dynamic-clamp-methods-to-investigate-impaired-neuronal-excitability

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 ...

Miami University

The resurgent sodium current (I) activates on membrane repolarization, such as during the downstroke of neuronal action potentials. Due to its unique activation properties, I is thought to drive high rates of repetitive neuronal firing. However, I is often studied in combination with the persistent or non-inactivating portion of sodium currents (I). We used dynamic clamp to test how I and I individually affect repetitive firing in adult cerebellar Purkinje neurons from male and female mice. We learned I does not scale repetitive firing rates due to its rapid decay at subthreshold voltages, and that subthreshold I is critical in regulating neuronal firing rate. Adjustments to the Nav conductance model used in these studies revealed I and I can be inversely scaled by adjusting occupancy in the slow inactivated kinetic state. Together with additional dynamic clamp experiments, these data suggest the regulation of sodium channel slow inactivation can fine-tune I and Purkinje neuron repetitive firing rates.

A Reinterpretation of the Relationship Between Persistent and Resurgent Sodium Currents.

The resurgent sodium current (I) activates on membrane repolarization, such as during the downstroke of neuronal action potentials. Due to its unique activation properties, I is thought to drive high rates of repetitive neuronal firing. However, I is often studied in combination with the persistent or noninactivating portion of sodium currents (I). We used dynamic clamp to test how I and I individually affect repetitive firing in adult cerebellar Purkinje neurons from male and female mice. We learned I does not scale repetitive firing rates due to its rapid decay at subthreshold voltages and that subthreshold I is critical in regulating neuronal firing rate. Adjustments to the voltage-gated sodium conductance model used in these studies revealed I and I can be inversely scaled by adjusting occupancy in the slow-inactivated kinetic state. Together with additional dynamic clamp experiments, these data suggest the regulation of sodium channel slow inactivation can fine-tune I and Purkinje neuron repetitive firing rates.

A Reinterpretation of the Relationship between Persistent and Resurgent Sodium Currents.

There is a striking sex bias in the prevalence and severity of autism spectrum disorder (ASD) with 80% of diagnoses occurring in males. Because the molecular etiology of ASD is likely combinatorial, including interactions across multiple genetic and environmental factors, it is difficult to investigate the physiological mechanisms driving sex-specific differences. Loss of function mutations in  result in dysregulated mTORC1 signaling and underlie a multi-system disorder known as tuberous sclerosis (TSC). Interestingly, more than 50% of individuals diagnosed with TSC are also diagnosed with ASD, making TSC mutations one of the most prevalent monogenic causes of ASD. Mice harboring targeted deletion of  selectively in cerebellar Purkinje neurons, referred to here as  , have multiple ASD-linked behavioral impairments, including deficits in social interactions, motor coordination, and vocalizations. However, these ASD-linked behavioral deficits have only been investigated using male  animals. Here, we used cohorts of male and female  animals to determine if behavioral impairments, previously identified in this model, are similar across sex. Specifically, we measured balance and motor coordination and social interaction behaviors in two age groups across sex. We determined balance and motor coordination deficits are similar in male and female  mice, and that deficits in the firing of  Purkinje neurons located in the cerebellar vermis are also similar across sex. However, impairments in social approach behavior were found to be significantly more severe in  males compared to females. These results indicate the selective deletion of  in Purkinje neurons differentially impairs cerebellar circuits based on sex.

Selective deletion of  from mouse cerebellar Purkinje neurons drives sex-specific behavioral impairments linked to autism.

Loss-of-function mutations in tuberous sclerosis 1 () are prevalent monogenic causes of autism spectrum disorder (ASD). Selective deletion of  from mouse cerebellar Purkinje neurons has been shown to cause several ASD-linked behavioral impairments, which are linked to reduced Purkinje neuron repetitive firing rates. We used electrophysiology methods to investigate why Purkinje neuron-specific  deletion (  ) impairs Purkinje neuron firing. These studies revealed a depolarized shift in action potential threshold voltage, an effect that we link to reduced expression of the fast-transient voltage-gated sodium (Nav) current in   Purkinje neurons. The reduced Nav currents in these cells was associated with diminished secondary immunofluorescence from anti-pan Nav channel labeling at Purkinje neuron axon initial segments (AIS). Interestingly, anti-ankyrinG immunofluorescence was also found to be significantly reduced at the AIS of   Purkinje neurons, suggesting Tsc1 is necessary for the organization and functioning of the Purkinje neuron AIS. An analysis of the 1 and 2 derivative of the action potential voltage-waveform supported this hypothesis, revealing spike initiation and propagation from the AIS of   Purkinje neurons is impaired compared to age-matched control Purkinje neurons. Heterozygous  deletion resulted in no significant changes in the firing properties of adult Purkinje neurons, and slight reductions in anti-pan Nav and anti-ankyrinG labeling at the Purkinje neuron AIS, revealing deficits in Purkinje neuron firing due to  haploinsufficiency are delayed compared to age-matched   Purkinje neurons. Together, these data reveal the loss of  impairs Purkinje neuron firing and membrane excitability through the dysregulation of proteins necessary for AIS organization and function.

 Deletion in Purkinje Neurons Disrupts the Axon Initial Segment, Impairing Excitability and Cerebellar Function.

Loss-of-function mutations in tuberous sclerosis 1 (TSC1) are prevalent monogenic causes of autism spectrum disorder (ASD). Selective deletion of Tsc1 from mouse cerebellar Purkinje neurons has been shown to cause several ASD-linked behavioral impairments, which are linked to reduced Purkinje neuron repetitive firing rates. We used electrophysiology methods to investigate why Purkinje neuron-specific Tsc1 deletion (Tsc1) impairs Purkinje neuron firing. These studies revealed a depolarized shift in action potential threshold voltage, an effect that we link to reduced expression of the fast-transient voltage-gated sodium (Nav) current in Tsc1 Purkinje neurons. The reduced Nav currents in these cells was associated with diminished secondary immunofluorescence from anti-pan Nav channel labeling at Purkinje neuron axon initial segments (AIS). Anti-ankyrinG immunofluorescence was also found to be significantly reduced at the AIS of Tsc1 Purkinje neurons, suggesting Tsc1 is necessary for the organization and functioning of the Purkinje neuron AIS. An analysis of the 1st and 2nd derivative of the action potential voltage-waveform supported this hypothesis, revealing spike initiation and propagation from the AIS of Tsc1 Purkinje neurons is impaired compared to age-matched control Purkinje neurons. Heterozygous Tsc1 deletion resulted in no significant changes in the firing properties of adult Purkinje neurons, and slight reductions in anti-pan Nav and anti-ankyrinG labeling at the Purkinje neuron AIS, revealing deficits in Purkinje neuron firing due to Tsc1 haploinsufficiency are delayed compared to age-matched Tsc1 Purkinje neurons. Together, these data reveal that the loss of Tsc1 impairs Purkinje neuron firing and membrane excitability through the dysregulation of proteins essential for AIS organization and function.

Article	Year
A Reinterpretation of the Relationship Between Persistent and Resurgent Sodium Currents. bioRxiv : the preprint server for biology\| PubMed ID: 38187680	2024
A Reinterpretation of the Relationship between Persistent and Resurgent Sodium Currents. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 38858080	2024
Selective deletion of from mouse cerebellar Purkinje neurons drives sex-specific behavioral impairments linked to autism. Frontiers in behavioral neuroscience\| PubMed ID: 39741564	2024
Deletion in Purkinje Neurons Disrupts the Axon Initial Segment, Impairing Excitability and Cerebellar Function. bioRxiv : the preprint server for biology\| PubMed ID: 39974887	2025
Tsc1 deletion in Purkinje neurons disrupts the axon initial segment, impairing excitability and cerebellar function. Neurobiology of disease\| PubMed ID: 40015654	2025

Samuel P. Brown

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