K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons.

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Forward Genetic Screen in Caenorhabditis elegans Suggests F57A10.2 and acp-4 As Suppressors of C9ORF72 Related Phenotypes.

An abnormally expanded GGGGCC repeat in C9ORF72 is the most frequent causal mutation associated with amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration (FTLD). Both gain-of-function (gf) and loss-of-function (lf) mechanisms have been involved in C9ORF72 related ALS/FTLD. The gf mechanism of C9ORF72 has been studied in various animal models but not in C. elegans. In the present study, we described mutant C9ORF72 modeling in C. elegans and report the finding of two suppressor genes. We made transgenes containing 9 or 29 repeats of GGGGCC in C9ORF72, driven by either the hsp-16 promoters or the unc-119 promoter. Transgenic worms were made to carry such transgenes. Phenotypic analysis of those animals revealed that Phsp-16::(G4C2)29::GFP transgenic animals (EAB 135) displayed severe paralysis by the second day of adulthood, followed by lethality, which phenotypes were less severe in Phsp-16::(G4C2)9::GFP transgenic animals (EAB242), and absent in control strains expressing empty vectors. Suppressor genes of this locomotor phenotype were pursued by introducing mutations with ethyl methanesulfonate in EAB135, screening mutant strains that moved faster than EAB135 by a food-ring assay, identifying mutations by whole-genome sequencing and testing the underlying mechanism of the suppressor genes either by employing RNA interference studies or C. elegans genetics. Three mutant strains, EAB164, EAB165 and EAB167, were identified. Eight suppressor genes carrying nonsense/canonical splicing site mutations were confirmed, among which a nonsense mutation of F57A10.2/VAMP was found in all three mutant strains, and a nonsense mutation of acp-4/ACP2 was only found in EAB164. Knock down/out of those two genes in EAB135 animals by feeding RNAi/introducing a known acp-4 null allele phenocopied the suppression of the C9ORF72 variant related movement defect in the mutant strains. Translational conformation in a mammalian system is required, but our worm data suggest that altering acp-4/ACP2 encoding lysosomal acid phosphatase may provide a potential therapeutic method of reducing acp-4/ACP2 levels, as opposed or complementary to directly reducing C9ORF72, to relieve C9ORF72-ALS phenotypes. It also suggests that the C9ORF72-ALS/FTLD may share a pathophysiologic mechanism with vesicle-associated membrane protein-associated protein B, a homolog of F57A10.2/VAMP, which is a proven ALS8 gene.

Acute and chronic effects of clozapine on cholinergic transmission in cultured mouse superior cervical ganglion neurons.

Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia, possibly through modulation of the cholinergic system. However, little is known about specific underlying mechanisms. We investigated the acute and chronic effects of clozapine on cholinergic synaptic transmission in cultured superior cervical ganglion (SCG) neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected and were reversibly inhibited by the nicotinic receptor antagonist d-tubocurarine, confirming that the synaptic responses were primarily mediated by nicotinic receptors. Bath application of clozapine at therapeutic concentrations rapidly and reversely inhibited both the amplitude and frequency of sEPSCs in a concentration-dependent manner, without changing either rise or decay time, suggesting that clozapine effects have both presynaptic and postsynaptic origins. The acute effects of clozapine on sEPSCs were recapitulated by chronic treatment of SCG cultures with similar concentrations of clozapine, as clozapine treatment for 4 d reduced the frequency and amplitude of sEPSCs without affecting their kinetics. Cell survival analysis indicated that SCG neuron cell counts after chronic clozapine treatment were comparable to the control group. These results demonstrate that therapeutic concentrations of clozapine suppress nicotinic synaptic transmission in SCG cholinergic synapses, a simple in vitro preparation of cholinergic transmission.

Dopamine-enabled anti-Hebbian timing-dependent plasticity in prefrontal circuitry.

Spike timing-dependent plasticity (STDP) of glutamatergic synapses is a Hebbian associative plasticity that may underlie certain forms of learning. A cardinal feature of STDP is its dependence on the temporal order of presynaptic and postsynaptic spikes during induction: pre-post (positive) pairings induce t-LTP (timing-dependent long-term potentiation) whereas post-pre (negative) pairings induce t-LTD (timing-dependent long-term depression). Dopamine (DA), a reward signal for behavioral learning, is believed to exert powerful modulations on synapse strength and plasticity, but its influence on STDP has remained incompletely understood. We previously showed that DA extends the temporal window of t-LTP in the prefrontal cortex (PFC) from +10 to +30 ms, gating Hebbian t-LTP. Here, we examined DA modulation of synaptic plasticity induced at negative timings in layer V pyramidal neurons on mouse medial PFC slices. Using a negative timing STDP protocol (60 post-pre pairings at 0.1 Hz, δt = -30 ms), we found that DA applied during post-pre pairings did not produce LTD, but instead enabled robust LTP. This anti-Hebbian t-LTP depended on GluN2B-containing NMDA receptors. Blocking D1- (D1Rs), but not D2- (D2Rs) class DA receptors or disrupting cAMP/PKA signaling in pyramidal neurons also abolished this atypical t-LTP, indicating that it was mediated by postsynaptic D1R-cAMP/PKA signaling in excitatory synapses. Unlike DA-enabled Hebbian t-LTP that requires suppression of GABAergic inhibition and cooperative actions of both D1Rs and D2Rs in separate PFC excitatory and inhibitory circuits, DA-enabled anti-Hebbian t-LTP occurred under intact inhibitory transmission and only required D1R activation in excitatory circuit. Our results establish DA as a potent modulator of coincidence detection during associative synaptic plasticity and suggest a mechanism by which DA facilitates input-target association during reward learning and top-down information processing in PFC circuits.

Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95.

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

A genome-wide RNAi screen in Caenorhabditis elegans identifies the nicotinic acetylcholine receptor subunit ACR-7 as an antipsychotic drug target.

We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.