Biology subjects

Clarke, V. R. J.

Publications and source records attributed to Clarke, V. R. J..

3 recordsLinked to original sources

bioRxiv2024

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme CaMKII plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on runaway synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

neuroscience↗

bioRxiv2023

State-dependent GABAergic regulation of striatal spiny projection neuron excitability

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

neuroscience↗

bioRxiv2022

Semaphorin 4D induced inhibitory synaptogenesis restores benzodiazepine sensitivity in a mouse model of Status Epilepticus

Previously we demonstrated that intra-hippocampal infusion of purified, Semaphorin 4D (Sema4D) extracellular domain into the mouse hippocampus rapidly promotes formation of GABAergic synapses and decreases seizure susceptibility in mice. Given the relatively fast action of Sema4D treatment revealed by these studies, we sought to determine the time course of Sema4D treatment on hippocampal network activity using an acute hippocampal slice preparation. We performed long-term extracellular recordings from area CA1 encompassing a 2 hour application of Sema4D and found that hippocampal excitation is suppressed hours following treatment. We also asked if Sema4D treatment could ameliorate seizures in an acute seizure model: the kainic acid (KA) mouse model of Status Epilepticus (SE). We demonstrate that Sema4D treatment suppresses seizures and lessens benzodiazepine (BZD) insensitivity which is a hallmark of refractory SE. Lastly, we sought to explore alternative methods of Sema4D delivery to hippocampus and thus created an Adeno Associated Virus expressing the extracellular domain of Sema4D. Our data reveal that virally delivered, chronically overexpressed Sema4D extracellular domain promotes GABAergic synapse formation and suppresses seizure activity similar to the actions of purified Sema4D protein. These results provide proof of concept that Sema4D-mediated GABAergic synapse development could combat BZD insensitivity in intractable forms of epilepsy, and that viral delivery of Sema4D is an efficacious and promising delivery method.

neuroscience↗