Anjali M. Rajadhyaksha

Enhanced CREB and DARPP-32 phosphorylation in the nucleus accumbens and CREB, ERK, and GluR1 phosphorylation in the dorsal hippocampus is associated with cocaine–conditioned place preference behavior

Environment‐induced relapse is a major concern in drug addiction because of the strong associations formed between drug reward and environment. Cocaine‐conditioned place preference is an ideal experimental tool to examine adaptations in the molecular pathways that are activated upon re‐exposure to an environment previously paired with drug reward. To better understand the mechanism of cocaine‐conditioned place preference we have used western blot analysis to examine changes in phosphorylation of cAMP‐response element binding protein (CREB), dopamine‐ and cyclic AMP‐regulated phosphoprotein 32 (DARPP‐32), extracellular signal‐regulated kinase (ERK) and GluR1, key molecular substrates altered by cocaine, in the nucleus accumbens (NAc) and dorsal hippocampus (DHC) of C57BL/6 mice. Our studies revealed that re‐exposing mice to an environment in which they were previously given cocaine resulted in increased levels of Ser133 phospho‐CREB and Thr34 phospho‐DARPP‐32 with a corresponding decrease in Thr75 phospho‐DARPP‐32 in the NAc. In DHC there were increased levels of phospho‐CREB, Thr183/Tyr185 phospho‐ERK, and Ser845 phospho‐GluR1. These data suggest that the formation of contextual drug reward associations involves recruitment of the DHC‐NAc circuit with activation of the DARPP‐32/CREB pathway in the NAc and the ERK/CREB pathway in the DHC.

Cav1.2 L-type Ca²? channels mediate cocaine-induced GluA1 trafficking in the nucleus accumbens, a long-term adaptation dependent on ventral tegmental area Ca(v)1.3 channels.

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization, we have examined phosphorylation of GluA1 at key residues serine 845 (S845) and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine-preexposed mice and the role of brain-specific Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs), therein. We found higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we found that a cocaine challenge that elicits expression of the cocaine-sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Cav1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Cav1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors expressing Cav1.3 and ERK2 siRNA further indicate that recruitment of the Cav1.2 pathway in the NAc is dependent on ventral tegmental area Cav1.3 LTCCs and ERK2. Together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.

L-Type Ca2+ Channels Mediate Adaptation of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation in the Ventral Tegmental Area after Chronic Amphetamine Treatment

L-type Ca2+ channels (LTCCs) play an important role in chronic psychostimulant-induced behaviors. However, the Ca2+ second messenger pathways activated by LTCCs after acute and recurrent psychostimulant administration that contribute to drug-induced molecular adaptations are poorly understood. Using a chronic amphetamine treatment paradigm in rats, we have examined the role of LTCCs in activating the mitogen-activated protein (MAP) kinase pathway in the ventral tegmental area (VTA), a primary target for the reinforcing properties of psychostimulants. Using immunoblot and immunohistochemical analyses, we find that in chronic saline-treated rats a challenge injection of amphetamine increases phosphorylation of MAP [extracellular signal-regulated kinase 1/2 (ERK1/2)] kinase in the VTA that is independent of LTCCs. However, in chronic amphetamine-treated rats there is no increase in amphetamine-mediated ERK1/2 phosphorylation unless LTCCs are blocked, in which case there is robust phosphorylation in VTA dopamine neurons. Examination of the expression of phosphatases reveals an increase in calcineurin [protein phosphatase 2B (PP2B)] and MAP kinase phosphatase-1 (MKP-1) in the VTA. Using in situ hybridization histochemistry and immunoblot analyses, we further examined the mRNA and protein expression of the LTCC subtypes Cav1.2 and Cav1.3 in VTA dopamine neurons in drug-naive animals and in rats after chronic amphetamine treatment. We found an increase in Cav1.2 mRNA and protein levels, with no change in Cav1.3. Together, our results suggest that one aspect of LTCC-induced changes in second messenger pathways after chronic amphetamine exposure involves activation of the MAP kinase phosphatase pathway by upregulation of Cav1.2 in VTA dopaminergic neurons.

Markers of Celiac Disease and Gluten Sensitivity in Children with Autism

Objective Gastrointestinal symptoms are a common feature in children with autism, drawing attention to a potential association with celiac disease or gluten sensitivity. However, studies to date regarding the immune response to gluten in autism and its association with celiac disease have been inconsistent. The aim of this study was to assess immune reactivity to gluten in pediatric patients diagnosed with autism according to strict criteria and to evaluate the potential link between autism and celiac disease. Methods Study participants included children (with or without gastrointestinal symptoms) diagnosed with autism according to both the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview, Revised (ADI-R) (n = 37), their unaffected siblings (n = 27), and age-matched healthy controls (n = 76). Serum specimens were tested for antibodies to native gliadin, deamidated gliadin, and transglutaminase 2 (TG2). Affected children were genotyped for celiac disease associated HLA-DQ2 and -DQ8 alleles. Results Children with autism had significantly higher levels of IgG antibody to gliadin compared with unrelated healthy controls (p<0.01). The IgG levels were also higher compared to the unaffected siblings, but did not reach statistical significance. The IgG anti-gliadin antibody response was significantly greater in the autistic children with gastrointestinal symptoms in comparison to those without them (p<0.01). There was no difference in IgA response to gliadin across groups. The levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between patients and controls. An association between increased anti-gliadin antibody and presence of HLA-DQ2 and/or -DQ8 was not observed. Conclusions A subset of children with autism displays increased immune reactivity to gluten, the mechanism of which appears to be distinct from that in celiac disease. The increased anti-gliadin antibody response and its association with GI symptoms points to a potential mechanism involving immunologic and/or intestinal permeability abnormalities in affected children.

Mutations in FLVCR1 Cause Posterior Column Ataxia and Retinitis Pigmentosa

The study of inherited retinal diseases has advanced our knowledge of the cellular and molecular mechanisms involved in sensory neural signaling. Dysfunction of two specific sensory modalities, vision and proprioception, characterizes the phenotype of the rare, autosomal-recessive disorder posterior column ataxia and retinitis pigmentosa (PCARP). Using targeted DNA capture and high-throughput sequencing, we analyzed the entire 4.2 Mb candidate sequence on chromosome 1q32 to find the gene mutated in PCARP in a single family. Employing comprehensive bioinformatic analysis and filtering, we identified a single-nucleotide coding variant in the feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), a gene encoding a heme-transporter protein. Sanger sequencing confirmed the FLVCR1 mutation in this family and identified different homozygous missense mutations located within the proteins transmembrane channel segment in two other unrelated families with PCARP. To determine whether the selective pathologic features of PCARP correlated with FLVCR1 expression, we examined wild-type mouse Flvcr1 mRNA levels in the posterior column of the spinal cord and the retina via quantitative real-time reverse-transcriptase PCR. The Flvcr1 mRNA levels were most abundant in the retina, followed by the posterior column of the spinal cord and other brain regions. These results suggest that aberrant FLVCR1 causes a selective degeneration of a subpopulation of neurons in the retina and the posterior columns of the spinal cord via dysregulation of heme or iron homeostasis. This finding broadens the molecular basis of sensory neural signaling to include common mechanisms that involve proprioception and vision.

Forebrain elimination of cacna1c mediates anxiety-like behavior in mice.

The CACNA1C gene encoding the Cav1.2 subunit of the L-type calcium channel has emerged as a new candidate gene for neuropsychiatric disease, including bipolar disorder, major depression, schizophrenia and autism.1, 2, 3 We report that global haploinsufficiency, forebrain-specific elimination and prefrontal cortex (PFC)-specific knockdown of cacna1c all increase anxiety-related behavior in mice, a prominent component of the forms of neuropsychiatric disease in which aberrations in CACNA1C have been implicated, without affecting compulsive behavior. Constitutive cacna1c heterozygous mice (HET) were evaluated in three behavioral assays related to anxiety: open field test, light–dark conflict test and elevated plus maze (EPM). HETs displayed anxiety-like behavior in the EPM (Figure 1a), spending significantly less time exploring the open arms compared with wild-type littermate controls (WT; F1,19=6.437; P<0.05). However, no differences were observed between HETs and WTs in the open field and light–dark conflict test, (Figures 1a and b, Supplementary Material). We also observed a similar statistically significant effect of increased anxiety-like behavior compared with WTs in EPM in adult female HETs (Figure 1d, Supplementary Material) and adolescent male HETs (Figure 1e, Supplementary Material). To more specifically investigate the function of cacna1c in the brain, we generated forebrain-specific conditional cacna1c-deficient mice (forebrain-cacna1c cKO) by crossing cacna1c-floxed mice with mice harboring alphaCaM Kinase II promoter-driven expression of Cre recombinase.4 Relative to WTs, this strategy achieved ∼70% elimination of cacna1c mRNA in the hippocampus, PFC, basolateral amygdala, striatum and nucleus accumbens, as assessed by quantitative PCR (Table 1, Supplementary Material). Cacna1c mRNA levels were unaffected in the ventral tegmental area and cerebellum. With this greater reduction in cacna1c in forebrain than could be achieved in HETs, significantly increased anxiety-like behavior was observed across all three behavioral assays. In EPM, forebrain-cacna1c cKO mice spent significantly less time exploring the open arms compared with WTs (Figure 1b, F1,16=68.587; P<0.0001 and Figure 2c, Supplementary Material). In the open field test, forebrain-cacna1c cKO mice spent less time exploring the center of the chamber compared with WTs (Figures 2a and 3a, Supplementary Material). In the light-dark conflict test, forebrain-cacna1c cKO mice spent significantly less time in the brightly lit side compared with WTs (Figures 2b and 3b, Supplementary Material). Figure 1 Anxiety-like behavior as measured in the elevated plus maze (EPM) assay is shown for (a) cacna1c haplosufficient (cacna1c HET; n=10) and wild-type (WT; n=11) littermates, (b) forebrain-specific cacna1c knockout (forebrain-cacna1c cKO; n=8) and WT controls ... Clinically, anxiety is often accompanied by compulsive behavior, such as in obsessive-compulsive disorder (OCD), in which patients seek alleviation from recurrent bouts of anxiety-inducing intrusive thoughts by engaging in compulsively repetitive behaviors. Experimental models for OCD, such as SAPAP3-5 or SLITRK5-deficient6 mice, display pathologically high compulsive grooming that is readily quantified by the spray-induced grooming test. Compared with respective WTs, we did not observe elevated grooming in either HETs or forebrain-cacna1c cKO mice (Figures 1c and 3c, Supplementary Material). Thus, the form of anxiety associated with cacna1c function is distinct from that associated with OCD spectrum illnesses. Some genetic variations in CACNA1C have been associated with altered PFC function7, 8, 9 in neuropsychiatric disease, so we next generated focal elimination of cacna1c in the PFC with adeno-associated viral (AAV) vector-expressing Cre recombinase (AAV-Cre).10 AAV-Cre was stereotaxically delivered bilaterally into the PFC of floxed cacna1c mice, and regional elimination of Cav1.2 was immunohistochemically confirmed (Figures 1d and e). Following elimination of cacna1c in the PFC, mice showed no differences in basal locomotor activity compared with AAV-GFP control injected mice (Figure 4, Supplementary Material). However, selective elimination of cacna1c in the PFC was associated with less time spent exploring open arms of the EPM, compared with control AAV-GFP injected mice (Figure 1c, F1,16=5.477; P<0.05). To evaluate the specificity of PFC cacna1c knockdown in mediating anxiety, we used AAV-expressing cacna1d siRNA10 to selectively eliminate cacna1d in the PFC, the other L-type Ca2+ channel isoform expressed in brain. Selective knockdown of cacna1d in the PFC had no effect on locomotor behavior (Figure 5a, Supplementary Material) or time spent in open arms in the EPM (Figure 5b, Supplementary Material). In summary, we report here the first direct evidence for a role of forebrain cacna1c in regulating anxiety. Mice harboring forebrain-specific elimination of cacna1c may thus provide a useful tool for studying the pathophysiology of anxiety in forms of neuropsychiatric diseases in which CACNA1C is implicated.

Dysregulation of large-conductance Ca2+-activated K+ channel expression in nonsyndromal mental retardation due to a cereblon p.R419X mutation.

A nonsense mutation (R419X) in the human cereblon gene [mutation (mut) CRBN] causes a mild type of autosomal recessive nonsyndromal mental retardation (ARNSMR). CRBN, a cytosolic protein, regulates the assembly and neuronal surface expression of large-conductance Ca2+-activated K+ channels (BKCa) in brain regions involved in memory and learning. Using the real-time quantitative polymerase chain reaction, we show that mut CRBN disturbs the development of adult brain BKCa isoforms. These changes are predicted to result in BKCa channels with a higher intracellular Ca2+ sensitivity, faster activation, and slower deactivation kinetics. Such alterations may contribute to cognitive impairments in patients with mild ARNSMR.

Molecular Switch from L-Type Cav1.3 to Cav1.2 Ca2+ Channel Signaling Underlies Long-Term Psychostimulant-Induced Behavioral and Molecular Plasticity

L-type Ca2+ channel (LTCC)-activated signaling cascades contribute significantly to psychostimulant-induced locomotor sensitization; however, the precise contribution of the two brain-specific subunits Cav1.2 and Cav1.3 remains mostly unknown. In this study, by using amphetamine and cocaine locomotor sensitization in mutant mice expressing dihydropyridine (DHP)-insensitive Cav1.2 LTCCs (Cav1.2DHP−/−), we find that, as opposed to a previously identified role of the Cav1.3 subunit of LTCCs in development of sensitization, the Cav1.2 subunit mediates expression of amphetamine and cocaine sensitization when examined after a 14 d drug-free period. Molecular studies to further elucidate the role of Cav1.2 versus Cav1.3 LTCCs in activating signaling pathways in the nucleus accumbens (NAc) of drug-naive versus drug-preexposed mice examined 14 d later revealed that an acute amphetamine and cocaine challenge in drug-naive mice increases Ser133 cAMP response element-binding protein (CREB) phosphorylation in the NAc via Cav1.3 channels and via a dopamine D1-dependent mechanism, independent of the extracellular signal-regulated kinase (ERK) pathway, an important mediator of psychostimulant-induced plasticity. In contrast, in amphetamine- and cocaine-preexposed mice, an amphetamine or cocaine challenge no longer activates CREB unless Cav1.2 LTCCs are blocked. This Cav1.2-dependent blunting of CREB activation that underlies expression of locomotor sensitization occurs only after extended drug-free periods and involves recruitment of D1 receptors and the ERK pathway. Thus, our results demonstrate that specific LTCC subunits are required for the development (Cav1.3) versus expression (Cav1.2) of psychostimulant sensitization and that subunit-specific signaling pathways recruited by psychostimulants underlies long-term drug-induced behavioral responses.

Up-regulation of dopamine D2L mRNA levels in the ventral tegmental area and dorsal striatum of amphetamine-sensitized C57BL/6 mice : role of Cav1.3 L-type Ca2+ channels

Dopamine D2 long (D2L) and D2 short (D2S) isoforms of the D2 receptor play an important role in psychostimulant‐induced neuronal adaptations. In this study, we used quantitative real‐time PCR to specifically amplify these two splice variants to examine their mRNA expression in the dorsal striatum (dStr), nucleus accumbens (NAc) and the ventral tegmental area (VTA) of amphetamine‐sensitized C57BL/6 mice. We found a significant increase in D2L mRNA in the VTA and dStr of amphetamine‐treated mice that positively correlated with the sensitized locomotor response. We also found a significant increase in D2S mRNA in the VTA. We further examined the role of the Cav1.3 subtype of L‐type Ca2+ channels in up‐regulation of D2L and D2S mRNA in the VTA. Amphetamine‐pretreated Cav1.3 wild‐type (Cav1.3+/+) mice exhibited sensitized behavior and a significant increase in D2L and D2S mRNA compared with saline‐pretreated mice Amphetamine‐pretreated homozygous Cav1.3 knockout (Cav1.3–/–) mice did not exhibit sensitized behavior. There was a significant increase in D2S mRNA, but not D2L mRNA. In conclusion, our results find that amphetamine increases D2L mRNA expression in the dStr and the VTA, an adaptation that correlates with expression of sensitized behavior and dependence on Cav1.3 Ca2+ channels.

Energy deficit in parvalbumin neurons leads to circuit dysfunction, impaired sensory gating and social disability.

Parvalbumin-expressing, fast spiking interneurons have high-energy demands, which make them particularly susceptible to energy impairment. Recent evidence suggests a link between mitochondrial dysfunction in fast spiking cortical interneurons and neuropsychiatric disorders. However, the effect of mitochondrial dysfunction restricted to parvalbumin interneurons has not been directly addressed in vivo. To investigate the consequences of mitochondrial dysfunction in parvalbumin interneurons in vivo, we generated conditional knockout mice with a progressive decline in oxidative phosphorylation by deleting cox10 gene selectively in parvalbumin neurons (PV-Cox10 CKO). Cox10 ablation results in defective assembly of cytochrome oxidase, the terminal enzyme of the electron transfer chain, and leads to mitochondrial bioenergetic dysfunction. PV-Cox10 CKO mice showed a progressive loss of cytochrome oxidase in cortical parvalbumin interneurons. Cytochrome oxidase protein levels were significantly reduced starting at postnatal day 60, and this was not associated with a change in parvalbumin interneuron density. Analyses of intrinsic electrophysiological properties in layer 5 primary somatosensory cortex revealed that parvalbumin interneurons could not sustain their typical high frequency firing, and their overall excitability was enhanced. An increase in both excitatory and inhibitory input onto parvalbumin interneurons was observed in PV-Cox10 CKO mice, resulting in a disinhibited network with an imbalance of excitation/inhibition. Investigation of network oscillations in PV-Cox10 CKO mice, using local field potential recordings in anesthetized mice, revealed significantly increased gamma and theta frequency oscillation power in both medial prefrontal cortex and hippocampus. PV-Cox10 CKO mice did not exhibit muscle strength or gross motor activity deficits in the time frame of the experiments, but displayed impaired sensory gating and sociability. Taken together, these data reveal that mitochondrial dysfunction in parvalbumin interneurons can alter their intrinsic physiology and network connectivity, resulting in behavioral alterations similar to those observed in neuropsychiatric disorders, such as schizophrenia and autism.