HCN hyperpolarization-activated cation channels strengthen virtual nicotinic EPSPs and thereby elevate synaptic amplification in rat sympathetic neurons.

The influence of hyperpolarization-activated cation current (h-current; Ih) upon synaptic integration in paravertebral sympathetic neurons was studied together with expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunit isoforms. All four HCN subunits were detected in homogenates of the rat superior cervical ganglion (SCG) using the PCR to amplify reverse-transcribed messenger RNAs (RT-PCR) and using quantitative PCR. Voltage clamp recordings from dissociated SCG neurons at 35°C detected Ih in all cells, with a maximum hyperpolarization-activated cation conductance of 1.2 ± 0.1 nS, half-maximal activation at -87.6 mV, and reversal potential of -31.6 mV. Interaction between Ih and synaptic potentials was tested with virtual fast nicotinic excitatory postsynaptic potentials (EPSPs) created with dynamic clamp. The blocking of Ih with 15 µM ZD7288 hyperpolarized cells by 4.7 mV and increased the virtual synaptic conductance required to stimulate an action potential from 7.0 ± 0.9 nS to 12.1 ± 0.9 nS. In response to stimulation with 40 s long trains of virtual EPSPs, ZD7288 reduced postsynaptic firing from 2.2 to 1.7 Hz and the associated synaptic amplification from 2.2 ± 0.1 to 1.7 ± 0.2. Cyclic nucleotide binding to HCN channels was simulated by blocking native Ih with ZD7288, followed by reconstitution with virtual Ih using a dynamic clamp model of the voltage clamp data. Over a 30-mV range, shifting the half-activation voltage for Ih in 10 mV depolarizing increments always increased synaptic gain. These results indicate that Ih, in sympathetic neurons, can strengthen nicotinic EPSPs and increase synaptic amplification, while also working as a substrate for cyclic nucleotide-dependent modulation.

Alterations in cerebellar physiology are associated with a stiff-legged gait in Atcay(ji-hes) mice.

Recent evidence suggests that dystonia, a movement disorder characterized by sustained involuntary muscle contractions, can be associated with cerebellar abnormalities. The basis for how functional changes in the cerebellum can cause dystonia is poorly understood. Here we identify alterations in physiology in Atcay(ji-hes) mice which in addition to ataxia, have an abnormal gait with hind limb extension and toe walking, reminiscent of human dystonic gait. No morphological abnormalities in the brain accompany the dystonia, but partial cerebellectomy causes resolution of the stiff-legged gait, suggesting that cerebellar dysfunction contributes to the dystonic gait of Atcay(ji-hes) mice. Recordings from Purkinje and deep cerebellar nuclear (DCN) neurons in acute brain slices were used to determine the physiological correlates of dystonia in the Atcay(ji-hes) mice. Approximately 50% of cerebellar Purkinje neurons fail to display the normal repetitive firing characteristic of these cells. In addition, DCN neurons exhibit increased intrinsic firing frequencies with a subset of neurons displaying bursts of action potentials. This increased intrinsic excitability of DCN neurons is accompanied by a reduction in after-hyperpolarization currents mediated by small-conductance calcium-activated potassium (SK) channels. An activator of SK channels reduces DCN neuron firing frequency in acute cerebellar slices and improves the dystonic gait of Atcay(ji-hes) mice. These results suggest that a combination of reduced Purkinje neuron activity and increased DCN intrinsic excitability can result in a combination of ataxia and a dystonia-like gait in mice.

Expression of Caytaxin protein in Cayman Ataxia mouse models correlates with phenotype severity.

Caytaxin is a highly-conserved protein, which is encoded by the Atcay/ATCAY gene. Mutations in Atcay/ATCAY have been identified as causative of cerebellar disorders such as the rare hereditary disease Cayman ataxia in humans, generalized dystonia in the dystonic (dt) rat, and marked motor defects in three ataxic mouse lines. While several lines of evidence suggest that Caytaxin plays a critical role in maintaining nervous system processes, the physiological function of Caytaxin has not been fully characterized. In the study presented here, we generated novel specific monoclonal antibodies against full-length Caytaxin to examine endogenous Caytaxin expression in wild type and Atcay mutant mouse lines. Caytaxin protein is absent from brain tissues in the two severely ataxic Atcay(jit) (jittery) and Atcay(swd) (sidewinder) mutant lines, and markedly decreased in the mildly ataxic/dystonic Atcay(ji-hes) (hesitant) line, indicating a correlation between Caytaxin expression and disease severity. As the expression of wild type human Caytaxin in mutant sidewinder and jittery mice rescues the ataxic phenotype, Caytaxin's physiological function appears to be conserved between the human and mouse orthologs. Across multiple species and in several neuronal cell lines Caytaxin is expressed as several protein isoforms, the two largest of which are caused by the usage of conserved methionine translation start sites. The work described in this manuscript presents an initial characterization of the Caytaxin protein and its expression in wild type and several mutant mouse models. Utilizing these animal models of human Cayman Ataxia will now allow an in-depth analysis to elucidate Caytaxin's role in maintaining normal neuronal function.