Diminished GAD67 Terminals and Ambient GABA Inhibition Associated with Reduced Motor Neuron Recruitment Threshold in the SOD1G93A ALS mouse

Pre-symptomatic studies in mouse models of the neurodegenerative motor neuron (MN) disease, Amyotrophic Lateral Sclerosis (ALS) highlight early alterations in intrinsic and synaptic excitability and have supported an excitotoxic theory of MN death. However, a role for synaptic inhibition in disease development is not sufficiently explored among other mechanisms. Since inhibition plays a role in both regulating motor output and in neuroprotection, we examined the age-dependent anatomical changes in inhibitory presynaptic terminals on MN cell bodies using fluorescent immunohistochemistry for GAD67 (GABA) and GlyT2 (glycine) presynaptic proteins comparing ALS-vulnerable trigeminal jaw closer (JC) motor pools with the ALS-resistant extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Our results indicate differential patterns of temporal changes of these terminals in vulnerable versus resilient MNs and relative differences between SOD1G93A and wild-type (WT) MNs. Notably, we found pre-symptomatic up-regulation in inhibitory terminals in the EO MNs while the vulnerable JC MNs mostly showed a decrease in inhibitory terminals. Specifically, there was a statistically significant decrease in the GAD67 somatic abuttal in the SOD1G93A JC MNs compared to WT around P12. Using in vitro patch-clamp electrophysiology, we found a parallel decrease in the ambient GABA-dependent tonic inhibition in the SOD1G93A JC MNs. While it is unclear if the two mechanisms are directly related, pharmacological blockade of specific subtype of GABAA-a5 receptors suggests that tonic inhibition can control MN recruitment threshold. Furthermore, reduction in tonic GABA current as observed here in the mutant, identifies a putative molecular mechanism explaining our observations of hyperexcitable shifts in JC MN recruitment threshold in the SOD1G93A mouse. Lastly, we showcase non-parametric resampling-based bootstrap statistics for data analyses, and provide the Python code on GitHub for wider reuse.

Activation of the α1β2γ2L GABAA Receptor by Physiological Agonists

The Cl− permeable GABAA receptor is a major contributor to cellular inhibition in the brain. The receptor is normally activated by synaptically-released or ambient GABA but is sensitive to a number of physiological compounds such as β-alanine, taurine, and neurosteroids that, to various degrees, activate the receptor and modulate responses either to the transmitter or to each other. Here, we describe α1β2γ2L GABAA receptor activation and modulation by combinations of orthosteric and allosteric activators. The overall goal was to gain insight into how changes in the levels of endogenous agonists modulate receptor activity and influence cellular inhibition. Experimental observations and simulations are described in the framework of a cyclic concerted transition model. We also provide general analytical solutions for the analysis of electrophysiological data collected in the presence of combinations of active compounds.

Presynaptic GABAB receptor–mediated network excitation in the medial prefrontal cortex of Tsc2+/- mice

The TSC1 and TSC2 tumor suppressor genes control the activity of mechanistic target of rapamycin (mTOR) pathway. Elevated activity of this pathway in Tsc2+/- mouse model leads to reduction of postsynaptic GABAB receptor–mediated inhibition and hyperexcitability in the medial prefrontal cortex (mPFC). In this study, we asked whether presynaptic GABAB receptors (GABABRs) can compensate this shift of hyperexcitability. Experiments were performed in brain slices from adolescent wild-type (WT) and Tsc2+/- mice. Miniature and spontaneous postsynaptic currents (m/sPSCs) were recorded from layer 2/3 pyramidal neurons in mPFC using patch-clamp technique using a Cs+-based intrapipette solution. Presynaptic GABABRs were activated by baclofen (10 µM) or blocked by CGP55845 (1 µM). Independent on genotype, GABABR modulators bidirectionally change miniature excitatory postsynaptic current (mEPSC) frequency by about 10%, indicating presynaptic GABABR-mediated effects on glutamatergic transmission are comparable in both genotypes. In contrast, frequencies of both mIPSCs and sIPCSs were suppressed by baclofen stronger in Tsc2+/- neurons than in WT ones, whereas CGP55845 significantly increased (m/s)IPSC frequencies only in WT cells. Effects of baclofen and CGP55845 on the amplitudes of evoked (e)IPSCs confirmed these observations. These data indicate (1) that GABAergic synapses are inhibited by ambient GABA in WT but not in Tsc2+/- slices, and (2) that baclofen shifts the E/I ratio, determined as the ratio of (m/s)EPSC frequency to (m/s)IPSC frequency, towards excitation only in Tsc2+/- cells. This excitatory presynaptic GABABR-mediated action has to be taken into account for a possible medication of mental disorders using baclofen. Graphical abstract

Tonic GABAA conductance favors temporal over rate coding in the rat hippocampus

Synaptic plasticity is triggered by different patterns of neuronal network activity. Network activity leads to an increase in ambient GABA concentration and tonic activation of GABAA receptors. How tonic GABAA conductance affects synaptic plasticity during temporal and rate-based coding is poorly understood. Here, we show that tonic GABAA conductance differently affects long-term potentiation (LTP) induced by different stimulation patterns. The LTP based on a temporal spike - EPSP order (spike-timing-dependent [st] LTP) was not affected by exogenous GABA application. Backpropagating action potential, which enables Ca2+ entry through N-methyl-D-aspartate receptors (NMDARs) during stLTP induction, was only slightly reduced by the tonic conductance. In contrast, GABA application impeded LTP dependent on spiking rate (theta-burst-induced [tb] LTP) by reducing the EPSP bust response and, hence, NMDAR-mediated Ca2+ entry during tbLTP induction. Our results may explain the changes in different forms of memory under physiological and pathological conditions that affect tonic GABAA conductance.

Opening of KATP Channel Regulates Tonic Currents From Pyramidal Neurons in Rat Brain

Background: ATP-sensitive K+ (KATP) channels couple metabolic state to cellular excitability. Activation of neuronal and astrocytic mitochondrial KATP (mitoKATP) channels regulates a variety of neuronal functions. However, less is known about the impact of mitoKATP on tonic γ-aminobutyric acid (GABA) inhibition. Tonic GABA inhibition is mediated by the binding of ambient GABA on extrasynaptic GABA A-type receptors (GABAARs) and is involved in regulating neuronal excitability. Methods: We determined the impact of activation of KATP channels with diazoxide (DIZ) on tonic inhibition and recorded tonic current from rat cortical layer 5 pyramidal cells by patch-clamp recordings. Results: We found that neonatal tonic current increased with an increase in GABA concentration, which was partially mediated by the GABA A-type receptor (GABAAR) α5, and likely the δ subunits. Activation of KATP channels resulted in decreased tonic current in newborns, but there was increased tonic current during the second postnatal week. Conclusions: These findings suggest that activation of KATP channels with DIZ regulates GABAergic transmission in neocortical pyramidal cells during development.

Reduction of Trial-to-Trial Perceptual Variability by Intracortical Tonic Inhibition

Variability is a prominent characteristic of cognitive brain function. For instance, different trials of presentation of the same stimulus yield higher variability in its perception: subjects sometimes fail in perceiving the same stimulus. Perceptual variability could be attributable to ongoing-spontaneous fluctuation in neuronal activity prior to sensory stimulation. Simulating a cortical neural network model, we investigated the underlying neuronal mechanism of perceptual variability in relation to variability in ongoing-spontaneous neuronal activity. In the network model, populations of principal cells (cell assemblies) encode information about sensory features. Each cell assembly is sensitive to one particular feature stimulus. Transporters on GABAergic interneurons regulate ambient GABA concentration in a neuronal activity-dependent manner. Ambient GABA molecules activate extrasynaptic GABA[Formula: see text] receptors on principal cells and interneurons, and provide them with tonic inhibitory currents. We controlled the variability of ongoing-spontaneous neuronal activity by manipulating the basal level of ambient GABA and assessed the perceptual performance of the network: detection of a feature stimulus. In an erroneous response, stimulus-irrelevant but not stimulus-relevant principal cells were activated, generating trains of action potentials. Perceptual variability, reflected in error rate in detecting the same stimulus that was presented repeatedly to the network, was increased as the variability in ongoing-spontaneous membrane potential among cell assemblies increased. Frequent, transient membrane depolarization below firing threshold was the major cause of the increased neuronal variability, for which a decrease in basal ambient GABA concentration was responsible. We suggest that ambient GABA in the brain may have a role in reducing the variability in ongoing-spontaneous neuronal activity, leading to a decrease in perceptual variability and therefore to reliable sensory perception.

Regulation of Local Ambient GABA Levels via Transporter-Mediated GABA Import and Export for Subliminal Learning

Perception of supraliminal stimuli might in general be reflected in bursts of action potentials (spikes), and their memory traces could be formed through spike-timing-dependent plasticity (STDP). Memory traces for subliminal stimuli might be formed in a different manner, because subliminal stimulation evokes a fraction (but not a burst) of spikes. Simulations of a cortical neural network model showed that a subliminal stimulus that was too brief (10 msec) to perceive transiently (more than about 500 msec) depolarized stimulus-relevant principal cells and hyperpolarized stimulus-irrelevant principal cells in a subthreshold manner. This led to a small increase or decrease in ongoing-spontaneous spiking activity frequency (less than 1 Hz). Synaptic modification based on STDP during this period effectively enhanced relevant synaptic weights, by which subliminal learning was improved. GABA transporters on GABAergic interneurons modulated local levels of ambient GABA. Ambient GABA molecules acted on extrasynaptic receptors, provided principal cells with tonic inhibitory currents, and contributed to achieving the subthreshold neuronal state. We suggest that ongoing-spontaneous synaptic alteration through STDP following subliminal stimulation may be a possible neuronal mechanism for leaving its memory trace in cortical circuitry. Regulation of local ambient GABA levels by transporter-mediated GABA import and export may be crucial for subliminal learning.