A cytoskeleton-membrane interaction conserved in fast-spiking neurons controls movement, emotion, and memory.

The pathogenesis of schizophrenia is believed to involve combined dysfunctions of many proteins including microtubule-associated protein 6 (MAP6) and Kv3.1 voltage-gated K+ (Kv) channel, but their relationship and functions in behavioral regulation are often not known. Here we report that MAP6 stabilizes Kv3.1 channels in parvalbumin-positive (PV+ ) fast-spiking GABAergic interneurons, regulating behavior. MAP6-/- and Kv3.1-/- mice display similar hyperactivity and avoidance reduction. Their proteins colocalize in PV+ interneurons and MAP6 deletion markedly reduces Kv3.1 protein level. We further show that two microtubule-binding modules of MAP6 bind the Kv3.1 tetramerization domain with high affinity, maintaining the channel level in both neuronal soma and axons. MAP6 knockdown by AAV-shRNA in the amygdala or the hippocampus reduces avoidance or causes hyperactivity and recognition memory deficit, respectively, through elevating projection neuron activity. Finally, knocking down Kv3.1 or disrupting the MAP6-Kv3.1 binding in these brain regions causes avoidance reduction and hyperactivity, consistent with the effects of MAP6 knockdown. Thus, disrupting this conserved cytoskeleton-membrane interaction in fast-spiking neurons causes different degrees of functional vulnerability in various neural circuits.

Dissociable effects of oxycodone on behavior, calcium transient activity, and excitability of dorsolateral striatal neurons.

Opioids are the most common medications for moderate to severe pain. Unfortunately, they also have addictive properties that have precipitated opioid misuse and the opioid epidemic. In the present study, we examined the effects of acute administration of oxycodone, a µ-opioid receptor (MOR) agonist, on Ca2+ transient activity of medium-sized spiny neurons (MSNs) in freely moving animals. Ca2+ imaging of MSNs in dopamine D1-Cre mice (expressing Cre predominantly in the direct pathway) or adenosine A2A-Cre mice (expressing Cre predominantly in the indirect pathway) was obtained with the aid of miniaturized microscopes (Miniscopes) and a genetically encoded Cre-dependent Ca2+ indicator (GCaMP6f). Systemic injections of oxycodone (3 mg/kg) increased locomotor activity yet, paradoxically, reduced concomitantly the number of active MSNs. The frequency of Ca2+ transients was significantly reduced in MSNs from A2A-Cre mice but not in those from D1-Cre mice. For comparative purposes, a separate group of mice was injected with a non-Cre dependent Ca2+ indicator in the cerebral cortex and the effects of the opioid also were tested. In contrast to MSNs, the frequency of Ca2+ transients in cortical pyramidal neurons was significantly increased by oxycodone administration. Additional electrophysiological studies in brain slices confirmed generalized inhibitory effects of oxycodone on MSNs, including membrane hyperpolarization, reduced excitability, and decreased frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results demonstrate a dissociation between locomotion and striatal MSN activity after acute administration of oxycodone.

Calcium imaging: A versatile tool to examine Huntington's disease mechanisms and progression.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that causes chorea, cognitive deficits, and psychiatric symptoms. It is characterized by accumulation of mutant Htt protein, which primarily impacts striatal medium-sized spiny neurons (MSNs), as well as cortical pyramidal neurons (CPNs), causing synapse loss and eventually cell death. Perturbed Ca2+ homeostasis is believed to play a major role in HD, as altered Ca2+ homeostasis often precedes striatal dysfunction and manifestation of HD symptoms. In addition, dysregulation of Ca2+ can cause morphological and functional changes in MSNs and CPNs. Therefore, Ca2+ imaging techniques have the potential of visualizing changes in Ca2+ dynamics and neuronal activity in HD animal models. This minireview focuses on studies using diverse Ca2+ imaging techniques, including two-photon microscopy, fiber photometry, and miniscopes, in combination of Ca2+ indicators to monitor activity of neurons in HD models as the disease progresses. We then discuss the future applications of Ca2+ imaging to visualize disease mechanisms and alterations associated with HD, as well as studies showing how, as a proof-of-concept, Ca2+imaging using miniscopes in freely-behaving animals can help elucidate the differential role of direct and indirect pathway MSNs in HD symptoms.

Synaptic pathology in Huntington's disease: Beyond the corticostriatal pathway.

Huntington's disease (HD) is a heritable, fatal neurodegenerative disorder caused by a mutation in the Huntingtin gene. It is characterized by chorea, as well as cognitive and psychiatric symptoms. Histopathologically, there is a massive loss of striatal projection neurons and less but significant loss in other areas throughout the cortico-basal ganglia-thalamocortical (CBGTC) loop. The mutant huntingtin protein has been implicated in numerous functions, including an important role in synaptic transmission. Most studies on anatomical and physiological alterations in HD have focused on striatum and cerebral cortex. However, based on recent CBGTC projectome evidence, the need to study other pathways has become increasingly clear. In this review, we examine the current status of our knowledge of morphological and electrophysiological alterations of those pathways in animal models of HD. Based on recent studies, there is accumulating evidence that synaptic disconnection, particularly along excitatory pathways, is pervasive and almost universal in HD, thus supporting a critical role of the huntingtin protein in synaptic transmission.

The mouse cortico-basal ganglia-thalamic network.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

Mechanisms underlying the enhancement of γ-aminobutyric acid responses in the external globus pallidus of R6/2 Huntington's disease model mice.

In Huntington's disease (HD), the output of striatal indirect pathway medium-sized spiny neurons (MSNs) is altered in its target region, the external globus pallidus (GPe). In a previous study we demonstrated that selective optogenetic stimulation of indirect pathway MSNs induced prolonged decay time of γ-aminobutyric acid (GABA) responses in GPe neurons. Here we identified the mechanism underlying this alteration. Electrophysiological recordings in slices from symptomatic R6/2 and wildtype (WT) mice were used to evaluate, primarily, the effects of GABA transporter (GAT) antagonists on responses evoked by optogenetic activation of indirect pathway MSNs. In addition, immunohistochemistry (IHC) and Western blots (WBs) were used to examine GAT-3 expression in HD and WT mice. A GAT-3 blocker (SNAP5114) increased decay time of GABA responses in WT and HD GPe neurons, but the effect was significantly greater in WT neurons. In contrast, a GAT-1 antagonist (NO-711) or a GABAB receptor antagonist (CGP 54626) produced small increases in decay time but no differential effects between genotypes. IHC and WBs showed reduction of GAT-3 expression in the GPe of HD mice. Thus, reduced expression or dysfunction of GAT-3 could underlie alterations of GPe responses to GABA inputs from striatum and could be a target for therapeutic intervention.

Paroxysmal Discharges in Tissue Slices From Pediatric Epilepsy Surgery Patients: Critical Role of GABAB Receptors in the Generation of Ictal Activity.

In the present study, we characterized the effects of bath application of the proconvulsant drug 4-aminopyridine (4-AP) alone or in combination with GABAA and/or GABAB receptor antagonists, in cortical dysplasia (CD type I and CD type IIa/b), tuberous sclerosis complex (TSC), and non-CD cortical tissue samples from pediatric epilepsy surgery patients. Whole-cell patch clamp recordings in current and voltage clamp modes were obtained from cortical pyramidal neurons (CPNs), interneurons, and balloon/giant cells. In pyramidal neurons, bath application of 4-AP produced an increase in spontaneous synaptic activity as well as rhythmic membrane oscillations. In current clamp mode, these oscillations were generally depolarizing or biphasic and were accompanied by increased membrane conductance. In interneurons, membrane oscillations were consistently depolarizing and accompanied by bursts of action potentials. In a subset of balloon/giant cells from CD type IIb and TSC cases, respectively, 4-AP induced very low-amplitude, slow membrane oscillations that echoed the rhythmic oscillations from pyramidal neurons and interneurons. Bicuculline reduced the amplitude of membrane oscillations induced by 4-AP, indicating that they were mediated principally by GABAA receptors. 4-AP alone or in combination with bicuculline increased cortical excitability but did not induce seizure-like discharges. Ictal activity was observed in pyramidal neurons and interneurons from CD and TSC cases only when phaclofen, a GABAB receptor antagonist, was added to the 4-AP and bicuculline solution. These results emphasize the critical and permissive role of GABAB receptors in the transition to an ictal state in pediatric CD tissue and highlight the importance of these receptors as a potential therapeutic target in pediatric epilepsy.

Pathological high frequency oscillations associate with increased GABA synaptic activity in pediatric epilepsy surgery patients.

Pathological high-frequency oscillations (HFOs), specifically fast ripples (FRs, >250 Hz), are pathognomonic of an active epileptogenic zone. However, the origin of FRs remains unknown. Here we explored the correlation between FRs recorded with intraoperative pre-resection electrocorticography (ECoG) and spontaneous synaptic activity recorded ex vivo from cortical tissue samples resected for the treatment of pharmacoresistant epilepsy. The cohort included 47 children (ages 0.22-9.99 yr) with focal cortical dysplasias (CD types I and II), tuberous sclerosis complex (TSC) and non-CD pathologies. Whole-cell patch clamp recordings were obtained from pyramidal neurons and interneurons in cortical regions that were positive or negative for pathological HFOs, defined as FR band oscillations (250-500 Hz) at ECoG. The frequency of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and IPSCs, respectively) was compared between HFO+ and HFO- regions. Regardless of pathological substrate, regions positive for FRs displayed significantly increased frequencies of sIPSCs compared with regions negative for FRs. In contrast, the frequency of sEPSCs was similar in both regions. In about one third of cases (n = 17), pacemaker GABA synaptic activity (PGA) was observed. In the vast majority (n = 15), PGA occurred in HFO+ areas. Further, fast-spiking interneurons displayed signs of hyperexcitability exclusively in HFO+ areas. These results indicate that, in pediatric epilepsy patients, increased GABA synaptic activity is associated with interictal FRs in the epileptogenic zone and suggest an active role of GABAergic interneurons in the generation of pathological HFOs. Increased GABA synaptic activity could serve to dampen excessive excitability of cortical pyramidal neurons in the epileptogenic zone, but it could also promote neuronal network synchrony.

Developmental origins of cortical hyperexcitability in Huntington's disease: Review and new observations.

Huntington's disease (HD), an inherited neurodegenerative disorder that principally affects striatum and cerebral cortex, is generally thought to have an adult onset. However, a small percentage of cases develop symptoms before 20 years of age. This juvenile variant suggests that brain development may be altered in HD. Indeed, recent evidence supports an important role of normal huntingtin during embryonic brain development and mutations in this protein cause cortical abnormalities. Functional studies also demonstrated that the cerebral cortex becomes hyperexcitable with disease progression. In this review, we examine clinical and experimental evidence that cortical development is altered in HD. We also provide preliminary evidence that cortical pyramidal neurons from R6/2 mice, a model of juvenile HD, are hyperexcitable and display dysmorphic processes as early as postnatal day 7. Further, some symptomatic mice present with anatomical abnormalities reminiscent of human focal cortical dysplasia, which could explain the occurrence of epileptic seizures in this genetic mouse model and in children with juvenile HD. Finally, we discuss recent treatments aimed at correcting abnormal brain development.

Pumas as ecosystem engineers: ungulate carcasses support beetle assemblages in the Greater Yellowstone Ecosystem.

Ecosystem engineers create physical changes in abiotic and biotic material, and through this process control the availability of resources for other species. Predators that abandon large portions of their prey may be ecosystem engineers that create habitat for carrion-dependent invertebrates that utilize carcasses during critical life-history periods. Between 04-May-2016 and 04-Oct-2016, we sampled beetle assemblages at 18 carcasses of prey killed by pumas and matching control sites in the southern Greater Yellowstone Ecosystem, USA, to measure the extent to which beetle families utilized these carcass "habitats". We used generalized linear-mixed models and linear-mixed effect models to examine changes in beetle abundance, species richness, and Simpson's Index of Diversity. We estimated kill rates and carrion production rates for individual pumas to better assess the impact of pumas on invertebrate communities. We collected 24,209 beetles representing 215 species. We identified eight beetle families that had significantly higher abundance at carcasses than control sites. Carcasses had a statistically large to very large effect (determined using Cohen's d) on beetle abundance, richness, and diversity for the initial 8 weeks of sampling. Our research revealed strong effects of an ecosystem engineer on beetle assemblages while highlighting the potential role of apex predators in creating and modifying physical habitats for carrion-dependent species. This suggests that there may be consequences for invertebrate communities where apex predators exist at reduced numbers or have been eradicated. The ecological role of invertebrates is often overlooked, yet they are essential taxa that provide critical ecological services upon which we depend.

Mutant huntingtin reduction in astrocytes slows disease progression in the BACHD conditional Huntington's disease mouse model.

Neuronal and non-neuronal cells express the huntingtin (HTT) protein, yet neurodegeneration in Huntington's disease (HD) is largely selective, affecting most prominently striatal medium spiny neurons and cortical pyramidal neurons. Selective toxicity of full-length human mutant HTT (fl-mHTT) may be due in part to its expression in non-neuronal cells. While studies suggest neuronal-glial interactions are important in HD and fl-mHTT is expressed in astrocytes, it has not been determined whether the expression of fl-mHTT in astrocytes is necessary for HD pathogenesis. To directly assess the necessity of fl-mHTT in astrocytes for HD pathogenesis, we used a mouse genetic approach and bred the conditional mHTT-expressing BACHD mouse model with GFAP-CreERT2 mice. We show that GFAP-CreERT2 expression in these mice is highly selective for astrocytes, and we are able to significantly reduce the expression of fl-mHTT protein in the striatum and cortex of BACHD/GFAP-CreERT2-tam mice. We performed behavioral, electrophysiological and neuropathological analyses of BACHD and BACHD/GFAP-CreERT2-tam mice. Behavioral analyses of BACHD/GFAP-CreERT2-tam mice demonstrate significant improvements in motor and psychiatric-like phenotypes. We observe improvements in neuropathological and electrophysiological phenotypes in BACHD/GFAP-CreERT2-tam mice compared to BACHD mice. We observed a restoration of the normal level αB-crystallin in the striatum of the BACHD/GFAP-CreERT2 mice, indicating a cell autonomous effect of mHTT on its expression. Taken together, this work indicates that astrocytes are important contributors to the progression of the behavioral and neuropathological phenotypes observed in HD.

Cellular antiseizure mechanisms of everolimus in pediatric tuberous sclerosis complex, cortical dysplasia, and non-mTOR-mediated etiologies.

The present study was designed to examine the potential cellular antiseizure mechanisms of everolimus, a mechanistic target of rapamycin (mTOR) pathway blocker, in pediatric epilepsy cases. Cortical tissue samples obtained from pediatric patients (n = 11, ages 0.67-6.75 years) undergoing surgical resections for the treatment of their pharmacoresistant epilepsy were examined electrophysiologically in ex vivo slices. The cohort included mTOR-mediated pathologies (tuberous sclerosis complex [TSC] and severe cortical dysplasia [CD]) as well as non-mTOR-mediated pathologies (tumor and perinatal infarct). Bath application of everolimus (2 µm) had practically no effect on spontaneous inhibitory postsynaptic activity. In contrast, long-term application of everolimus reduced spontaneous excitatory postsynaptic activity, burst discharges induced by blockade of γ-aminobutyric acid A (GABAA) receptors, and epileptiform activity generated by 4-aminopyridine, a K+ channel blocker. The antiseizure effects were more pronounced in TSC and CD cases, whereas in non-mTOR-mediated pathologies, the effects were subtle at best. These results support further clinical trials of everolimus in mTOR pathway-mediated pathologies and emphasize that the effects require sustained exposure over time.

Striatal Direct and Indirect Pathway Output Structures Are Differentially Altered in Mouse Models of Huntington's Disease.

The present study examined synaptic communication between direct and indirect output pathway striatal medium-sized spiny neurons (MSNs) and their target structures, the substantia nigra pars reticulata (SNr) and the external globus pallidus (GPe) in two mouse models of Huntington's disease (HD). Cre recombination, optogenetics, and whole-cell patch-clamp recordings were used to determine alterations in intrinsic and synaptic properties of SNr and GPe neurons from both male and female symptomatic R6/2 (>60 d) and presymptomatic (2 months) or symptomatic (10-12 months) YAC128 mice. Cell membrane capacitance was decreased, whereas input resistance was increased in SNr neurons from R6/2, but not YAC128 mice. The amplitude of GABAergic responses evoked by optogenetic stimulation of direct pathway terminals was reduced in SNr neurons of symptomatic mice of both models. A decrease in spontaneous GABA synaptic activity, in particular large-amplitude events, in SNr neurons also was observed. Passive membrane properties of GPe neurons were not different between R6/2 or YAC128 mice and their control littermates. Similarly, the amplitude of GABA responses evoked by activation of indirect pathway MSN terminals and the frequency of spontaneous GABA synaptic activity were similar in HD and control animals. In contrast, the decay time of the evoked GABA response was significantly longer in cells from HD mice. Interestingly, activation of indirect pathway MSNs within the striatum evoked larger-amplitude responses in direct pathway MSNs. Together, these results demonstrate differential alterations in responses evoked by direct and indirect pathway terminals in SNr and GPe leading to striatal output imbalance and motor dysfunction.SIGNIFICANCE STATEMENT Previous work on Huntington's disease (HD) focused on striatal medium-sized spiny neurons (MSNs) almost exclusively. Little is known about the effects that alterations in the striatum have on output structures of the direct and indirect pathways, the substantia nigra pars reticulata (SNr) and the external segment of the globus pallidus (GPe), respectively. We combined electrophysiological and optogenetic methods to examine responses evoked by selective activation of terminals of direct and indirect pathway MSNs in SNr and GPe neurons in two mouse models of HD. We show a differential disruption of synaptic communication between the direct and indirect output pathways of the striatum with their target regions leading to an imbalance of striatal output, which will contribute to motor dysfunction.

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.

Altered membrane properties and firing patterns of external globus pallidus neurons in the R6/2 mouse model of Huntington's disease.

In mouse models of Huntington's disease (HD), striatal neuron properties are significantly altered. These alterations predict changes in striatal output regions. However, little is known about alterations in those regions. The present study examines changes in passive and active membrane properties of neurons in the external globus pallidus (GPe), the first relay station of the indirect pathway, in the R6/2 mouse model of juvenile HD at presymptomatic (1 month) and symptomatic (2 month) stages. In GPe, two principal types of neurons can be distinguished based on firing properties and the presence (type A) or absence (type B) of Ih currents. In symptomatic animals (2 month), cell membrane capacitance and input resistance of type A neurons were increased compared with controls. In addition, action potential afterhyperpolarization amplitude was reduced. Although the spontaneous firing rate of GPe neurons was not different between control and R6/2 mice, the number of spikes evoked by depolarizing current pulses was significantly reduced in symptomatic R6/2 animals. In addition, these changes were accompanied by altered firing patterns evidenced by increased interspike interval variation and increased number of bursts. Blockade of GABAA receptors facilitated bursting activity in R6/2 mice but not in control littermates. Thus, alterations in firing patterns could be caused by changes in intrinsic membrane conductances and modulated by synaptic inputs. © 2016 Wiley Periodicals, Inc.

Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons.

Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav, but not Kv1.2, channels in mouse cerebellum. These results indicate that AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport before anchoring them to the AIS and nodes of Ranvier.

Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels.

The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

Coupling mechanical forces to electrical signaling: molecular motors and the intracellular transport of ion channels.

Proper localization of various ion channels is fundamental to neuronal functions, including postsynaptic potential plasticity, dendritic integration, action potential initiation and propagation, and neurotransmitter release. Microtubule-based forward transport mediated by kinesin motors plays a key role in placing ion channel proteins to correct subcellular compartments. PDZ- and coiled-coil-domain proteins function as adaptor proteins linking ionotropic glutamate and GABA receptors to various kinesin motors, respectively. Recent studies show that several voltage-gated ion channel/transporter proteins directly bind to kinesins during forward transport. Three major regulatory mechanisms underlying intracellular transport of ion channels are also revealed. These studies contribute to understanding how mechanical forces are coupled to electrical signaling and illuminating pathogenic mechanisms in neurodegenerative diseases.

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency.

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.