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PUBLICATIONS
Full list of publications can be found here:
Dynamic clamp electrophysiology can establish causal links between neuronal firing deficits and ion channel dysfunction. We describe dynamic clamp methods and protocols in mouse cerebellar Purkinje neurons, noting useful resources and technical considerations. In a representative study, we add modeled sodium conductance to Tsc1-/- Purkinje neurons to rescue repetitive firing.
Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with deficits in motor coordination and cognitive function. Mice lacking iFGF14 (Fgf14-/-) display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to a hyperpolarizing shift in the voltage-dependence of voltage-gated Na+ (Nav) current steady-state inactivation. Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal pyramidal neurons. Current-clamp recordings from CA1 pyramidal neurons in acute in vitro slices, however, revealed that evoked firing rates were higher in Fgf14-/- than in wild type (WT) cells. Voltage-clamp recordings demonstrated the loss of iFGF14 did not affect the voltage dependence of steady-state inactivation of Nav currents in CA1 pyramidal neurons. In addition, in contrast with results reported for neonatal (rat) hippocampal pyramidal neurons in dissociated cell culture, immunohistochemical experiments revealed that loss of iFGF14 does not disrupt the localization or alter the normalized distribution of α-Nav1.6 or α-ankyrin G labeling along the axon initial segments (AIS) of mature hippocampal CA1 neurons in situ. However, the integrated intensities of α-Nav1.6 labeling were significantly higher along the AIS of Fgf14-/-, compared with WT, adult hippocampal CA1 pyramidal neurons, consistent with the marked increase in the excitability of CA1 neurons with the loss of iFGF14.
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 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 mutant Purkinje neurons. Reduced Nav current 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 mutant Purkinje neurons, suggesting Tsc1 is necessary for the organization and functioning of the Purkinje neuron AIS. 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.
There is a striking sex bias in the prevalence and severity of autism spectrum disorder (ASD) with 80% of diagnoses occurring in males. It is difficult, however, to investigate the physiological mechanisms driving sex-specific differences in ASD. Loss of function mutations in TSC1 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 Tsc1 selectively in cerebellar Purkinje neurons, referred to here as Tsc1 mutants, have multiple ASD-linked behavioral impairments. However, these ASD-linked behavioral deficits have only been investigated using male Tsc1 mutant animals. Here, we used cohorts of male and female Tsc1 mutant animals to determine if behavioral impairments are similar across sexes. Specifically, we measured balance and motor coordination and social interaction behaviorsacross sexes. We determined balance and motor coordination deficits are similar in male and female mutants, and that deficits in the firing of Tsc1 mutant Purkinje neurons are also similar across sex. However, impairments in social approach behavior were significantly more severe in Tsc1 mutant males than females. These results indicate the selective deletion of Tsc1 in Purkinje neurons differentially impairs cerebellar circuits based on sex.
The resurgent sodium current (INaR) activates on membrane repolarization, such as during the downstroke of neuronal action potentials. Due to its unique activation properties, INaR is thought to drive high rates of repetitive neuronal firing. However, INaR is often studied in combination with the persistent or noninactivating portion of sodium currents (INaP). We used dynamic clamp to test how INaR and INaP individually affect repetitive firing in adult cerebellar Purkinje neurons from male and female mice. We learned INaR does not scale repetitive firing rates due to its rapid decay at subthreshold voltages and that subthreshold INaP is critical in regulating neuronal firing rate.
The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state.
Purkinje neurons, the sole output of the cerebellar cortex, deliver GABA-mediated inhibition to the deep cerebellar nuclei. To subserve this critical function, Purkinje neurons fire repetitively, and at high frequencies, features that have been linked to the unique properties of the voltage-gated sodium (Nav) channels expressed. In addition to the rapidly activating and inactivating, or transient, component of the Nav current (INaT) present in many types of central and peripheral neurons, Purkinje neurons, also expresses persistent (INaP) and resurgent (INaR) Nav currents.
The resurgent component of voltage-gated Na+ (Nav) currents, INaR, has been suggested to provide the depolarizing drive for high-frequency firing and to be generated by voltage-dependent Nav channel block (at depolarized potentials) and unblock (at hyperpolarized potentials) by the accessory Navβ4 subunit. To test these hypotheses, we examined the effects of the targeted deletion of Scn4b (Navβ4) on INaR and on repetitive firing in cerebellar Purkinje neurons.
