NPAS2 Regulation of Anxiety-Like Behavior and GABAA Receptors

“Diazepam has been shown to potentiate GABAa-receptor mediated currents by binding specific subunit combinations of synaptic GABAa receptors and promoting the binding of GABA, which in turn increases total conductance of chloride (Macdonald and Olsen, 1994; Xu and Sastry, 2005).”

“This phenotype is similar to that of Clock∆19 mutant mice, which exhibit reduced anxiety-like behaviors and is the opposite of the Per1/Per2 double mutant, which exhibit increased anxiety-like behavior (Roybal et al., 2007; Spencer et al., 2013).”

“…are strongly associated with psychiatric diseases like seasonal affective disorder, bipolar disorder, major depression and drug addiction (Mukherjee et al., 2010; McClung, 2011; SalgadoDelgado et al., 2011; Hasler et al., 2012; McCarthy and Welsh, 2012; Li et al., 2013; McCarthy et al., 2013).”

“In fact, nearly all psychiatric disorders involve some disruption to the normal sleep/wake cycle and this is often one of the criteria used for diagnosis (McClung, 2013).”

“Although it would be preferential to determine the effects of diazepam, an anxiolytic drug, in a behavioral test that measures anxiety-like behavior, evidence from several laboratories suggests the genetic background (C57BL/6J) of these mice are not responsive to the anxiolytic effects of diazepam in the assays we use…”

“We chose to employ UCMS, a paradigm well-known to alter mood-related behaviors, and in our hands reliably induces an increase in anxiety- and depressive-like behavior (Guilloux et al., 2011; Logan et al., 2014).”

“SNPs in Npas2 are associated with seasonal affective disorder and major depressive disorder (Partonen et al., 2007; Soria et al., 2010).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“PER and CRY proteins are phosphorylated by casein kinase epsilon 1 (CKE1), form homomers or heteromers and translocate to the nucleus where they can inhibit CLOCK:BMAL1 or NPAS2:BMAL1mediated transcription (Lowrey and Takahashi, 2000; Wang et al., 2007; Mohawk and Takahashi, 2011).”

“Frontiers in Molecular Neuroscience | www.frontiersin.org 4 November 2017 | Volume 10 | Article 360 experimenter, location, apparatus and test, et cetera (Wahlsten et al., 2006; Roybal et al., 2007; Mukherjee et al., 2010 Coque et al., 2011; Arey et al., 2013; Spencer et al., 2013).”

“…are strongly associated with psychiatric diseases like seasonal affective disorder, bipolar disorder, major depression and drug addiction (Mukherjee et al., 2010; McClung, 2011; SalgadoDelgado et al., 2011; Hasler et al., 2012; McCarthy and Welsh, 2012; Li et al., 2013; McCarthy et al., 2013).”

“SNPs in Npas2 are associated with seasonal affective disorder and major depressive disorder (Partonen et al., 2007; Soria et al., 2010).”

“An inter-test interval of 1–2 days was adopted for this study in line with our previously published work (Roybal et al., 2007; Mukherjee et al., 2010; Spencer et al., 2013).”

“PER and CRY proteins are phosphorylated by casein kinase epsilon 1 (CKE1), form homomers or heteromers and translocate to the nucleus where they can inhibit CLOCK:BMAL1 or NPAS2:BMAL1mediated transcription (Lowrey and Takahashi, 2000; Wang et al., 2007; Mohawk and Takahashi, 2011).”

“Previous studies indicate that there is little to no effect on behavioral performance between inter-test intervals of 1 week compared with 1–2 days allowing for rapid and semi-high-throughput screening of behavior (Paylor et al., 2006).”

“The generation of Npas2 null mutant (−/−) mice is described in Garcia et al. (2000).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“, 2017) and that repeated exposure to FSS results in increased time spent immobile (Serchov et al., 2015; Mul et al., 2016).”

“…are strongly associated with psychiatric diseases like seasonal affective disorder, bipolar disorder, major depression and drug addiction (Mukherjee et al., 2010; McClung, 2011; SalgadoDelgado et al., 2011; Hasler et al., 2012; McCarthy and Welsh, 2012; Li et al., 2013; McCarthy et al., 2013).”

“It is noteworthy to mention that many of the therapies used to treat these disorders are known to modulate the circadian clock (Bunney and Bunney, 2013).”

“Clinical and pre-clinical studies provide strong evidence that circadian rhythms and the genes that make up the molecular clock play a key role in the expression of mood-related symptoms in psychiatric disorders (Falcón and McClung, 2009; Karatsoreos, 2014; Landgraf et al., 2014; Logan et al., 2014).”

“…are strongly associated with psychiatric diseases like seasonal affective disorder, bipolar disorder, major depression and drug addiction (Mukherjee et al., 2010; McClung, 2011; SalgadoDelgado et al., 2011; Hasler et al., 2012; McCarthy and Welsh, 2012; Li et al., 2013; McCarthy et al., 2013).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“A number of studies show that the striatum is important for behavioral responses to stressful stimuli (Kim et al., 2016; Han and Nestler, 2017).”

“…in conjunction with the studies presented here, support a model whereby VTA Clock is important for negatively regulating reward and anxiety and NAc Npas2 is important for positively regulating reward and anxiety (Mukherjee et al., 2010; Coque et al., 2011; Ozburn et al., 2012; Sidor et al., 2015).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“Male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were utilized for Npas2 knockdown (behavioral assays or qPCR), gene expression following acute Forced Swim Stress (FSS) or Unpredictable Chronic Mild Stress (UCMS), Chromatin Immunoprecipitation (ChIP) experiments and electrophysiological experiments (ca…”

“We chose to employ UCMS, a paradigm well-known to alter mood-related behaviors, and in our hands reliably induces an increase in anxiety- and depressive-like behavior (Guilloux et al., 2011; Logan et al., 2014).”

“Genetic manipulation of Drd3 in mice has not yielded reports of consistent effects on anxiety-like behavior (Steiner et al., 1997; Xing et al., 2013; Moraga-Amaro et al., 2014; Leggio et al., 2015).”

“…are strongly associated with psychiatric diseases like seasonal affective disorder, bipolar disorder, major depression and drug addiction (Mukherjee et al., 2010; McClung, 2011; SalgadoDelgado et al., 2011; Hasler et al., 2012; McCarthy and Welsh, 2012; Li et al., 2013; McCarthy et al., 2013).”

“…and pre-clinical studies provide strong evidence that circadian rhythms and the genes that make up the molecular clock play a key role in the expression of mood-related symptoms in psychiatric disorders (Falcón and McClung, 2009; Karatsoreos, 2014; Landgraf et al., 2014; Logan et al., 2014).”

“Studies have revealed that circadian genes and rhythms significantly contribute to mood, anxiety and depression, as well as reward and motivation (Roybal et al., 2007; Ozburn et al., 2012, 2013, 2015; McClung, 2013; Spencer et al., 2013; Logan et al., 2014; Parekh et al., 2015).”

“Serial testing began with the least stressful and ended with most stressful, as previously characterized (Tarantino and Bucan, 2000; Spencer et al., 2013).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“Previous reports, in conjunction with the studies presented here, support a model whereby VTA Clock is important for negatively regulating reward and anxiety and NAc Npas2 is important for positively regulating reward and anxiety (Mukherjee et al., 2010; Coque et al., 2011; Ozburn et al., 2012; Sidor et al., 2015).”

“This phenotype is similar to that of Clock∆19 mutant mice, which exhibit reduced anxiety-like behaviors and is the opposite of the Per1/Per2 double mutant, which exhibit increased anxiety-like behavior (Roybal et al., 2007; Spencer et al., 2013).”

“They exhibit brain region and cell-type specific expression differences, as well as many unique transcriptional targets (Ozburn et al., 2015).”

“Genetic manipulation of Drd3 in mice has not yielded reports of consistent effects on anxiety-like behavior (Steiner et al., 1997; Xing et al., 2013; Moraga-Amaro et al., 2014; Leggio et al., 2015).”

“Studies have revealed that circadian genes and rhythms significantly contribute to mood, anxiety and depression, as well as reward and motivation (Roybal et al., 2007; Ozburn et al., 2012, 2013, 2015; McClung, 2013; Spencer et al., 2013; Logan et al., 2014; Parekh et al., 2015).”

“Additionally, single nucleotide polymorphisms (SNP) in a number of circadian genes have been associated with mood disorders (Desan et al., 2000; Benedetti et al., 2004; Nievergelt et al., 2006; Kishi et al., 2008; Kripke et al., 2009; Lavebratt et al., 2010a,b).”

“Frontiers in Molecular Neuroscience | www.frontiersin.org 4 November 2017 | Volume 10 | Article 360 experimenter, location, apparatus and test, et cetera (Wahlsten et al., 2006; Roybal et al., 2007; Mukherjee et al., 2010 Coque et al., 2011; Arey et al., 2013; Spencer et al., 2013).”

“…an anxiolytic drug, in a behavioral test that measures anxiety-like behavior, evidence from several laboratories suggests the genetic background (C57BL/6J) of these mice are not responsive to the anxiolytic effects of diazepam in the assays we used (Griebel et al., 2000; Thompson et al., 2015).”

“Genetic manipulation of Drd3 in mice has not yielded reports of consistent effects on anxiety-like behavior (Steiner et al., 1997; Xing et al., 2013; Moraga-Amaro et al., 2014; Leggio et al., 2015).”

“Genetic manipulation of Drd3 in mice has not yielded reports of consistent effects on anxiety-like behavior (Steiner et al., 1997; Xing et al., 2013; Moraga-Amaro et al., 2014; Leggio et al., 2015).”

“Diazepam has been shown to potentiate GABAa-receptor mediated currents by binding specific subunit combinations of synaptic GABAa receptors and promoting the binding of GABA, which in turn increases total conductance of chloride (Macdonald and Olsen, 1994; Xu and Sastry, 2005).”

“Further, it has been shown that a single experience with FSS induces immediate early genes in the ventral striatum (Morello et al., 2017) and that repeated exposure to FSS results in increased time spent immobile (Serchov et al., 2015; Mul et al., 2016).”

“PER and CRY proteins are phosphorylated by casein kinase epsilon 1 (CKE1), form homomers or heteromers and translocate to the nucleus where they can inhibit CLOCK:BMAL1 or NPAS2:BMAL1mediated transcription (Lowrey and Takahashi, 2000; Wang et al., 2007; Mohawk and Takahashi, 2011).”

“However, Drd3 antagonism has been shown to reduce (pre-adolescent) stress-induced depression-like behavior in adult mice (Seo and Kuzhikandathil, 2015).”

“This raises the possibility that the NPAS2-related changes in the adolescent WIN – cocaine group act in concert with ERK and gephyrin to regulate the availability of GABRA1 and the composition of GABA(A) receptors in response to cocaine.”

“…in D1 MSNs (Ozburn et al., 2015; Parekh et al., 2019; Becker-Krail et al., 2022b); Npas2 mutant mice actually show significant increases in locomotor response to novelty, exploratory drive, cocaine self-administration and selfadministration motivation (Ozburn et al., 2017; DePoy et al., 2020).”

“Stereotaxic surgery was performed as previously described (Ozburn et al., 2017).”

“Interestingly, deleting the Clock homologue Npas2 118 reduced anxiety similarly to the Clock Δ 19 mutation 119 .”

“Many of these targets contain E-Box and/or CRE sites in their promoters, further supporting direct transcriptional control by circadian transcription factors [19,37].”

“…regions, such as the striatum, have been targeted over the past years to elaborate the molecular and neurophysiological mechanism driving the temporal coordination and execution of behaviors related to mood, reward and motor control (Landgraf et al., 2016b; Ozburn et al., 2017; Porcu et al., 2020).”

“…clock is likely (Franken and Dijk 2009) and may be preferentially involved in modulating arousal and wakefulness through actions in the striatum (Becker-Krail et al. 2022; DePoy et al. 2021; Garcia et al. 2000; Luo et al. 2018; Ozburn et al. 2015, 2017; Parekh et al. 2019; Zhang et al. 2021).”

“These data suggest that Npas2 is crucially involved in the response to stress and the development of anxiety and takes part in the regulation of GABAergic neurotransmission in the ventral striatum [73].”

“NPAS2-deficient mice show deficiencies in complex emotional long-term memory and behavior, including decreased reactions to both cued and contextual fear tasks and decreased anxiety-like behaviors (Garcia, 2000; Ozburn et al., 2017).”

“NPAS2isoneofthecoregenesthatcontrolthe rhythm of the biological clock 43 , which is associated with anxiety and cancer 44,45 .”

“Ozburn and colleagues found that Npas2-KO mice displayed anxiety-like behavior in elevated plus maze, light/dark box, and open field tests compared to WT mice (Ozburn et al., 2017).”

“…and a selective and functional marker gene for glutamatergic 33 and GABAergic synapses 34 in inhibitory neurons and interneurons; DAOA associated with schizophrenia in an Asian cohort 35 ; and NPAS2 , conferring neuropsychiatric anxiety disorder and regulating GABAergic signal transmissions 36 .”

“NPAS2 regulates anxiety-like behavior and non-rapid eye movement sleep[45,46].”

“In addition to its role in circadian rhythms, NPAS2 is also responsible for regulating fear and anxiety behavior along with memory learning [41,42], which are some of the primary functions of the AMYG [43–45].”

“13 In pain research, NPAS2 knockout (NPAS2 −/− ) mice have been shown to exhibit no significant differences in baseline thermal pain thresholds or acute analgesia.”

“Alterations in the NPAS2 may contribute to shifts in circadian rhythm types among patients with MDD. Notably, NPAS2 expression is further modulated by stress, suggesting that environmental stressors may exacerbate the genetic effects described above (Ozburn et al. 2017).”

“47 MYC promotes neuronal differentiation in developing neural tube, 48 and VSX1 contributes to interneuron development.”

“However, population-level candidate gene studies have identi ed multiple links between clock genes and depressive disorders, including anxiety (Lavebratt et al., 2010b; Liberman et al., 2017; Ozburn et al., 2017b; Partonen, 2012; Silva et al., 2020).”

“The VTA mRNA is isolated and quantified using a method described by (Ozburn et al., 2017).”

“Studies on the dorsal striatum (DS), the major input structure of the basal ganglia, revealed a clear link between circadian dysregulation and altered behavioural phenotypes in animal models (de Zavalia et al., 2021; Landgraf et al., 2016a; Ozburn et al., 2017; Schoettner et al., 2022).”

“Taken together, this evidence suggests loss of Npas2 is anxiolytic and suggests a role for Npas2 in the regulation of cognition and anxiety 7,10,11 .”

“Although NPAS2 was initially described as a central component of the CR in the central nervous system [62– 64], increasing evidence demonstrates that its expression in peripheral tissues plays essential physiological roles, particularly in the regulation of the cell cycle, maintenance of genomic…”