Dopamine D1R-neuron cacna1c deficiency: a new model of extinction therapy-resistant post-traumatic stress.

Post-traumatic stress disorder (PTSD) is characterized by persistent fear memory of remote traumatic events, mental re-experiencing of the trauma, long-term cognitive deficits, and PTSD-associated hippocampal dysfunction. Extinction-based therapeutic approaches acutely reduce fear. However, many patients eventually relapse to the original conditioned fear response. Thus, understanding the underlying molecular mechanisms of this condition is critical to developing new treatments for patients. Mutations in the neuropsychiatric risk gene CACNA1C, which encodes the Cav1.2 isoform of the L-type calcium channel, have been implicated in both PTSD and highly comorbid neuropsychiatric conditions, such as anxiety and depression. Here, we report that male mice with global heterozygous loss of cacna1c exhibit exacerbated contextual fear that persists at remote time points (up to 180 days after shock), despite successful acute extinction training, reminiscent of PTSD patients. Because dopamine has been implicated in contextual fear memory, and Cav1.2 is a downstream target of dopamine D1-receptor (D1R) signaling, we next generated mice with specific deletion of cacna1c from D1R-expressing neurons (D1-cacna1cKO mice). Notably, D1-cacna1cKO mice also show the same exaggerated remote contextual fear, as well as persistently elevated anxiety-like behavior and impaired spatial memory at remote time points, reminiscent of chronic anxiety in treatment-resistant PTSD. We also show that D1-cacna1cKO mice exhibit elevated death of young hippocampal neurons, and that treatment with the neuroprotective agent P7C3-A20 eradicates persistent remote fear. Augmenting survival of young hippocampal neurons may thus provide an effective therapeutic approach for promoting durable remission of PTSD, particularly in patients with CACNA1C mutations or other genetic aberrations that impair calcium signaling or disrupt the survival of young hippocampal neurons.

Correction: D-cycloserine improves synaptic transmission in an animal mode of Rett syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0183026.].

D-cycloserine improves synaptic transmission in an animal model of Rett syndrome.

Rett syndrome (RTT), a leading cause of intellectual disability in girls, is predominantly caused by mutations in the X-linked gene MECP2. Disruption of Mecp2 in mice recapitulates major features of RTT, including neurobehavioral abnormalities, which can be reversed by re-expression of normal Mecp2. Thus, there is reason to believe that RTT could be amenable to therapeutic intervention throughout the lifespan of patients after the onset of symptoms. A common feature underlying neuropsychiatric disorders, including RTT, is altered synaptic function in the brain. Here, we show that Mecp2tm1.1Jae/y mice display lower presynaptic function as assessed by paired pulse ratio, as well as decreased long term potentiation (LTP) at hippocampal Schaffer-collateral-CA1 synapses. Treatment of Mecp2tm1.1Jae/y mice with D-cycloserine (DCS), an FDA-approved analog of the amino acid D-alanine with antibiotic and glycinergic activity, corrected the presynaptic but not LTP deficit without affecting deficient hippocampal BDNF levels. DCS treatment did, however, partially restore lower BDNF levels in the brain stem and striatum. Thus, treatment with DCS may mitigate the severity of some of the neurobehavioral symptoms experienced by patients with Rett syndrome.

From Gene to Behavior: L-Type Calcium Channel Mechanisms Underlying Neuropsychiatric Symptoms.

The L-type calcium channels (LTCCs) Cav1.2 and Cav1.3, encoded by the CACNA1C and CACNA1D genes, respectively, are important regulators of calcium influx into cells and are critical for normal brain development and plasticity. In humans, CACNA1C has emerged as one of the most widely reproduced and prominent candidate risk genes for a range of neuropsychiatric disorders, including bipolar disorder (BD), schizophrenia (SCZ), major depressive disorder, autism spectrum disorder, and attention deficit hyperactivity disorder. Separately, CACNA1D has been found to be associated with BD and autism spectrum disorder, as well as cocaine dependence, a comorbid feature associated with psychiatric disorders. Despite growing evidence of a significant link between CACNA1C and CACNA1D and psychiatric disorders, our understanding of the biological mechanisms by which these LTCCs mediate neuropsychiatric-associated endophenotypes, many of which are shared across the different disorders, remains rudimentary. Clinical studies with LTCC blockers testing their efficacy to alleviate symptoms associated with BD, SCZ, and drug dependence have provided mixed results, underscoring the importance of further exploring the neurobiological consequences of dysregulated Cav1.2 and Cav1.3. Here, we provide a review of clinical studies that have evaluated LTCC blockers for BD, SCZ, and drug dependence-associated symptoms, as well as rodent studies that have identified Cav1.2- and Cav1.3-specific molecular and cellular cascades that underlie mood (anxiety, depression), social behavior, cognition, and addiction.

Cacna1c in the Prefrontal Cortex Regulates Depression-Related Behaviors via REDD1.

The CACNA1C gene that encodes the L-type Ca2+ channel (LTCC) Cav1.2 subunit has emerged as a candidate risk gene for multiple neuropsychiatric disorders including bipolar disorder, major depressive disorder, and schizophrenia, all marked with depression-related symptoms. Although cacna1c heterozygous (HET) mice have been previously reported to exhibit an antidepressant-like phenotype, the molecular and circuit-level dysfunction remains unknown. Here we report that viral vector-mediated deletion of cacna1c in the adult prefrontal cortex (PFC) of mice recapitulates the antidepressant-like effect observed in cacna1c HET mice using the sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST). Molecular studies identified lower levels of REDD1, a protein previously linked to depression, in the PFC of HET mice, and viral-mediated REDD1 overexpression in the PFC of these HET mice reversed the antidepressant-like effect in SPT and TST. Examination of downstream REDD1 targets found lower levels of active/phosphorylated Akt (S473) with no change in mTORC1 phosphorylation. Examination of the transcription factor FoxO3a, previously linked to depression-related behavior and shown to be regulated in other systems by Akt, revealed higher nuclear levels in the PFC of cacna1c HET mice that was further increased following REDD1-mediated reversal of the antidepressant-like phenotype. Collectively, these findings suggest that REDD1 in cacna1c HET mice may influence depression-related behavior via regulation of the FoxO3a pathway. Cacna1c HET mice thus serve as a useful mouse model to further study cacna1c-associated molecular signaling and depression-related behaviors relevant to human CACNA1C genetic variants.

Sensory-Derived Glutamate Regulates Presynaptic Inhibitory Terminals in Mouse Spinal Cord.

Circuit function in the CNS relies on the balanced interplay of excitatory and inhibitory synaptic signaling. How neuronal activity influences synaptic differentiation to maintain such balance remains unclear. In the mouse spinal cord, a population of GABAergic interneurons, GABApre, forms synapses with the terminals of proprioceptive sensory neurons and controls information transfer at sensory-motor connections through presynaptic inhibition. We show that reducing sensory glutamate release results in decreased expression of GABA-synthesizing enzymes GAD65 and GAD67 in GABApre terminals and decreased presynaptic inhibition. Glutamate directs GAD67 expression via the metabotropic glutamate receptor mGluR1ß on GABApre terminals and regulates GAD65 expression via autocrine influence on sensory terminal BDNF. We demonstrate that dual retrograde signals from sensory terminals operate hierarchically to direct the molecular differentiation of GABApre terminals and the efficacy of presynaptic inhibition. These retrograde signals comprise a feedback mechanism by which excitatory sensory activity drives GABAergic inhibition to maintain circuit homeostasis.

The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons.

Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract.

Effects of prenatal exposure to cocaine on brain structure and function.

Drug abuse during pregnancy affects the mother and has adverse effects on the unborn child. This chapter highlights our recent findings at the neuroanatomical, molecular, and behavioral levels in a prenatal cocaine exposure mouse model. In the embryonic brains of prenatally cocaine-exposed mice, we observed a delay in the tangential migration of GABA neurons to the cerebral cortex as a result of a significant but transient decrease in the expression of the neurotrophin brain-derived neurotrophic factor (BDNF). These developmental changes lead to lasting deficits in the numerical density of GABA neurons in the mature medial prefrontal cortex (mPFC). In adult prenatally cocaine-exposed mice, we observed a behavioral deficit in the recall of an extinguished cue-conditioned fear, which was rescued by administration of exogenous recombinant BDNF protein directly into the infralimbic cortex of the mPFC, which may result from altered activity-driven transcriptional regulation of BDNF.

Effects of prenatal cocaine exposure on social development in mice.

Prenatal cocaine exposure (PCE) in humans and animals has been shown to impair social development. Molecules that mediate synaptic plasticity and learning in the medial prefrontal cortex (mPFC), specifically brain-derived neurotrophic factor (BDNF) and its downstream signaling molecule, early growth response protein 1 (egr1), have been shown to affect the regulation of social interactions (SI). In this study we determined the effects of PCE on SI and the corresponding ultrasonic vocalizations (USVs) in developing mice. Furthermore, we studied the PCE-induced changes in the constitutive expression of BDNF, egr1 and their transcriptional regulators in the mPFC as a possible molecular mechanism mediating the altered SI. In prenatal cocaine-exposed (PCOC) mice we identified increased SI and USV production at postnatal day (PD) 25, and increased SI but not USVs at PD35. By PD45 the expression of both social behaviors normalized in PCOC mice. At the molecular level, we found increased BDNF exon IV and egr1 mRNA in the mPFC of PCOC mice at PD30 that normalized by PD45. This was concurrent with increased EGR1 protein in the mPFC of PCOC mice at PD30, suggesting a role of egr1 in the enhanced SI observed in juvenile PCOC mice. Additionally, by measuring the association of acetylation of histone 3 at lysine residues 9 and 14 (acH3K9,14) and MeCP2 at the promoters of BDNF exons I and IV and egr1, our results provide evidence of promoter-specific alterations in the mPFC of PCOC juvenile mice, with increased association of acH3K9,14 only at the BDNF exon IV promoter. These results identify a potential PCE-induced molecular alteration as the underlying neurobiological mechanism mediating the altered social development in juvenile mice.

Molecular mechanisms mediating a deficit in recall of fear extinction in adult mice exposed to cocaine in utero.

Prenatal cocaine exposure has been shown to alter cognitive processes of exposed individuals, presumed to be a result of long-lasting molecular alterations in the brain. In adult prenatal cocaine exposed (PCOC) mice we have identified a deficit in recall of fear extinction, a behavior that is dependent on the medial prefrontal cortex (mPFC) and the hippocampus. While we observed no change in the constitutive expression of brain derived neurotrophic factor (BDNF) protein and mRNA in the mPFC and hippocampus of adult PCOC mice, we observed blunted BDNF signaling in the mPFC of adult PCOC mice after fear extinction compared to the control animals. Specifically, during the consolidation phase of the extinction memory, we observed a decrease in BDNF protein and it's phospho-TrkB receptor expression. Interestingly, at this same time point there was a significant increase in total Bdnf mRNA levels in the mPFC of PCOC mice as compared with controls. In the Bdnf gene, we identified decreased constitutive binding of the transcription factors, MeCP2 and P-CREB at the promoters of Bdnf exons I and IV in the mPFC of PCOC mice, that unlike control mice remained unchanged when measured during the behavior. Finally, bilateral infusion of recombinant BDNF protein into the infralimbic subdivision of the mPFC during the consolidation phase of the extinction memory rescued the behavioral deficit in PCOC mice. In conclusion, these findings extend our knowledge of the neurobiologic impact of prenatal cocaine exposure on the mPFC of mice, which may lead to improved clinical recognition and treatment of exposed individuals.

Cup of Joe: a brain development "no"?

Treating pregnant mice with adenosine receptor antagonists including caffeine results in delayed migration of cortical γ-aminobutyric acid neurons and altered brain development in mouse offspring (Silva et al.).

Brain-derived neurotrophic factor genotype impacts the prenatal cocaine-induced mouse phenotype.

Prenatal cocaine exposure leads to persistent alterations in the growth factor brain-derived neurotrophic factor (BDNF), particularly in the medial prefrontal cortex (mPFC) and hippocampus, brain regions important in cognitive functioning. BDNF plays an important role in the strengthening of existing synaptic connections as well as in the formation of new contacts during learning. A single nucleotide polymorphism in the BDNF gene (Val66Met), leading to a Met substitution for Val at codon 66 in the prodomain, is common in human populations, with an allele frequency of 20-30% in Caucasians. To study the interaction between prenatal cocaine exposure and BDNF, we have utilized a line of BDNF Val66Met transgenic mice on a Swiss Webster background in which BDNF(Met) is endogenously expressed. Examination of baseline levels of mature BDNF protein in the mPFC of prenatally cocaine-treated wild-type (Val66Val) and Val66Met mice revealed significantly lower levels compared to prenatally saline-treated mice. In contrast, in the hippocampus of prenatally saline- and cocaine-treated adult Val66Met mice, there were significantly lower levels of mature BDNF protein compared to Val66Val mice. In extinction of a conditioned fear, we found that prenatally cocaine-treated Val66Met mice had a deficit in recall of extinction. Examination of mature BDNF protein levels immediately after the test for extinction recall revealed lower levels in the mPFC of prenatally cocaine-treated Val66Met mice compared to saline-treated mice. However, 2 h after the extinction test, there was increased BDNF exons I, IV, and IX mRNA expression in the prelimbic cortex of the mPFC in the prenatally cocaine-treated BDNF Val66Met mice compared to prenatally saline-treated mice. Taken together, our results suggest the possibility that prenatal cocaine-induced constitutive alterations in BDNF mRNA and protein expression in the mPFC differentially poises animals for alterations in behaviorally induced gene activation, which are interactive with BDNF genotype and differentially impact those behaviors. Such findings in our prenatal cocaine mouse model suggest a gene X environment interaction of potential clinical relevance.

Enhanced dopamine D1 and BDNF signaling in the adult dorsal striatum but not nucleus accumbens of prenatal cocaine treated mice.

Previous work from our group and others utilizing animal models have demonstrated long-lasting structural and functional alterations in the meso-cortico-striatal dopamine pathway following prenatal cocaine (PCOC) treatment. We have shown that PCOC treatment results in augmented D1-induced cyclic AMP (cAMP) and cocaine-induced immediate-early gene expression in the striatum of adult mice. In this study we further examined basal as well as cocaine or D1-induced activation of a set of molecules known to be mediators of neuronal plasticity following psychostimulant treatment, with emphasis in the dorsal striatum (Str) and nucleus accumbens (NAc) of adult mice exposed to cocaine in utero. Basally, in the Str of PCOC treated mice there were significantly higher levels of (1) CREB and Ser133 P-CREB (2) Thr34 P-DARPP-32 and (3) GluA1 and Ser 845 P-GluA1 when compared to prenatal saline (PSAL) treated mice. In the NAc there were significantly higher basal levels of (1) CREB and Ser133 P-CREB, (2) Thr202/Tyr204 P-ERK2, and (3) Ser845 P-GluA1. Following acute administration of cocaine (15 mg/kg, i.p.) or D1 agonist (SKF 82958; 1 mg/kg, i.p.) there were significantly higher levels of Ser133 P-CREB, Thr34 P-DARPP-32, and Thr202/Tyr204 P-ERK2 in the Str that were evident in all animals tested. However, these cocaine-induced increases in phosphorylation were significantly augmented in PCOC mice compared to PSAL mice. In sharp contrast to the observations in the Str, in the NAc, acute administration of cocaine or D1 agonist significantly increased P-CREB and P-ERK2 in PSAL mice, a response that was not evident in PCOC mice. Examination of Ser 845 P-GluA1 revealed that cocaine or D1 agonist significantly increased levels in PSAL mice, but significantly decreased levels in the PCOC mice in both the Str and NAc. We also examined changes in brain-derived neurotrophic factor (BDNF). Our studies revealed significantly higher levels of the BDNF precursor, pro-BDNF, and one of its receptors, TrkB in the Str of PCOC mice compared to PSAL mice. These results suggest a persistent up-regulation of molecules critical to D1 and BDNF signaling in the Str of adult mice exposed to cocaine in utero. These molecular adaptations may underlie components of the behavioral deficits evident in exposed animals and a subset of exposed humans, and may represent a therapeutic target for ameliorating aspects of the PCOC-induced phenotype.