An optogenetic feedback controller for clamping firing rates in dissociated cortical networks Jon Newman, Ming-fai Fong, Riley Zeller-Townson, Neal Laxpati, Ted French, and Steve M. Potter Synchronized bouts of network firing are characteristic of developing neural tissue, both in-vivo and in-vitro. In the intact nervous system, synchronized activity gives way to sparse irregular firing patterns as functional microcircuits are formed. In contrast, synchronized network bursting in long term, in-vitro preparations continues indefinitely (up to 2 years in our lab). We hypothesize that the persistence of synchronized activity in cortical cultures is due primarily to a lack of afferent drive during the development of chemical synapses, which severely limits dissociated cortical cultures' usefulness as simplified models of functional cortical processing. Therefore, we sought a method for replacing afferent drive to in-vitro cortical networks during synapse development to study and control the effects of deafferentation on developing synapses. We created an optogenetic feedback controller capable of clamping population firing rates to set-points by controlling photocurrents in ChR2- expressing pyramidal cells. This tool provides a non-pharmacological method for clamping network activity levels that is useful for deducing the triggers of homeostatic plasticity at developing synapses. Additionally, it may provide a means for normalizing the synaptic state of deafferented in-vitro cortical networks in order to increase their ability to respond appropriately to externally provided signals. Pathological effects of synaptic homeostasis in cases of deafferentation It is likely that there is a common etiology for a large class of neurological disorders that involve persistent, low-frequency network discharges; namely, counterproductive homeostatic plasticity in response to deafferentation. Homeostatic plasticity is a set of biochemical mechanisms that alter the intrinsic excitability of neurons or the strength of synapses in a way that counters deviations in long-time average activity levels [1]. One form of homeostatic plasticity is synaptic scaling, a phenomenon whereby synaptic connections of a single neuron are strengthened or weakened by a multiplicative factor in order to maintain a target activity level [2]. For instance, chronic pharmacological blockade of network activity (either excitatory synaptic or spiking) causes a compensatory increase in excitatory synaptic strength, whereas chronically elevating activity (by dis-inhibition) weakens excitatory synapses. When afferent drive to a cortical network is reduced due to deafferentation, homeostatic compensation can lead to network instability. Modeling studies have indicated that aberrant synaptic scaling is a likely mechanism for the development of cortical instability following deafferentation [3]. When afferent drive is lost, excitatory synaptic strength (amplitude of post-synaptic events) is increased to compensate. As excitatory synaptic strength is increased, network firing levels are recovered, but activity becomes confined to low frequency population discharges as the network enters a state of intrinsic instability. In this state, the information processing capabilities of the network are severely reduced due to strong recurrent input overshadowing relatively weak signals from the outside world [3]. Experimental evidence supporting these results has been provided by studies that cut white matter tracks under the suprasylvian gyrus in cats while performing EEG and intracellular recordings. In the weeks following cortical undercut, periodic paroxysmal discharges develop in deafferented tissue and signal propagation speed over the deafferented area is increased. Additionally, in-vitro preparations of intact sensory-motor cortical islands have shown increased miniature and evoked EPSC amplitude and frequency following cortical undercut [4]. Given the importance of synaptic scaling in the development of pathological activity patterns in cases of deafferentation, we sought to develop a tool (1) to better understand the aspects of network activity that triggers the induction of synaptic scaling in pyramidal cells, which remains hotly debated [1], and (2) to reverse the effects of pathological synaptic scaling in dissociated networks in order to increase their ability to respond appropriately to externally provided signals. This is relevant to cases where cortical cultures are used as biological computers for time series prediction or classification. Optogenetic feedback controller for clamping population firing level Fig 1. Real-time, multichannel electrophysiology system. (A) NR software; (B) Closed-loop DLL 'plugin' ; (C) 4 channel LED driver (D) LED- fiber coupler; (E) Köhler illumination train; (F) 59 channel MEA amplifier and temperature control system; (G) MEA showing culturing well.