Physiological Perspectives on Molecular Mechanisms and Regulation of Vesicular Glutamate Transport: Lessons From Calyx of Held Synapses

Accumulation of glutamate, the primary excitatory neurotransmitter in the mammalian central nervous system, into presynaptic synaptic vesicles (SVs) depends upon three vesicular glutamate transporters (VGLUTs). Since VGLUTs are driven by a proton electrochemical gradient across the SV membrane generated by vacuolar-type H+-ATPases (V-ATPases), the rate of glutamate transport into SVs, as well as the amount of glutamate in SVs at equilibrium, are influenced by activities of both VGLUTs and V-ATPase. Despite emerging evidence that suggests various factors influencing glutamate transport by VGLUTs in vitro, little has been reported in physiological or pathological contexts to date. Historically, this was partially due to a lack of appropriate methods to monitor glutamate loading into SVs in living synapses. Furthermore, whether or not glutamate refilling of SVs can be rate-limiting for synaptic transmission is not well understood, primarily due to a lack of knowledge concerning the time required for vesicle reuse and refilling during repetitive stimulation. In this review, we first introduce a unique electrophysiological method to monitor glutamate refilling by VGLUTs in a giant model synapse from the calyx of Held in rodent brainstem slices, and we discuss the advantages and limitations of the method. We then introduce the current understanding of factors that potentially alter the amount and rate of glutamate refilling of SVs in this synapse, and discuss open questions from physiological viewpoints.

Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability

Intrinsic membrane excitability (IME) sets up neuronal responsiveness to synaptic drive. Several neurotransmitters and neuromodulators, acting through G-protein-coupled receptors (GPCRs), fine-tune motoneuron (MN) IME by modulating background K+ channels TASK1. However, intracellular partners linking GPCRs to TASK1 modulation are not yet well-known. We hypothesized that isoform 2 of rho-kinase (ROCK2), acting as downstream GPCRs, mediates adjustment of MN IME via TASK1. Electrophysiological recordings were performed in hypoglossal MNs (HMNs) obtained from adult and neonatal rats, neonatal knockout mice for TASK1 (task1–/–) and TASK3 (task3–/–, the another highly expressed TASK subunit in MNs), and primary cultures of embryonic spinal cord MNs (SMNs). Small-interfering RNA (siRNA) technology was also used to knockdown either ROCK1 or ROCK2. Furthermore, ROCK activity assays were performed to evaluate the ability of various physiological GPCR ligands to stimulate ROCK. Microiontophoretically applied H1152, a ROCK inhibitor, and siRNA-induced ROCK2 knockdown both depressed AMPAergic, inspiratory-related discharge activity of adult HMNs in vivo, which mainly express the ROCK2 isoform. In brainstem slices, intracellular constitutively active ROCK2 (aROCK2) led to H1152-sensitive HMN hyper-excitability. The aROCK2 inhibited pH-sensitive and TASK1-mediated currents in SMNs. Conclusively, aROCK2 increased IME in task3–/–, but not in task1–/– HMNs. MN IME was also augmented by the physiological neuromodulator lysophosphatidic acid (LPA) through a mechanism entailing Gαi/o-protein stimulation, ROCK2, but not ROCK1, activity and TASK1 inhibition. Finally, two neurotransmitters, TRH, and 5-HT, which are both known to increase MN IME by TASK1 inhibition, stimulated ROCK2, and depressed background resting currents via Gαq/ROCK2 signaling. These outcomes suggest that LPA and several neurotransmitters impact MN IME via Gαi/o/Gαq-protein-coupled receptors, downstream ROCK2 activation, and subsequent inhibition of TASK1 channels.

Microtubule assembly by soluble tau impairs vesicle endocytosis and excitatory neurotransmission via dynamin sequestration in Alzheimer's disease synapse model

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in brainstem slices. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 μM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Glutamatergic Control Of A Pattern-Generating Central Nucleus In A Gymnotiform Fish

The activity of central pattern-generating networks (CPGs) may change under the control exerted by various neurotransmitters and modulators to adapt its behavioral outputs to different environmental demands. Although the mechanisms underlying this control have been well established in invertebrates, most of their synaptic and cellular bases are not yet well understood in vertebrates. Gymnotus omarorum, a pulse-type gymnotiform electric fish, provides a well-suited vertebrate model to investigate these mechanisms. G. omarorum emits rhythmic and stereotyped electric organ discharges (EODs), which function in both perception and communication, under the command of an electromotor CPG. This nucleus is composed of electrotonically coupled intrinsic pacemaker cells, which pace the rhythm, and bulbospinal projecting relay cells that contribute to organize the pattern of the muscle-derived effector activation that produce the EOD. Descending influences target CPG neurons to produce adaptive behavioral electromotor responses to different environmental challenges. We used electrophysiological and pharmacological techniques in brainstem slices of G. omarorum to investigate the underpinnings of the fast transmitter control of its electromotor CPG. We demonstrate that pacemaker, but not relay cells, are endowed with ionotropic and metabotropic glutamate receptor subtypes. We also show that glutamatergic control of the CPG likely involves two types of synapses contacting pacemaker cells, one type containing both AMPAR-NMDAR receptors and the other one only-NMDAR. Fast neurotransmitter control of vertebrate CPGs seems to exploit the kinetics of the involved postsynaptic receptors to command different behavioral outputs. The prospect of common neural designs to control CPG activity in vertebrates is discussed.

Effects of Neurotrophin-3 on Intrinsic Neuronal Properties at a Central Auditory Structure

Neurotrophins, a class of growth factor proteins that control neuronal proliferation, morphology, and apoptosis, are found ubiquitously throughout the nervous system. One particular neurotrophin (NT-3) and its cognate tyrosine receptor kinase (TrkC) have recently received attention as a possible therapeutic target for synaptopathic sensorineural hearing loss. Additionally, research shows that NT-3-TrkC signaling plays a role in establishing the sensory organization of frequency topology (ie, tonotopic order) in the cochlea of the peripheral inner ear. However, the neurotrophic effects of NT-3 on central auditory properties are unclear. In this study we examined whether NT-3-TrkC signaling affects the intrinsic electrophysiological properties at a first-order central auditory structure in chicken, known as nucleus magnocellularis (NM). Here, the expression pattern of specific neurotrophins is well known and tightly regulated. By using whole-cell patch-clamp electrophysiology, we show that NT-3 application to brainstem slices does not affect intrinsic properties of high-frequency neuronal regions but had robust effects for low-frequency neurons, altering voltage-dependent potassium functions, action potential repolarization kinetics, and passive membrane properties. We suggest that NT-3 may contribute to the precise establishment and organization of tonotopy in the central auditory pathway by playing a specialized role in regulating the development of intrinsic neuronal properties of low-frequency NM neurons.

Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem

Neuron–glia communication contributes to the fine-tuning of synaptic functions. Oligodendrocytes near synapses detect and respond to neuronal activity, but their role in synapse development and plasticity remains largely unexplored. We show that oligodendrocytes modulate neurotransmitter release at presynaptic terminals through secretion of brain-derived neurotrophic factor (BDNF). Oligodendrocyte-derived BDNF functions via presynaptic tropomyosin receptor kinase B (TrkB) to ensure fast, reliable neurotransmitter release and auditory transmission in the developing brain. In auditory brainstem slices from Bdnf+/– mice, reduction in endogenous BDNF significantly decreased vesicular glutamate release by reducing the readily releasable pool of glutamate vesicles, without altering presynaptic Ca2+ channel activation or release probability. Using conditional knockout mice, cell-specific ablation of BDNF in oligodendrocytes largely recapitulated this effect, which was recovered by BDNF or TrkB agonist application. This study highlights a novel function for oligodendrocytes in synaptic transmission and their potential role in the activity-dependent refinement of presynaptic properties.

Capsaicin causes robust reduction in glycinergic transmission to rat hypoglossal motor neurons via a TRPV1-independent mechanism

The effect of capsaicin on glycinergic synaptic transmission to juvenile rat hypoglossal motor neurons in acute brainstem slices was evaluated in the presence of TTX. Capsaicin caused a robust decrease in miniature IPSC frequency, amplitude, and half-width, showing that this effect is independent of action potential generation. In the presence of capsazepine, a classic TRPV1 antagonist, capsaicin was still able to reduce spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. We further investigated whether the effect of capsaicin on glycinergic transmission to hypoglossal motor neurons is pre- or postsynaptic in nature by recording pairs of evoked IPSCs. Interestingly, capsaicin also reduced evoked IPSC amplitude without affecting paired-pulse ratio, indicating a postsynaptic mechanism of action. Significant reduction was also observed in evoked IPSC half-width, rise time, and decay tau. We also show that capsaicin does not have any effect on either transient (It) or sustained (Is) potassium currents. Finally, we also show that the hyperpolarization-activated cationic current (Ih) also remains unchanged after capsaicin application. NEW & NOTEWORTHY Capsaicin reduces the amplitude of quantal and evoked glycinergic inhibitory neurotransmission to brainstem motor neurons without altering activity-dependent transmitter release. This effect of capsaicin is not due to activation of TRPV1 receptors, as it is not blocked by capsazepine, a TRPV1 receptor antagonist. Capsaicin does not alter voltage-dependent potassium current or the hyperpolarization-activated cationic current in brainstem motor neurons.

Involvement of the cAMP-Dependent Pathway in Dextromethorphan-Induced Inhibition of Spontaneous Glutamate Transmission in the Nucleus Tractus Solitarius Neurons of Guinea Pigs

Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism.