Dynamic Clamp Methods to Investigate Impaired Neuronal Excitability Associated with Autism

Autism spectrum disorder (ASD) arises from a wide range of genetic and environmental factors. While numerous ASD-linked mutations disrupt synapse development or plasticity, an increasing number have been shown to alter the expression and functioning of voltage-gated ion channels, resulting in deficits in neuronal intrinsic excitability. Whole-cell voltage-clamp recordings can be used to characterize how ASD-related mutations affect ion channel function. However, these experiments fail to directly assess how an altered ionic conductance affects neuronal action potential firing. Dynamic clamp electrophysiology bridges this gap by enabling real-time injection of user-defined ionic conductances into living neurons. This allows causal testing of how changes in ion channel properties affect the electrical activity of a given cell type. In this methods article, we describe how to implement dynamic clamp electrophysiology in adult mouse Purkinje neurons recorded under physiological conditions in acutely prepared cerebellar brain slices. Purkinje neurons are a particularly relevant model for this work because they have an intrinsic capacity to fire repetitive, high-frequency (20-100 Hz) action potentials and are consistently implicated in ASD-related cerebellar circuit dysfunction. We focus on Tsc1, a gene whose loss-of-function mutations are among the most common monogenic causes of ASD. In mouse Purkinje neurons, Tsc1 deletion has also been linked to reduced voltage-gated sodium (Nav) channel expression. We utilize Markov kinetic state models to simulate and reproduce Purkinje neuron Nav conductance properties and go on to use dynamic clamp to directly assess how changes in the Nav conductance impact the intrinsic firing of intact cerebellar Purkinje neurons. We provide instructions and resources for modifying and tuning ionic conductance models. By integrating ionic conductance modeling, dynamic clamp, and conventional patch-clamp techniques, this approach provides a powerful and flexible framework for linking genetic perturbations to physiological outcomes in ASD-relevant neurons.