Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing

Abstract

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered three days after slices were released from five days of deprivation, they were less able to recover after ten days of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39% and 56% following five and ten days of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55% and 73% respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.Significance StatementWhen cortical activity is silenced during development, lifelong seizures often result. Here we explored whether these seizures result from overcompensation of homeostatic recovery mechanisms. Prior work showed that neurons briefly deprived of the ability to fire action potentials compensate by becoming more excitable, increasing synaptic drive and intrinsic excitability. We found that prolonged silencing of cortex produced a profound loss of synapse density, especially for inhibitory synapses, pointing to a circuit unable to maintain excitatory/inhibitory balance. These results show that homeostatic responses, which are normally restorative, can result in maladaptive circuit configurations when brought to extremes.

Prolonged Activity Deprivation Causes Pre- and Postsynaptic Compensatory Plasticity at Neocortical Excitatory Synapses

“…The results of this and other recent studies employing activity manipulations in slice cultures ( Valakh et al, 2023 ; Wise et al, 2024 ) reveal that most of the homeostatic mechanisms identified in prior studies of dissociated cultures, including increased intrinsic excitability, changes in inhibition, and pre- and postsynaptic changes in excitatory synaptic transmission can be readily studied in this more intact preparation in which many histological and anatomical features of cortical circuits are preserved but which are broadly amenable to pharmacological and genetic manipulation. This may provide a useful platform for identifying candidate molecules involved in the signal transduction pathways and transcriptional networks regulating homeostatic plasticity.…”

“…The loss of excitatory synapses may reflect an inability to maintain transient synapses under silenced conditions when coincident firing cannot contribute to stabilization ( Hedrick et al, 2024 ). Nevertheless, the presence of rebound hyperexcitability measured previously in cultures subjected to the same activity deprivation ( Koch et al, 2010 ; Wise et al, 2024 ) suggests the reductions in synapse number are not sufficient to overcome other changes in circuit excitability, including the observed increases in excitatory synaptic strength.…”

“…To broadly block activity, we applied the voltage-gated sodium channel blocker TTX (0.5 µM) to a set of cultures throughout the last 5 d of this 10 d period and compared properties of excitatory synapses to age-matched controls with uninhibited spontaneous activity. This manipulation induces a robust homeostatic program that profoundly shifts network dynamics toward hyperexcitability ( Koch et al, 2010 ; Wise et al, 2024 ). We obtained whole-cell patch-clamp recordings from layer 5 pyramidal neurons in somatosensory cortex.…”

“…Although not directly measured here, we know that after 5 d of deprivation, there is a strong activity rebound reflecting circuit hyperexcitability ( Wise et al, 2024 ). Our experiments suggest that one factor contributing to this hyperexcitability is likely enhanced excitatory synaptic transmission, as evidenced by increased frequency and amplitude of mEPSCs and enlarged pre- and postsynaptic size.…”