed as unmyelinated C-fibers. All ST afferents release the fast neurotransmitter glutamate with remarkably similar, high-probability release characteristics. Second order NTS neurons receive surprisingly limited primary afferent information with one or two individual inputs converging on single second order NTS neurons. A- and C-fiber afferents never mix at NTS second order neurons. Many transmitters modify the basic glutamatergic excitatory postsynaptic current often by reducing glutamate release or interrupting terminal depolarization. Thus, a distinguishing feature of ST transmission is presynaptic expression of G-protein coupled receptors for peptides common to peripheral or forebrain (e.g., hypothalamus) neuron sources. Presynaptic receptors for angiotensin (AT1), vasopressin (V1a), oxytocin, opioid (MOR), ghrelin (GHSR1), and cholecystokinin differentially control glutamate release on particular subsets of neurons with most other ST afferents unaffected. Lastly, lipid-like signals are transduced by two key ST presynaptic receptors, the transient receptor potential vanilloid type 1 and the cannabinoid receptor that oppositely control glutamate release. Increasing evidence suggests that peripheral nervous signaling mechanisms are repurposed at central terminals to control excitation and are major sites of signal integration of peripheral and central inputs particularly from the hypothalamus.

Distinct Calcium Sources Support Multiple Modes of Synaptic Release from Cranial Sensory Afferents

Most craniosensory afferents have unmyelinated axons expressing TRP Vanilloid 1 (TRPV1) receptors in synaptic terminals at the solitary tract nucleus (NTS). Neurotransmission from these synapses is characterized by substantial asynchronous EPSCs following action potential-synched EPSCs and high spontaneous rates that are thermally sensitive. The present studies blocked voltage-activated calcium channels (CaV) using the nonselective CaV blocker Cd2+ or the specific N-type blocker ω-conotoxin GVIA to examine the calcium dependence of the synchronous, asynchronous, spontaneous, and thermally gated modes of release. In rat brainstem slices containing caudal NTS, shocks to the solitary tract (ST) triggered synchronous ST-EPSCs and trailing asynchronous EPSCs. Cd2+ or GVIA efficiently reduced both synchronous and asynchronous EPSCs without altering spontaneous or thermal-evoked transmission. Activation of TRPV1 with either the selective agonist resiniferatoxin (150 pm) or temperature augmented basal sEPSC rates but failed to alter the synchronous or asynchronous modes of release. These data indicate that calcium sourced through TRPV1 has no access to the synchronous or asynchronous release mechanism(s) and conversely that CaV-sourced calcium does not interact with the thermally evoked mode of release. Buffering intracellular calcium with EGTA-AM or BAPTA-AM reduced asynchronous EPSC rates earlier and to a greater extent than synchronous ST-EPSC amplitudes without altering sEPSCs or thermal sensitivity. Buffering therefore distinguishes asynchronous vesicles as possessing a highly sensitive calcium sensor located perhaps more distant from CaV than synchronous vesicles or thermally evoked vesicles from TRPV1. Together, our findings suggest separate mechanisms of release for spontaneous, asynchronous and synchronous vesicles that likely reside in unique, spatially separated vesicle domains. SIGNIFICANCE STATEMENT Most craniosensory fibers release glutamate using calcium entry from two sources: CaVs and TRPV1. We demonstrate that calcium segregation distinguishes three vesicle release mechanisms. Most surprisingly, asynchronous release is associated with CaV and not TRPV1 calcium entry. This reveals that asynchronous release is an additional and separate phenotypic marker of unmyelinated afferents rather than operated by TRPV1. The functional independence of the two calcium sources expands the regulatory repertoire of transmission and imbues these inputs with additional modulation targets for synaptic release not present at conventional CaV synapses. Peptides and lipid mediators may target one or both of these calcium sources at afferent terminals within the solitary tract nucleus to independently modify release from distinct, functionally segregated vesicle pools.

External QX-314 inhibits evoked cranial primary afferent synaptic transmission independent of TRPV1

The cell-impermeant lidocaine derivative QX-314 blocks sodium channels via intracellular mechanisms. In somatosensory nociceptive neurons, open transient receptor potential vanilloid type 1 (TRPV1) receptors provide a transmembrane passageway for QX-314 to produce long-lasting analgesia. Many cranial primary afferents express TRPV1 at synapses on neurons in the nucleus of the solitary tract and caudal trigeminal nucleus (Vc). Here, we investigated whether QX-314 interrupts neurotransmission from primary afferents in rat brain-stem slices. Shocks to the solitary tract (ST) activated highly synchronous evoked excitatory postsynaptic currents (ST-EPSCs). Application of 300 μM QX-314 increased the ST-EPSC latency from TRPV1+ ST afferents, but, surprisingly, it had similar actions at TRPV1- ST afferents. Continued exposure to QX-314 blocked evoked ST-EPSCs at both afferent types. Neither the time to onset of latency changes nor the time to ST-EPSC failure differed between responses for TRPV1+ and TRPV1- inputs. Likewise, the TRPV1 antagonist capsazepine failed to prevent the actions of QX-314. Whereas QX-314 blocked ST-evoked release, the frequency and amplitude of spontaneous EPSCs remained unaltered. In neurons exposed to QX-314, intracellular current injection evoked action potentials suggesting a presynaptic site of action. QX-314 acted similarly at Vc neurons to increase latency and block EPSCs evoked from trigeminal tract afferents. Our results demonstrate that QX-314 blocked nerve conduction in cranial primary afferents without interrupting the glutamate release mechanism or generation of postsynaptic action potentials. The TRPV1 independence suggests that QX-314 either acted extracellularly or more likely entered these axons through an undetermined pathway common to all cranial primary afferents.

Temperature differentially facilitates spontaneous but not evoked glutamate release from cranial visceral primary afferents.

Temperature is fundamentally important to all biological functions including synaptic glutamate release. Vagal afferents from the solitary tract (ST) synapse on second order neurons in the nucleus of the solitary tract, and glutamate release at this first central synapse controls autonomic reflex function. Expression of the temperature-sensitive Transient Receptor Potential Vanilloid Type 1 receptor separates ST afferents into C-fibers (TRPV1+) and A-fibers (TRPV1-). Action potential-evoked glutamate release is similar between C- and A-fiber afferents, but TRPV1 expression facilitates a second form of synaptic glutamate release in C-fibers by promoting substantially more spontaneous glutamate release. The influence of temperature on different forms of glutamate release is not well understood. Here we tested how temperature impacts the generation of evoked and spontaneous release of glutamate and its relation to TRPV1 expression. In horizontal brainstem slices of rats, activation of ST primary afferents generated synchronous evoked glutamate release (ST-eEPSCs) at constant latency whose amplitude reflects the probability of evoked glutamate release. The frequency of spontaneous EPSCs in these same neurons measured the probability of spontaneous glutamate release. We measured both forms of glutamate from each neuron during ramp changes in bath temperature of 4–5°C. Spontaneous glutamate release from TRPV1+ closely tracked with these thermal changes indicating changes in the probability of spontaneous glutamate release. In the same neurons, temperature changed axon conduction registered as latency shifts but ST-eEPSC amplitudes were constant and independent of TRPV1 expression. These data indicate that TRPV1-operated glutamate release is independent of action potential-evoked glutamate release in the same neurons. Together, these support the hypothesis that evoked and spontaneous glutamate release originate from two pools of vesicles that are independently modulated and are distinct processes.