Knockdown of the Non-canonical Wnt Gene Prickle2 Leads to Cerebellar Purkinje Cell Abnormalities While Cerebellar-Mediated Behaviors Remain Intact.

Autism spectrum disorders (ASD) involve brain wide abnormalities that contribute to a constellation of symptoms including behavioral inflexibility, cognitive dysfunction, learning impairments, altered social interactions, and perceptive time difficulties. Although a single genetic variation does not cause ASD, genetic variations such as one involving a non-canonical Wnt signaling gene, Prickle2, has been found in individuals with ASD. Previous work looking into phenotypes of Prickle2 knock-out (Prickle2-/-) and heterozygous mice (Prickle2-/+) suggest patterns of behavior similar to individuals with ASD including altered social interaction and behavioral inflexibility. Growing evidence implicates the cerebellum in ASD. As Prickle2 is expressed in the cerebellum, this animal model presents a unique opportunity to investigate the cerebellar contribution to autism-like phenotypes. Here, we explore cerebellar structural and physiological abnormalities in animals with Prickle2 knockdown using immunohistochemistry, whole-cell patch clamp electrophysiology, and several cerebellar-associated motor and timing tasks, including interval timing and eyeblink conditioning. Histologically, Prickle2-/- mice have significantly more empty spaces or gaps between Purkinje cells in the posterior lobules and a decreased propensity for Purkinje cells to fire action potentials. These structural cerebellar abnormalities did not impair cerebellar-associated behaviors as eyeblink conditioning and interval timing remained intact. Therefore, although Prickle-/- mice show classic phenotypes of ASD, they do not recapitulate the involvement of the adult cerebellum and may not represent the pathophysiological heterogeneity of the disorder.

Effects of acid-sensing ion channel-1A (ASIC1A) on cocaine-induced synaptic adaptations.

Chronic drug abuse is thought to induce synaptic changes in nucleus accumbens medium spiny neurons (MSNs) that promote subsequent craving and drug-seeking behavior. Accumulating data suggest acid-sensing ion channels (ASICs) may play a critical role. In drug naïve mice, disrupting the ASIC1A subunit produced a variety of synaptic changes reminiscent of wild-type mice following cocaine withdrawal, including increased AMPAR/NMDAR ratio, increased AMPAR rectification, and increased dendrite spine density. Importantly, these changes in Asic1a -/- mice were normalized by a single dose of cocaine. Here we sought to understand the temporal effects of cocaine exposure in Asic1a -/- mice and the cellular site of ASIC1A action. Six hours after cocaine exposure, there was no effect. However, 15 h, 24 h and 4 days after cocaine exposure there was a significant reduction in AMPAR/NMDAR ratio in Asic1a -/- mice. Within 7 days the AMPAR/NMDAR ratio had returned to baseline levels. Cocaine-evoked changes in AMPAR rectification and dendritic spine density followed a similar time course with significant reductions in rectification and dendritic spines 24 h after cocaine exposure in Asic1a -/- mice. To test the cellular site of ASIC1A action on these responses, we disrupted ASIC1A specifically in a subpopulation of MSNs. We found that effects of ASIC1A disruption were cell autonomous and restricted to neurons in which the channels are disrupted. We further tested whether ASIC1A disruption differentially affects MSNs subtypes and found AMPAR/NMDAR ratio was elevated in dopamine receptor 1-expressing MSNs, suggesting a preferential effect for these cells. Finally, we tested if protein synthesis was involved in synaptic adaptations that occurred after ASIC1A disruption, and found the protein synthesis inhibitor anisomycin normalized AMPAR-rectification and AMPAR/NMDAR ratio in drug-naïve Asic1a -/- mice to control levels, observed in wild-type mice. Together, these results provide valuable mechanistic insight into the effects of ASICs on synaptic plasticity and drug-induced effects and raise the possibility that ASIC1A might be therapeutically manipulated to oppose drug-induced synaptic changes and behavior.

Intoxicating effects of alcohol depend on acid-sensing ion channels.

Persons at risk for developing alcohol use disorder (AUD) differ in their sensitivity to acute alcohol intoxication. Alcohol effects are complex and thought to depend on multiple mechanisms. Here, we explored whether acid-sensing ion channels (ASICs) might play a role. We tested ASIC function in transfected CHO cells and amygdala principal neurons, and found alcohol potentiated currents mediated by ASIC1A homomeric channels, but not ASIC1A/2 A heteromeric channels. Supporting a role for ASIC1A in the intoxicating effects of alcohol in vivo, we observed marked alcohol-induced changes on local field potentials in basolateral amygdala, which differed significantly in Asic1a-/- mice, particularly in the gamma, delta, and theta frequency ranges. Altered electrophysiological responses to alcohol in mice lacking ASIC1A, were accompanied by changes in multiple behavioral measures. Alcohol administration during amygdala-dependent fear conditioning dramatically diminished context and cue-evoked memory on subsequent days after the alcohol had cleared. There was a significant alcohol by genotype interaction. Context- and cue-evoked memory were notably worse in Asic1a-/- mice. We further examined acute stimulating and sedating effects of alcohol on locomotor activity, loss of righting reflex, and in an acute intoxication severity scale. We found loss of ASIC1A increased the stimulating effects of alcohol and reduced the sedating effects compared to wild-type mice, despite similar blood alcohol levels. Together these observations suggest a novel role for ASIC1A in the acute intoxicating effects of alcohol in mice. They further suggest that ASICs might contribute to intoxicating effects of alcohol and AUD in humans.

Carbonic anhydrase 4 disruption decreases synaptic and behavioral adaptations induced by cocaine withdrawal.

Cocaine use followed by withdrawal induces synaptic changes in nucleus accumbens (NAc), which are thought to underlie subsequent drug-seeking behaviors and relapse. Previous studies suggest that cocaine-induced synaptic changes depend on acid-sensing ion channels (ASICs). Here, we investigated potential involvement of carbonic anhydrase 4 (CA4), an extracellular pH-buffering enzyme. We examined effects of CA4 in mice on ASIC-mediated synaptic transmission in medium spiny neurons (MSNs) in NAc, as well as on cocaine-induced synaptic changes and behavior. We found that CA4 is expressed in the NAc and present in synaptosomes. Disrupting CA4 either globally, or locally, increased ASIC-mediated synaptic currents in NAc MSNs and protected against cocaine withdrawal-induced changes in synapses and cocaine-seeking behavior. These findings raise the possibility that CA4 might be a previously unidentified therapeutic target for addiction and relapse.

Ca2+-Binding Protein 1 Regulates Hippocampal-dependent Memory and Synaptic Plasticity.

Ca2+-binding protein 1 (CaBP1) is a Ca2+-sensing protein similar to calmodulin that potently regulates voltage-gated Ca2+ channels. Unlike calmodulin, however, CaBP1 is mainly expressed in neuronal cell-types and enriched in the hippocampus, where its function is unknown. Here, we investigated the role of CaBP1 in hippocampal-dependent behaviors using mice lacking expression of CaBP1 (C-KO). By western blot, the largest CaBP1 splice variant, caldendrin, was detected in hippocampal lysates from wild-type (WT) but not C-KO mice. Compared to WT mice, C-KO mice exhibited mild deficits in spatial learning and memory in both the Barnes maze and in Morris water maze reversal learning. In contextual but not cued fear-conditioning assays, C-KO mice showed greater freezing responses than WT mice. In addition, the number of adult-born neurons in the hippocampus of C-KO mice was ∼40% of that in WT mice, as measured by bromodeoxyuridine labeling. Moreover, hippocampal long-term potentiation was significantly reduced in C-KO mice. We conclude that CaBP1 is required for cellular mechanisms underlying optimal encoding of hippocampal-dependent spatial and fear-related memories.

Decalmodulation of Cav1 channels by CaBPs.

Ca(2+)-dependent inactivation (CDI) is a negative feedback regulation of voltage-gated Cav1 and Cav2 channels that is mediated by the Ca(2+) sensing protein, calmodulin (CaM), binding to the pore-forming Cav α1 subunit. David Yue and his colleagues made seminal contributions to our understanding of this process, as well as factors that regulate CDI. Important in this regard are members of a family of Ca(2+) binding proteins (CaBPs) that are related to calmodulin. CaBPs are expressed mainly in neural tissues and can antagonize CaM-dependent CDI for Cav1 L-type channels. This review will focus on the roles of CaBPs as Cav1-interacting proteins, and the significance of these interactions for vision, hearing, and neuronal Ca(2+) signaling events.

Synaptic depolarization is more effective than back-propagating action potentials during induction of associative long-term potentiation in hippocampal pyramidal neurons.

Long-term potentiation (LTP) requires postsynaptic depolarization that can result from EPSPs paired with action potentials or larger EPSPs that trigger dendritic spikes. We explored the relative contribution of these sources of depolarization to LTP induction during synaptically driven action potential firing in hippocampal CA1 pyramidal neurons. Pairing of a weak test input with a strong input resulted in large LTP (approximately 75% increase) when the weak and strong inputs were both located in the apical dendrites. This form of LTP did not require somatic action potentials. When the strong input was located in the basal dendrites, the resulting LTP was smaller (< or =25% increase). Pairing the test input with somatically evoked action potentials mimicked this form of LTP. Thus, back-propagating action potentials may contribute to modest LTP, but local synaptic depolarization and/or dendritic spikes mediate a stronger form of LTP that requires spatial proximity of the associated synaptic inputs.

Active and passive membrane properties and intrinsic kinetics shape synaptic inhibition in hippocampal CA1 pyramidal neurons.

The impact of synaptic inhibition depends on the passive and active properties of the neuronal membrane as well as on the characteristics of the underlying synaptic conductances. Here, we evaluated the contributions of these different factors to the IPSPs produced by two kinetically and anatomically distinct inhibitory synapses onto hippocampal CA1 pyramidal neurons: somatic GABA(A,fast) and dendritic GABA(A,slow). Using combined current-clamp and voltage-clamp recordings from neurons in hippocampal brain slices, we found that despite pronounced differences in kinetics and only weak voltage dependence of the underlying synaptic conductances, there were much smaller differences in duration but strong voltage dependence of IPSPs arising from somatic and dendritic synapses. Pharmacologic tests and compartmental modeling showed that these effects were produced by the hyperpolarization-activated cation current, I(H), which accelerated IPSP decay over a broad range of membrane potentials and reduced IPSP amplitudes at hyperpolarized potentials, and the persistent sodium current, I(NaP), which prolonged and amplified IPSPs at depolarized subthreshold potentials. The relative magnitudes of their influences depended on the kinetics of the underlying synaptic conductances: the effect on duration was greater for GABA(A,fast) and on amplitude was greater for GABA(A,slow). Passive and active factors thus influence the impact of synaptic inhibition in a location- and voltage-dependent manner.

Development of GABA(A) receptor-mediated inhibitory postsynaptic currents in hippocampus.

Hippocampal CA1 pyramidal cells receive two kinetic classes of GABA(A) receptor-mediated inhibition: slow dendritic inhibitory postsynaptic currents (GABA(A,slow) IPSCs) and fast perisomatic (GABA(A,fast)) IPSCs. These two classes of IPSCs are likely generated by two distinct groups of interneurons, and we have previously shown that the kinetics of the IPSCs have important functional consequences for generating synchronous firing patterns. Here, we studied developmental changes in the properties of GABA(A,fast) and GABA(A,slow) spontaneous, miniature, and evoked IPSCs (sIPSCs, mIPSCs, and eIPSCs, respectively) using whole cell voltage-clamp recordings in brain slices from animals aged P10-P35. We found that the rate of GABA(A,slow) sIPSCs increased by over 70-fold between P11 and P35 (from 0.0017 to 0.12 s(-1)). Over this same age range, we observed a >3.5-fold increase in the maximal amplitude of GABA(A,slow) eIPSCs evoked by stratum lacunosum-moleculare (SL-M) stimuli. However, the rate and amplitude of GABA(A,slow) mIPSCs remained unchanged between P10 and P30, suggesting that the properties of GABA(A,slow) synapses remained stable over this age range, and that the increase in sIPSC rate and in eIPSC amplitude was due to increased excitability or excitation of GABA(A,slow) interneurons. This hypothesis was tested using bath application of norepinephrine (NE), which we found at low concentrations (1 microM) selectively increased the rate of GABA(A,slow) sIPSCs while leaving GABA(A,fast) sIPSCs unchanged. This effect was observed in animals as young as P13 and was blocked by coapplication of tetrodotoxin, suggesting that NE was acting to increase the spontaneous firing rate of GABA(A,slow) interneurons and consistent with our hypothesis that developmental changes in GABA(A,slow) IPSCs are due to changes in presynaptic excitability. In contrast to the changes we observed in GABA(A,slow) IPSCs, the properties of GABA(A,fast) sIPSCs remained largely constant between P11 and P35, whereas the rate, amplitude, and kinetics of GABA(A,fast) mIPSCs showed significant changes between P10 and P30, suggesting counterbalancing changes in action potential-dependent GABA(A,fast) sIPSCs. These observations suggest differential developmental regulation of the firing properties of GABA(A,fast) and GABA(A,slow) interneurons in CA1 between P10 and P35.