Optogenetic Visualization of Presynaptic Tonic Inhibition of Cerebellar Parallel Fibers

Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABA A receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABA A receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABA A receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits.National Science Foundation (U.S.) (Grant 1512826)National Science Foundation (U.S.) (Grant MH106013

Tonic inhibition of accumbal spiny neurons by extrasynaptic 4 GABAA receptors modulates the actions of psychostimulants

Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors

Optogenetic visualization of presynaptic tonic inhibition of cerebellar parallel fibers

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 36 (2016): 5709-5723, doi:10.1523/JNEUROSCI.4366-15.2016.Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABAA receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABAA receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABAA receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits.This work was supported by National Institute of Mental Health Grants, Alfred P. Sloan Research Fellowship, Klingenstein Fellowship Award in the Neuroscience, Beckman Young Investigator Award, World Class Institute program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology WCI 2009-003, National Research Foundation of Singapore CRP Grant, National Science Foundation Grant 1512826, and BRAIN Initiative MH106013.2016-11-2

Multiple and plastic receptors mediate tonic GABAA receptor currents in the hippocampus

Persistent activation of GABAA receptors by extracellular GABA (tonic inhibition) plays a critical role in signal processing and network excitability in the brain. In hippocampal principal cells, tonic inhibition has been reported to be mediated by {alpha}5-subunit-containing GABAA receptors ({alpha}5GABAARs). Pharmacological or genetic disruption of these receptors improves cognitive performance, suggesting that tonic inhibition has an adverse effect on information processing. Here, we show that {alpha}5GABAARs contribute to tonic currents in pyramidal cells only when ambient GABA concentrations increase (as may occur during increased brain activity). At low ambient GABA concentrations, activation of {delta}-subunit-containing GABAA receptors predominates. In epileptic tissue, {alpha}5GABAARs are downregulated and no longer contribute to tonic currents under conditions of raised extracellular GABA concentrations. Under these conditions, however, the tonic current is greater in pyramidal cells from epileptic tissue than in pyramidal cells from nonepileptic tissue, implying substitution of {alpha}5GABAARs by other GABAA receptor subtypes. These results reveal multiple components of tonic GABAA receptor-mediated conductance that are activated by low GABA concentrations. The relative contribution of these components changes after the induction of epilepsy, implying an adaptive plasticity of the tonic current in the presence of spontaneous seizures

The amount of astrocytic GABA positively correlates with the degree of tonic inhibition in hippocampal CA1 and cerebellum

A tonic form of synaptic inhibition occurs in discrete regions of the central nervous system and has an important role in controlling neuronal excitability. Recently, we reported that GABA present in astrocyte is the major source of tonic inhibition in cerebellum and that GABA is released through Bestrophin-1 channel by direct permeation. In this study, we screened for the presence of astrocytic GABA in various brain regions such as hippocampus, thalamus, hypothalamus and cerebellum using immunohistochemistry. We found that astrocytic GABA was present in the regions that were reported to show tonic inhibition. Because the existence of tonic inhibition in hippocampal CA1 is somewhat controversial, we compared the amount of astrocytic GABA and tonic inhibition between the hippocampal CA1 pyramidal cell layer and the cerebellar granule cell layer. Unlike cerebellar glial cells, hippocampal astrocytes did not contain GABA. The tonic inhibition was also much lower in the pyramidal neurons of hippocampal CA1 compared to the granule cells of cerebellum. Nevertheless, most of the hippocampal astrocytes expressed Bestrophin-1 channel. These data indicate that the absence of astrocytic GABA results in a low level of tonic inhibition in hippocampal CA1 region

Tonic Inhibition Enhances Fidelity of Sensory Information Transmission in the Cerebellar Cortex

Tonic inhibition is a key regulator of neuronal excitability and network function in the brain, but its role in sensory information processing remains poorly understood. The cerebellum is a favorable model system for addressing this question as granule cells, which form the input layer of the cerebellar cortex, permit high-resolution patch-clamp recordings in vivo, and are the only neurons in the cerebellar cortex that express the α6δ-containing GABA(A) receptors mediating tonic inhibition. We investigated how tonic inhibition regulates sensory information transmission in the rat cerebellum by using a combination of intracellular recordings from granule cells and molecular layer interneurons in vivo, selective pharmacology, and in vitro dynamic clamp experiments. We show that blocking tonic inhibition significantly increases the spontaneous firing rate of granule cells while only moderately increasing sensory-evoked spike output. In contrast, enhancing tonic inhibition reduces the spike probability in response to sensory stimulation with minimal effect on the spontaneous spike rate. Both manipulations result in a reduction in the signal-to-noise ratio of sensory transmission in granule cells and of parallel fiber synaptic input to downstream molecular layer interneurons. These results suggest that under basal conditions the level of tonic inhibition in vivo enhances the fidelity of sensory information transmission through the input layer of the cerebellar cortex

Role of tonic inhibition in associative reward condiitoning in Lymnaea

Changes in the strength of excitatory synaptic connections are known to underlie associative memory formation in the molluscan nervous system but less is known about the role of synaptic inhibition. Tonic or maintained synaptic inhibition has an important function in controlling the Lymnaea feeding system and is known to suppress feeding in the absence of food or in satiated animals. Tonic inhibition to the feeding network is provided by the N3t interneuron that has inhibitory monosynaptic connection with the central pattern generator interneuron, the N1M. Here we asked whether a reduction in the level of tonic inhibition provided by the N3t cell could play a role in reward conditioning? Semi-intact preparations made from hungry snails were conditioned using a previously-developed one-trail chemical conditioning paradigm. We recorded electrical activity in a feeding motoneuron, the B3, at various time-points after conditioning. This allowed us to measure the frequency of spike activity in the N3t interneuron and monitor fictive feeding patterns that generate the rhythmic movements involved in food ingestion. We show that there is a reduction in N3t spiking at 1, 2, 3 and 4 hours after conditioning but not at 10 minutes and 30 minutes and the reduction in N3t firing inversely correlates with an increase in the conditioned fictive feeding response. Computer simulation of N3t-N1M interactions suggests that changes in N3t firing are sufficient to explain the increase in the fictive feeding activity produced by conditioning. A network model is presented that summarizes evidence suggesting that reward conditioning in Lymnaea is due to the combined effects of reduced tonic inhibition and enhanced excitatory synaptic connections between the CS pathway and feeding command neurons

Functional properties and pharmacology of extrasynaptic GABA-A receptors

The “ambient” GABA that is present in the extracellular space surrounding all neurons of the brain is believed to be capable of persistently activating high‐affinity extrasynaptic GABA-A receptors to generate a tonic membrane conductance. This generates a form of shunting inhibition that is capable of influencing cellular and network excitability. Extrasynaptic δ subunit-containing GABA-A receptors are known to generate this form of tonic inhibition in a number of defined brain regions and these are emerging as important clinical drug targets for the treatment of a number of neurological conditions. This thesis examines the functional and pharmacological properties of recombinant and native GABA-A receptors that allow them to function as ambient GABA detectors. Surprisingly, the data presented in this Thesis shows that the behaviour of these extrasynaptic GABA-A receptor populations is dramatically influenced by the steady-state GABA concentration they experience. For example, recombinant α4βδ and α6βδ receptor populations are shown to exhibit profound levels of desensitization in the presence of low ambient GABA levels that will limit their ability to respond to changes in ambient GABA. We also find that the action of certain sedative/hypnotic drugs on extrasynaptic GABA-A receptors expressed in cerebellar granule neurons is critically dependent upon the concentration of ambient GABA. For example, we show that the intravenous anaesthetic propofol will only enhance tonic inhibition when ambient GABA levels are below 100 nM. Similarly, we show that the GABA-A receptor agonist Gaboxadol is not capable of enhancing tonic inhibition when ambient GABA levels are high. In contrast to the behaviour of drugs like propofol and Gaboxadol, we find that neurosteroid enhancement of tonic inhibition will occur regardless of ambient GABA levels. This issue will be important when considering therapeutic strategies to target tonic inhibition in the treatment of neurological disorders. Furthermore, we show for the first time, that copper ions can potently block extrasynaptic GABA-A receptors, suggesting that copper may provide a means to selectively block tonic inhibition in the brain, and may even represent a novel source of extrasynaptic GABA-A receptor modulation in vivo

Differential control by 5-HT and 5-HT1A, 2A, 2C receptors of phasic and tonic GABAA inhibition in the visual thalamus

Thalamocortical (TC) neurons, including those of the dorsal lateral geniculate nucleus (dLGN), one of the visual sensory thalamic nuclei, exhibit two forms of GABAA receptor-mediated inhibition: phasic or classical inhibitory postsynaptic currents (IPSCs) generated by the activation of synaptic GABAA receptors (sGABAAR) and tonic inhibition generated by extra- or peri-synaptic GABAA receptors (eGABAAR). The source of GABA mediating tonic inhibition mostly arises from spillover out of the synaptic cleft, because tonic inhibition is blocked by TTX and removal of extracellular Ca2+ in adult murine dLGN TC neurons. Therefore, modulation of vesicular GABA release may not only affect phasic but also tonic inhibition. Previous work in the cat and rat dLGN has shown that several neurotransmitters, including acetylcholine, serotonin (5-HT), dopamine, and norepinephrine can modulate vesicular GABA release from inhibitory interneurons, resulting in changes in phasic inhibition (IPSC frequency), primarily through presynaptic modulation of GABA release from dendro-dendritic synapses [5]. However, except for dopamine in the somatosensory thalamus, the effect of these neurotransmitters on tonic GABAA inhibition in TC neurons has not been examined. Here, we investigated whether 5-HT and its 5-HT1A, 5-HT2A and 5-HT2C receptors exert a control over tonic and phasic GABAA currents in dLGN TC neurons. We used whole cell patch clamp recordings in coronal slices (300 mm) containing the dLGN from postnatal day 20–25 Wistar rats. Data analysis and experimental procedures were similar to those previously described and in accordance with the Animals (Scientific Procedures) Act 1986 (UK). Focal application of gabazine (GBZ, 100 mM) was used to reveal the presence of tonic GABAA current (Figure 1). All serotonergic drugs were dissolved in the recording solution, and their concentrations, co-administration, and effects on phasic and tonic GABAA current are shown in Table 1 and Figure 1. We found that 5-HT enhances phasic GABAA inhibition (i.e., spontaneous IPSCs), but has no action on tonic inhibition. These effects are identical to those observed following 5-HT1A/7R activation with 8-OH-DPAT. On the other hand, α-M-5-HT and mCPP enhances and reduces, respectively, both phasic and tonic GABAA inhibition. These effects are dependent on 5-HT2AR and 5-HT2CR activation, respectively, as they are blocked by co-perfusion with selective antagonists, ketanserin, and SB242084. Thus, the lack of 5-HT modulation of tonic inhibition might be explained by the counterbalance of co-activation of 5-HT2ARs and 5-HT2CRs by the endogenous ligand.peer-reviewe

The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABA_A receptor-mediated inhibition

Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described ‘spillover’ mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABA(A)Rs to the thalamic effects of etomidate, we performed whole-cell recordings from mouse TC neurons lacking synaptic (α1(0/0)) or extrasynaptic (δ(0/0)) GABA(A)Rs. Etomidate (3 μm) significantly inhibited action-potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABA(A)Rs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ-GABA(A)Rs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate-induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states