Astrocytic glutamate transporters reduce the neuronal and physiological influence of metabotropic glutamate receptors in nucleus tractus solitarii

Astrocytic excitatory amino acid transporters (EAATs) are critical to restraining synaptic and neuronal activity in the nucleus tractus solitarii (nTS). Relief of nTS EAAT restraint generates two opposing effects, an increase in neuronal excitability that reduces blood pressure and breathing and an attenuation in afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. Although the former is due, in part, to activation of ionotropic glutamate receptors, there remains a substantial contribution from another unidentified glutamate receptor. In addition, the mechanism(s) by which EAAT inhibition reduced TS-EPSC amplitude is unknown. Metabotropic glutamate receptors (mGluRs) differentially modulate nTS excitability. Activation of group I mGluRs on nTS neuron somas leads to depolarization, whereas group II/III mGluRs on sensory afferents decrease TS-EPSC amplitude. Thus we hypothesize that EAATs control postsynaptic excitability and TS-EPSC amplitude via restraint of mGluR activation. To test this hypothesis, we used in vivo recording, brain slice electrophysiology, and imaging of glutamate release and TS-afferent Ca2+. Results show that EAAT blockade in the nTS with (3 S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) induced group I mGluR-mediated depressor, bradycardic, and apneic responses that were accompanied by neuronal depolarization, elevated discharge, and increased spontaneous synaptic activity. Conversely, upon TS stimulation TFB-TBOA elevated extracellular glutamate to decrease presynaptic Ca2+ and TS-EPSC amplitude via activation of group II/III mGluRs. Together, these data suggest an important role of EAATs in restraining mGluR activation and overall cardiorespiratory function.

TRPV1 channels contribute to spontaneous glutamate release in nucleus tractus solitarii following chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) reduces afferent-evoked excitatory postsynaptic currents (EPSCs) but enhances basal spontaneous (s) and asynchronous (a) EPSCs in second-order neurons of nucleus tractus solitarii (nTS), a major area for cardiorespiratory control. The net result is an increase in synaptic transmission. The mechanisms by which this occurs are unknown. The N-type calcium channel and transient receptor potential cation channel TRPV1 play prominent roles in nTS sEPSCs and aEPSCs. The functional role of these channels in CIH-mediated afferent-evoked EPSC, sEPSC, and aEPSC was tested in rat nTS slices following antagonist inhibition and in mouse nTS slices that lack TRPV1. Block of N-type channels decreased aEPSCs in normoxic and, to a lesser extent, CIH-exposed rats. sEPSCs examined in the presence of TTX (miniature EPSCs) were also decreased by N-type block in normoxic but not CIH-exposed rats. Antagonist inhibition of TRPV1 reduced the normoxic and the CIH-mediated increase in sEPSCs, aEPSCs, and mEPSCs. As in rats, in TRPV1+/+ control mice, aEPSCs, sEPSCs, and mEPSCs were enhanced following CIH. However, none were enhanced in TRPV1−/− null mice. Normoxic tractus solitarii (TS)-evoked EPSC amplitude, and the decrease after CIH, were comparable in control and null mice. In rats, TRPV1 was localized in the nodose-petrosal ganglia (NPG) and their central branches. CIH did not alter TRPV1 mRNA but increased its protein in NPG consistent with an increased contribution of TRPV1. Together, our studies indicate TRPV1 contributes to the CIH increase in aEPSCs and mEPSCs, but the CIH reduction in TS-EPSC amplitude occurs via an alternative mechanism. NEW & NOTEWORTHY This study provides information on the underlying mechanisms responsible for the chronic intermittent hypoxia (CIH) increase in synaptic transmission that leads to exaggerated sympathetic nervous and respiratory activity at baseline and in response to low oxygen. We demonstrate that the CIH increase in asynchronous and spontaneous excitatory postsynaptic currents (EPSCs) and miniature EPSCs, but not decrease in afferent-driven EPSCs, is dependent on transient receptor potential vanilloid type 1 (TRPV1). Thus TRPV1 is important in controlling nucleus tractus solitarii synaptic activity during CIH.

5-Hydroxytryptamine 2C receptors tonically augment synaptic currents in the nucleus tractus solitarii

The nucleus tractus solitarii (nTS) is the primary termination and integration point for visceral afferents in the brain stem. Afferent glutamate release and its efficacy on postsynaptic activity within this nucleus are modulated by additional neuromodulators and transmitters, including serotonin (5-HT) acting through its receptors. The 5-HT2 receptors in the medulla modulate the cardiorespiratory system and autonomic reflexes, but the distribution of the 5-HT2C receptor and the role of these receptors during synaptic transmission in the nTS remain largely unknown. In the present study, we examined the distribution of 5-HT2C receptors in the nTS and their role in modulating excitatory postsynaptic currents (EPSCs) in monosynaptic nTS neurons in the horizontal brain stem slice. Real-time RT-PCR and immunohistochemistry identified 5-HT2C receptor message and protein in the nTS and suggested postsynaptic localization. In nTS neurons innervated by general visceral afferents, 5-HT2C receptor activation increased solitary tract (TS)-EPSC amplitude and input resistance and depolarized membrane potential. Conversely, 5-HT2C receptor blockade reduced TS-EPSC and miniature EPSC amplitude, as well as input resistance, and hyperpolarized membrane potential. Synaptic parameters in nTS neurons that receive sensory input from carotid body chemoafferents were also attenuated by 5-HT2C receptor blockade. Taken together, these data suggest that 5-HT2C receptors in the nTS are located postsynaptically and augment excitatory neurotransmission.

Endocannabinoids mediate synaptic plasticity at glutamatergic synapses on spiny neurons within a basal ganglia nucleus necessary for song learning

Activation of type 1 cannabinoid receptors (CB1R) in many central nervous system structures induces both short- and long-term changes in synaptic transmission. Within mammalian striatum, endocannabinoids (eCB) are one of several mechanisms that induce synaptic plasticity at glutamatergic terminals onto medium spiny neurons. Striatal synaptic plasticity may contribute a critical component of adaptive motor coordination and procedural learning. Songbirds are advantageous for studying the neural mechanisms of motor learning because they possess a neural pathway necessary for song learning and adult song plasticity that includes a striato-pallidal nucleus, area X (homologous to a portion of mammalian basal ganglia). Recent findings suggest that eCBs contribute to vocal development. For example, dense CB1R expression in song control nuclei peaks around the closure of the sensori-motor integration phase of song development. Also, systemic administration of a CB1R agonist during vocal development impairs song learning. Here we test whether activation of CB1R alters excitatory synaptic input on spiny neurons in area X of adult male zebra finches. Application of the CB1R agonist WIN55212–2 decreased excitatory postsynaptic current (EPSC) amplitude; that decrease was blocked by the CB1R antagonist AM251. Guided by eCB experiments in mammalian striatum, we tested and verified that at least two mechanisms indirectly activate CB1Rs through eCBs in area X. First, activation of group I metabotropic glutamate receptors with the agonist 3,5-dihydroxyphenylglycine (DHPG) induced a CB1R-mediated reduction in EPSC amplitude. Second, we observed that a 10 s postsynaptic depolarization induced a calcium-mediated, eCB-dependent decrease in synaptic strength that resisted rescue with late CB1R blockade. Together, these results show that eCB modulation occurs at inputs to area X spiny neurons and could influence motor learning and production.

Presynaptic GABAB Receptors Regulate Retinohypothalamic Tract Synaptic Transmission by Inhibiting Voltage-Gated Ca2+ Channels

Presynaptic GABAB receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABAB-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by ω-conotoxin GVIA (≤72%), mibefradil (≤52%), and ω-agatoxin TK (≤15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP–sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.

Synaptic and Somatic Effects of Axotomy in the Intact, Innervated Rat Sympathetic Neuron

A biophysical description of the axotomized rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. Multiple aspects of neuron functioning were tested. Synaptic conductance activated by the whole presynaptic input decreased to 29% of the control value (0.92 μS per neuron) 1 day after axotomy and to 18% after 3 days. Despite the decrease in amplitude of the macroscopic current, miniature excitatory postsynaptic current (mEPSC) mean conductance, acetylcholine (ACh) equilibrium potential, and EPSC decay time constant were unaffected. Synaptic efficacy was tested during paired-pulse or maintained stimulation (5, 10, and 15 Hz, 10-s duration). Quantal release in axotomized neurons was preserved during the tetanus despite the reduction of the initial EPSC amplitude, suggesting that ACh secretion depended on the number of surviving synapses; each of them exhibited dynamic behavior during trains similar to that of normal synapses. Facilitation of EPSC amplitude was noted in 2-day axotomized neurons during the first few impulses in the train. Voltage-dependent potassium currents (the delayed IKD and the transient IA) exhibited an early drastic decrease in peak amplitude; these effects persisted 7 days after axotomy. Marked changes in IA kinetics occurred after injury: the steady-state inactivation curve shifted by up to +17 mV toward positive potentials and the voltage sensitivity of inactivation removal became steeper. IA impairment was reflected in a reduced inward threshold charge for discharge and reduced spike repolarization rate. Synaptic and somatic data were applied in a mathematical model to describe the progressive decrease in the safety factor, and the eventual failure of ganglionic transmission after axotomy.

Enhanced Striatal NR2B-Containing N-Methyl-d-Aspartate Receptor-Mediated Synaptic Currents in a Mouse Model of Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract near the N terminus of the protein huntingtin, leading to dramatic loss of striatal medium-sized spiny GABAergic projection neurons (MSNs). Evidence suggests overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors (NMDARs) contributes to selective degeneration of MSNs in HD. Striatal MSNs are enriched in NR2B, and whole cell current and excitotoxicity mediated predominantly by the NR2B subtype of NMDARs is increased with expression of mutant huntingtin in transfected cell lines and striatal MSNs from mice models. To test whether synaptic NMDAR current is altered by mutant huntingtin expression, we recorded striatal MSN excitatory postsynaptic currents (EPSCs) evoked by stimulation of cortical afferents in corticostriatal slices from YAC72 mice and their wild-type (WT) littermates at age 21–31 days. The ratio of NMDAR- to AMPAR-mediated EPSC amplitude was significantly increased in YAC72 compared to WT mice. Furthermore, using a paired-pulse stimulation protocol as a measure of presynaptic glutamate release probability, we found no significant differences between YAC72 and WT striatal MSN responses. These data suggest selective potentiation of postsynaptic NMDAR activity at corticostriatal synapses in YAC72 mice. Measurements of EPSC decay kinetics, as well as the effects of NR2B-subtype selective antagonists and glycine concentration on EPSC amplitude, are consistent with the majority of postsynaptic NMDARs being triheteromers of NR1/NR2A/NR2B in both WT and YAC72 mice. Together with previous results, our data suggest that enhanced activity of NR2B-containing NMDARs is one of the earliest changes leading to neuronal degeneration in HD.