Alexandre Moreau

Serotoninergic Fine-Tuning of the Excitation–Inhibition Balance in Rat Visual Cortical Networks

Fundamental brain functions depend on a balance between excitation (E) and inhibition (I) that is highly adjusted to a 20-80% set point in layer 5 pyramidal neurons (L5PNs) of rat visual cortex. Dysregulations of both the E-I balance and the serotonergic system in neocortical networks lead to serious neuronal diseases including depression, schizophrenia, and epilepsy. However, no link between the activation of neuronal 5-hydroxytryptamine receptors (5-HTRs) and the cortical E-I balance has yet been reported. Here we used a combination of patch-clamp recordings of composite stimulus-locked responses in L5PN following local electrical stimulations in either layer 2/3 or 6, simultaneous measurement of excitatory and inhibitory conductance dynamics, together with selective pharmacological targeting and single-cell reverse transcriptase-polymerase chain reaction. We show that cortical serotonin shifts the E-I balance in favor of more E and we reveal fine and differential modulations of the E-I balance between 5-HTR subtypes, in relation to whether layer 2/3 or 6 was stimulated and in concordance with the specific expression pattern of these subtypes in pyramidal cells and deep interneurons. This first evidence for the functional segregation of 5-HTR subtypes sheds new light on their coherent functioning in polysynaptic sensory circuits.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery

The V0 membrane domain of the V-ATPase reversibly dissociates from V1 at acidic intragranular pH and is necessary for normal exocytosis and synaptic transmission.

Impaired GABAergic transmission disrupts normal homeostatic plasticity in rat cortical networks

In the cortex, homeostatic plasticity appears to be a key process for maintaining neuronal network activity in a functional range. This phenomenon depends on close interactions between excitatory and inhibitory circuits. We previously showed that application of a high frequency of stimulation (HFS) protocol in layer 2/3 induces parallel potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons, leading to an unchanged excitation/inhibition (E/I) balance. These coordinated long‐term potentiations of excitation and inhibition correspond to homeostatic plasticity of the neuronal networks. We showed here, on the rat visual cortex, that blockade (with gabazine) or overactivation (with 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol) of GABAA receptors enhanced the E/I balance and prevented the potentiation of excitatory and inhibitory inputs after an HFS protocol. These impairements of the GABAergic transmission led to a long‐term depression‐like effect after an HFS protocol. We also observed that the blockade of inhibition reduced excitation (by 60%), and conversely, the blockade of excitation decreased inhibition (by 90%). These results support the idea that inhibitory interneurons are critical for recurrent interactions underlying homeostatic plasticity in cortical networks.

Involvement of NR2A-or NR2B-containing N-methyl-D-aspartate receptors in the potentiation of cortical layer 5 pyramidal neurone inputs depends on the developmental stage

In the cortex, N‐methyl‐d‐aspartate receptors (NMDARs) play a critical role in the control of synaptic plasticity processes. We have previously shown in rat visual cortex that the application of a high‐frequency stimulation (HFS) protocol used to induce long‐term potentiation in layer 2/3 leads to a parallel potentiation of excitatory and inhibitory inputs received by cortical layer 5 pyramidal neurones without changing the excitation/inhibition balance of the pyramidal neurone, indicating a homeostatic control of this parameter. We show here that the blockade of NMDARs of the neuronal network prevents the potentiation of excitatory and inhibitory inputs, and this result leaves open to question the role of the NMDAR isoform involved in the induction of long‐term potentiation, which is actually being strongly debated. In postnatal day (P)18–23 rat cortical slices, the blockade of synaptic NR2B‐containing NMDARs prevents the induction of the potentiation induced by the HFS protocol, whereas the blockade of NR2A‐containing NMDARs reduced the potentiation itself. In P29–P32 cortical slices, the specific activation of NR2A‐containing receptors fully ensures the potentiation of excitatory and inhibitory inputs. These results constitute the first report of a functional shift in subunit composition of NMDARs during the critical period (P12–P36), which explains the relative contribution of both NR2B‐ and NR2A‐containing NMDARs in synaptic plasticity processes. These effects of the HFS protocol are mediated by the activation of synaptic NMDARs but our results also indicate that the homeostatic control of the excitation/inhibition balance is independent of NMDAR activation and is due to specialized recurrent interactions between excitatory and inhibitory networks.

p21-activated Kinase 3 (PAK3) Protein Regulates Synaptic Transmission through Its Interaction with the Nck2/Grb4 Protein Adaptor

Background: The mental retardation p21-activated kinase (PAK3) protein regulates synaptic plasticity through the regulation of cytoskeleton dynamics of dendritic spines. Results: PAK3 binds the Grb4/Nck2 adaptor in brain, and inhibition of this complex alters synaptic currents but not spine morphology. Conclusion: The PAK3-Nck2 complex regulates post-synaptic transmission independently of spine dynamics. Significance: This opens perspectives in understanding the PAK3 implication in synaptic plasticity and mental retardation. Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.

Roles of nitric oxide in the homeostatic control of the excitation-inhibition balance in rat visual cortical networks.

The level of excitability of cortical neurons depends on the balance between their excitatory and inhibitory inputs (excitation/inhibition [E/I] balance). In the cortex, the E/I balance received by a neuron is dynamically maintained through a coordinated regulation of the strength of these inputs, described in term of homeostatic plasticity. Using a method allowing the determination of the E/I balance in rat cortical layer 5 pyramidal neurons (L5-PNs, the main output stage of the cortex), while keeping the interactions between excitatory and inhibitory networks functional, we examined the effects of high or low frequency of stimulation (HFS or LFS) protocols in layer 4 (in order to mimic thalamo-cortical entries) on the E-I level of the neuronal network. We previously showed that the E/I balance of L5-PNs remains stable due to a dual potentiation or dual depression of E and I after HFS or LFS protocols. Here, using a specific neuronal nitric oxide synthase (nNOS) inhibitor, we show that the related potentiation or depression of E and I (underlying homeostatic plasticity processes) required nNOS activation. We also show that application of an unspecific blocker of nitric oxide synthase (NOS) or a nitric oxide (NO) scavenger induces an increase of the E/I balance suggesting a role for a tonic NO synthesis in the regulation of the network activity. It is concluded that, in the cortex, a phasic NO effect (due to activation of nNOS) is required for the induction of homeostatic plasticity processes whereas a tonic NO signal is involved in the regulation of a set-point value for the E/I balance.