GABA-mediated tonic inhibition differentially modulates gain in functional subtypes of cortical interneurons

The binding of GABA (gamma-aminobutyric acid) to extrasynaptic GABA(A) receptors generates tonic inhibition that acts as a powerful modulator of cortical network activity. Despite GABA being present throughout the extracellular space of the brain, previous work has shown that GABA may differentially modulate the excitability of neuron subtypes according to variation in chloride gradient. Here, using biophysically detailed neuron models, we predict that tonic inhibition can differentially modulate the excitability of neuron subtypes according to variation in electrophysiological properties. Surprisingly, tonic inhibition increased the responsiveness (or gain) in models with features typical for somatostatin interneurons but decreased gain in models with features typical for parvalbumin interneurons. Patch-clamp recordings from cortical interneurons supported these predictions, and further in silico analysis was then performed to seek a putative mechanism underlying gain modulation. We found that gain modulation in models was dependent upon the magnitude of tonic current generated at depolarized membrane potential-a property associated with outward rectifying GABA(A) receptors. Furthermore, tonic inhibition produced two biophysical changes in models of relevance to neuronal excitability: 1) enhanced action potential repolarization via increased current flow into the dendritic compartment, and 2) reduced activation of voltage-dependent potassium channels. Finally, we show theoretically that reduced potassium channel activation selectively increases gain in models possessing action potential dynamics typical for somatostatin interneurons. Potassium channels in parvalbumin-type models deactivate rapidly and are unavailable for further modulation. These findings show that GABA can differentially modulate interneuron excitability and suggest a mechanism through which this occurs in silico via differences of intrinsic electrophysiological properties.

Conformational dynamics of cationic cys-loop receptors

Davor Kosanic

Understanding the functional dynamics of ligand-gated ion-channels (LGIC's) on the molecular level is the aim of this thesis. Members of the protein family of LGIC's are involved in the fast synaptic signal transmission and thereby play a central role in inter-cellular communication in higher multi-cellular organisms. Here, two prototypical members of ligand-gated cys-loop receptors were studied: the homopentameric type 3 serotonin receptor (5HT3AR) and the muscle-type nicotinic acetylcholine receptor (nAchR). The single-channel kinetics of the 5HT3AR was investigated using single-channel patch-clamp and single-molecule fluorescence techniques. Our principal findings are that the 5HT3AR channel shows up to five, agonist activated, nearly equidistant conductance levels, by binding of up to five agonist molecules. Detailed analysis of data obtained using serotonin the native agonist and quipazine-TMR, a fluorescent partial agonist at different ionic conditions has been performed. An allosteric calcium modulation was observed with serotonin but not with the partial agonist indicating receptor activation with altered conformational pathways. In conclusion, a functional model is proposed, describing a two-way conformational activation. Binding of each agonist opens a "conductance filter" within the addressed subunit. Additionally, a global gate, which can be modulated by the presence of calcium ions, determines the channel kinetics. In order to identify at least one of the hypothesized conformational pathways of the 5HT3A receptor activation, a mutation of aspartate to cysteine at the position 298 was introduced into the receptor variant devoid of cysteine residues. Labelling the mutated residue with a fluorescent dye resulted in a fluorescence quenching upon receptor activation. This effect was interpreted as a functional conformational rearrangement. Although no specific time-scale of such a (local or global) change in conformation could be determined on the molecular level, the application of the described approach as a possible test for functionality upon receptor solubilisation is discussed. The simultaneous observation of the discrete ligand binding and the subsequent single-channel gating was discussed for years in the LGIC field as the method of choice for direct and unbiased examination of the channel activation process. Here, current recordings of single-channel gating events were combined with optical single-molecule detection of fluorescent agonist binding. The time delay Δt between ligand binding and gating events of a gain-of-function variant of the foetal and the wild-type adult nAchR were measured on the single molecule level on living cells. For times above approximately 1 ms, the distribution of Δt for both receptor types obeys a power-law with an exponent close to 3/2, corresponding to a normal conformational diffusion. Increasing the time resolution, a monoexponential function describes the obtained data best for times below 1 ms, suggesting that an activation barrier in the free energy landscape of the receptor activation can be overcome within one step. Finally, a novel method for the fast and simple induction of synapse formation was developed. Optical tweezers were used for pulling membrane-nanotubes out of the plasma membrane of a living cell. Connecting these nanotubes to the cell membrane of up to 100 µm distant cells, axonal or dendritic protrusions can be mimicked. Functional electrical synapses, based on gap junctions, were artificially created. The results obtained in this work contribute to the functional characterisation of LGIC's in living cells, yielding models that might be extrapolated as concepts of conformational dynamics of proteins in general.

EPFL2009

Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells

Henry Markram, Gilad Silberberg

Reliable activation of inhibitory pathways is essential for maintaining the balance between excitation and inhibition during cortical activity. Little is known, however, about the activation of these pathways at the level of the local neocortical microcircuit. We report a disynaptic inhibitory pathway among neocortical pyramidal cells (PCs). Inhibitory responses were evoked in layer 5 PCs following stimulation of individual neighboring PCs with trains of action potentials. The probability for inhibition between PCs was more than twice that of direct excitation, and inhibitory responses increased as a function of rate and duration of presynaptic discharge. Simultaneous somatic and dendritic recordings indicated that inhibition originated from PC apical and tuft dendrites. Multineuron whole-cell recordings from PCs and interneurons combined with morphological reconstructions revealed the mediating interneurons as Martinotti cells. Martinotti cells received facilitating synapses from PCs and formed reliable inhibitory synapses onto dendrites of neighboring PCs. We describe this feedback pathway and propose it as a central mechanism for regulation of cortical activity.

2007

A subpopulation of cortical VIP-expressing interneurons with highly dynamic spines

Jérôme Blanc, Christina Georgiou, Graham Knott, Kok Sin Lee

Structural synaptic plasticity may underlie experience and learning-dependent changes in cortical circuits. In contrast to excitatory pyramidal neurons, insight into the structural plasticity of inhibitory neurons remains limited. Interneurons are divided into various subclasses, each with specialized functions in cortical circuits. Further knowledge of subclass-specific structural plasticity of interneurons is crucial to gaining a complete mechanistic understanding of their contribution to cortical plasticity overall. Here, we describe a subpopulation of superficial cortical multipolar interneurons expressing vasoactive intestinal peptide (VIP) with high spine densities on their dendrites located in layer (L) 1, and with the electrophysiological characteristics of bursting cells. Using longitudinal imaging in vivo, we found that the majority of the spines are highly dynamic, displaying lifetimes considerably shorter than that of spines on pyramidal neurons. Using correlative light and electron microscopy, we confirmed that these VIP spines are sites of excitatory synaptic contacts, and are morphologically distinct from other spines in L1. Advanced light and electron microscopy identify a subset of cortical multipolar vasoactive intestinal peptide interneurons with high spine dynamics and characteristics that are greatly different from pyramidal neurons.

NATURE PORTFOLIO2022

Conformational dynamics of cationic cys-loop receptors

Davor Kosanic

EPFL2009