Correction: Cellular and molecular basis for stress-induced depression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Elevation of p11 in lateral habenula mediates depression-like behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

p11 in Cholinergic Interneurons of the Nucleus Accumbens Is Essential for Dopamine Responses to Rewarding Stimuli.

A recent study showed that p11 expressed in cholinergic interneurons (CINs) of the nucleus accumbens (NAc) is a key regulator of depression-like behaviors. Dopaminergic neurons projecting to the NAc are responsible for reward-related behaviors, and their function is impaired in depression. The present study investigated the role of p11 in NAc CINs in dopamine responses to rewarding stimuli. The extracellular dopamine and acetylcholine (ACh) levels in the NAc were determined in freely moving male mice using in vivo microdialysis. Rewarding stimuli (cocaine, palatable food, and female mouse encounter) induced an increase in dopamine efflux in the NAc of wild-type (WT) mice. The dopamine responses were attenuated (cocaine) or abolished (food and female mouse encounter) in constitutive p11 knock-out (KO) mice. The dopamine response to cocaine was accompanied by an increase in ACh NAc efflux, whereas the attenuated dopamine response to cocaine in p11 KO mice was restored by activation of nicotinic or muscarinic ACh receptors in the NAc. Dopamine responses to rewarding stimuli and ACh release in the NAc were attenuated in mice with deletion of p11 from cholinergic neurons (ChAT-p11 cKO mice), whereas gene delivery of p11 to CINs restored the dopamine responses. Furthermore, chemogenetic studies revealed that p11 is required for activation of CINs in response to rewarding stimuli. Thus, p11 in NAc CINs plays a critical role in activating these neurons to mediate dopamine responses to rewarding stimuli. The dysregulation of mesolimbic dopamine system by dysfunction of p11 in NAc CINs may be involved in pathogenesis of depressive states.

CK2 regulates 5-HT4 receptor signaling and modulates depressive-like behavior.

The serotonergic neurotransmitter system has been widely implicated in the pathophysiology of mood-related disorders such as anxiety and major depressive disorder (MDD). The onset of therapeutic efficacy of traditional antidepressants is delayed by several weeks. The 5-HT4 receptor has emerged as a new therapeutic target since agonists of this receptor induce rapid antidepressant-like responses in rodents. Here we show that the 5-HT4 receptor is regulated by CK2, at transcriptional and post-transcriptional levels. We present evidence, in two different CK2α knockout mouse lines, that this regulation is region-specific, with the 5-HT4 receptor upregulated in prefrontal cortex (PFC) but not striatum or hippocampus where CK2α is also ablated. 5-HT4 receptor signaling is enhanced in vitro, as evidenced by enhanced cAMP production or receptor plasma membrane localization in the presence of CK2 inhibitor or shRNA targeting CK2α. In vivo, 5-HT4 receptor signaling is also upregulated since ERK activation is elevated and sensitive to the inverse agonist, GR113808 in the PFC of CK2α KO mice. Behaviorally, KO mice as well as mice with AAV-mediated deletion of CK2α in the PFC show a robust 'anti-depressed-like' phenotype and display an enhanced response to antidepressant treatment when tested in paradigms for mood and anxiety. Importantly, it is sufficient to overexpress the 5-HT4 receptor in the mPFC to generate mice with a similar 'anti-depressed-like' phenotype. Our findings identify the mPFC as the region that mediates the effect of enhanced 5-HT4 receptor activity and CK2 as modulator of 5-HT4 receptor levels in this brain region that regulates mood-related phenotypes.

Elevation of p11 in lateral habenula mediates depression-like behavior.

The lateral habenula (LHb) is a key brain region involved in the pathophysiology of depression. It is activated by stimuli associated with negative experiences and is involved in encoding aversive signals. Hyperactivity of LHb is found in both rodent models of depression and human patients with depression. However, little is known about the underlying molecular mechanisms. Here we show that in LHb neurons, p11, a multifunctional protein implicated in depression, is significantly upregulated by chronic restraint stress. Knockdown of p11 expression in LHb alleviates the stress-induced depression-like behaviors. Moreover, chronic restraint stress induces bursting action potentials in LHb neurons, which are abolished by p11 knockdown. Overexpression of p11 in dopamine D2 receptor-containing LHb neurons of control mice induces depression-like behaviors. These results have identified p11 in LHb as a key molecular determinant regulating negative emotions, which may help to understand the molecular and cellular basis of depression.

Genetic evidence for role of integration of fast and slow neurotransmission in schizophrenia.

The most recent genome-wide association studies (GWAS) of schizophrenia (SCZ) identified hundreds of risk variants potentially implicated in the disease. Further, novel statistical methodology designed for polygenic architecture revealed more potential risk variants. This can provide a link between individual genetic factors and the mechanistic underpinnings of SCZ. Intriguingly, a large number of genes coding for ionotropic and metabotropic receptors for various neurotransmitters-glutamate, γ-aminobutyric acid (GABA), dopamine, serotonin, acetylcholine and opioids-and numerous ion channels were associated with SCZ. Here, we review these findings from the standpoint of classical neurobiological knowledge of neuronal synaptic transmission and regulation of electrical excitability. We show that a substantial proportion of the identified genes are involved in intracellular cascades known to integrate 'slow' (G-protein-coupled receptors) and 'fast' (ionotropic receptors) neurotransmission converging on the protein DARPP-32. Inspection of the Human Brain Transcriptome Project database confirms that that these genes are indeed expressed in the brain, with the expression profile following specific developmental trajectories, underscoring their relevance to brain organization and function. These findings extend the existing pathophysiology hypothesis by suggesting a unifying role of dysregulation in neuronal excitability and synaptic integration in SCZ. This emergent model supports the concept of SCZ as an 'associative' disorder-a breakdown in the communication across different slow and fast neurotransmitter systems through intracellular signaling pathways-and may unify a number of currently competing hypotheses of SCZ pathophysiology.

Cellular and molecular basis for stress-induced depression.

Chronic stress has a crucial role in the development of psychiatric diseases, such as anxiety and depression. Dysfunction of the medial prefrontal cortex (mPFC) has been linked to the cognitive and emotional deficits induced by stress. However, little is known about the molecular and cellular determinants in mPFC for stress-associated mental disorders. Here we show that chronic restraint stress induces the selective loss of p11 (also known as annexin II light chain, S100A10), a multifunctional protein binding to 5-HT receptors, in layer II/III neurons of the prelimbic cortex (PrL), as well as depression-like behaviors, both of which are reversed by selective serotonin reuptake inhibitors (SSRIs) and the tricyclic class of antidepressant (TCA) agents. In layer II/III of the PrL, p11 is highly concentrated in dopamine D2 receptor-expressing (D2+) glutamatergic neurons. Viral expression of p11 in D2+ PrL neurons alleviates the depression-like behaviors exhibited by genetically manipulated mice with D2+ neuron-specific or global deletion of p11. In stressed animals, overexpression of p11 in D2+ PrL neurons rescues depression-like behaviors by restoring glutamatergic transmission. Our results have identified p11 as a key molecule in a specific cell type that regulates stress-induced depression, which provides a framework for the development of new strategies to treat stress-associated mental illnesses.

Alteration by p11 of mGluR5 localization regulates depression-like behaviors.

Mood disorders and antidepressant therapy involve alterations of monoaminergic and glutamatergic transmission. The protein S100A10 (p11) was identified as a regulator of serotonin receptors, and it has been implicated in the etiology of depression and in mediating the antidepressant actions of selective serotonin reuptake inhibitors. Here we report that p11 can also regulate depression-like behaviors via regulation of a glutamatergic receptor in mice. p11 directly binds to the cytoplasmic tail of metabotropic glutamate receptor 5 (mGluR5). p11 and mGluR5 mutually facilitate their accumulation at the plasma membrane, and p11 increases cell surface availability of the receptor. Whereas p11 overexpression potentiates mGluR5 agonist-induced calcium responses, overexpression of mGluR5 mutant, which does not interact with p11, diminishes the calcium responses in cultured cells. Knockout of mGluR5 or p11 specifically in glutamatergic neurons in mice causes depression-like behaviors. Conversely, knockout of mGluR5 or p11 in GABAergic neurons causes antidepressant-like behaviors. Inhibition of mGluR5 with an antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), induces antidepressant-like behaviors in a p11-dependent manner. Notably, the antidepressant-like action of MPEP is mediated by parvalbumin-positive GABAergic interneurons, resulting in a decrease of inhibitory neuronal firing with a resultant increase of excitatory neuronal firing. These results identify a molecular and cellular basis by which mGluR5 antagonism achieves its antidepressant-like activity.

Bidirectional regulation of emotional memory by 5-HT1B receptors involves hippocampal p11.

Cognitive impairments are common in depression and involve dysfunctional serotonin neurotransmission. The 5-HT1B receptor (5-HT(1B)R) regulates serotonin transmission, via presynaptic receptors, but can also affect transmitter release at heterosynaptic sites. This study aimed at investigating the roles of the 5-HT(1B)R, and its adapter protein p11, in emotional memory and object recognition memory processes by the use of p11 knockout (p11KO) mice, a genetic model for aspects of depression-related states. 5-HT(1B)R agonist treatment induced an impairing effect on emotional memory in wild type (WT) mice. In comparison, p11KO mice displayed reduced long-term emotional memory performance. Unexpectedly, 5-HT(1B)R agonist stimulation enhanced memory in p11KO mice, and this atypical switch was reversed after hippocampal adeno-associated virus mediated gene transfer of p11. Notably, 5-HT(1B)R stimulation increased glutamatergic neurotransmission in the hippocampus in p11KO mice, but not in WT mice, as measured by both pre- and postsynaptic criteria. Magnetic resonance spectroscopy demonstrated global hippocampal reductions of inhibitory GABA, which may contribute to the memory enhancement and potentiation of pre- and post-synaptic measures of glutamate transmission by a 5-HT(1B)R agonist in p11KO mice. It is concluded that the level of hippocampal p11 determines the directionality of 5-HT(1B)R action on emotional memory processing and modulates hippocampal functionality. These results emphasize the importance of using relevant disease models when evaluating the role of serotonin neurotransmission in cognitive deficits related to psychiatric disorders.

Localization of dopamine- and cAMP-regulated phosphoprotein-32 and inhibitor-1 in area 9 of Macaca mulatta prefrontal cortex.

The actions of dopamine D1 family receptors (D1R) depend upon a signal transduction cascade that modulates the phosphorylation state of important effector proteins, such as glutamate receptors and ion channels. This is accomplished both through activation of protein kinase A (PKA) and the inhibition of protein phosphatase-1 (PP1). Inhibition of PP1 occurs through PKA-mediated phosphorylation of dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) or the related protein inhibitor-1 (I-1), and the availability of DARPP-32 is essential to the functional outcome of D1R activation in the basal ganglia. While D1R activation is critical for prefrontal cortex (PFC) function, especially working memory, the functional role played by DARPP-32 or I-1 is less clear. In order to examine this more thoroughly, we have utilized immunoelectron microscopy to quantitatively determine the localization of DARPP-32 and I-1 in the neuropil of the rhesus monkey PFC. Both were distributed widely in the different components of the neuropil, but were enriched in dendritic shafts. I-1 label was more frequently identified in axon terminals than was DARPP-32, and DARPP-32 label was more frequently identified in glia than was I-1. We also quantified the extent to which these proteins were found in dendritic spines. DARPP-32 and I-1 were present in small subpopulations of dendritic spines, (4.4% and 7.7% and respectively), which were substantially smaller than observed for D1R in our previous studies (20%). Double-label experiments did not find evidence for colocalization of D1R and DARPP-32 or I-1 in spines or terminals. Thus, at the least, not all prefrontal spines which contain D1R also contain I-1 or DARPP-32, suggesting important differences in D1R signaling in the PFC compared to the striatum.

Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear.

Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.

Phosphodiesterase 1B differentially modulates the effects of methamphetamine on locomotor activity and spatial learning through DARPP32-dependent pathways: evidence from PDE1B-DARPP32 double-knockout mice.

Mice lacking phosphodiesterase 1B (PDE1B) exhibit an exaggerated locomotor response to D-methamphetamine and increased in vitro phosphorylation of DARPP32 (dopamine- and cAMP-regulated phosphoprotein, M r 32 kDa) at Thr34 in striatal brain slices treated with the D1 receptor agonist, SKF81297. These results indicated a possible regulatory role for PDE1B in pathways involving DARPP32. Here, we generated PDE1B x DARPP32 double-knockout (double-KO) mice to test the role of PDE1B in DARPP32-dependent pathways in vivo. Analysis of the response to d-methamphetamine on locomotor activity showed that the hyperactivity experienced by PDE1B mutant mice was blocked in PDE1B-/- x DARPP32-/- double-KO mice, consistent with participation of PDE1B and DARPP32 in the same pathway. Further behavioral testing in the elevated zero-maze revealed that DARPP32-/- mice showed a less anxious phenotype that was nullified in double-mutant mice. In contrast, in the Morris water maze, double-KO mice showed deficits in spatial reversal learning not observed in either single mutant compared with wild-type mice. The data suggest a role for PDE1B in locomotor responses to psychostimulants through modulation of DARPP32-dependent pathways; however, this modulation does not necessarily impact other behaviors, such as anxiety or learning. Instead, the phenotype of double-KOs observed in these latter tasks may be mediated through independent pathways.

Role of adenosine A1 receptors in the modulation of dopamine D1 and adenosine A2A receptor signaling in the neostriatum.

Adenosine is known to modulate the function of neostriatal neurons. Adenosine acting on A(2A) receptors increases the phosphorylation of dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) at Thr34 (the cAMP-dependent protein kinase [PKA] site) in striatopallidal neurons, and opposes dopamine D2 receptor signaling. In contrast, the role of adenosine A(1) receptors in the regulation of dopamine/DARPP-32 signaling is not clearly understood. Here, we investigated the effect of adenosine A(1) receptors on D(1), D(2) and A(2A) receptor signaling using mouse neostriatal slices. An A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (100 nM), caused a transient increase, followed by a transient decrease, in DARPP-32 Thr34 phosphorylation. Our data support the following model for the actions of the A(1) receptor agonist. The A(1) receptor-induced early increase in Thr34 phosphorylation was mediated by presynaptic inhibition of dopamine release, and the subsequent removal of tonic inhibition by D(2) receptors of A(2A) receptor/G(olf)/cAMP/PKA signaling. The A(1) receptor-induced late decrease in Thr34 phosphorylation was mediated by a postsynaptic G(i) mechanism, resulting in inhibition of D(1) and A(2A) receptor-coupled G(olf)/cAMP/PKA signaling in direct and indirect pathway neurons, respectively. In conclusion, A(1) receptors play a major modulatory role in dopamine and adenosine receptor signaling.

Distinct roles for spinophilin and neurabin in dopamine-mediated plasticity.

Protein phosphatase 1 plays a major role in the governance of excitatory synaptic activity, and is subject to control via the neuromodulatory actions of dopamine. Mechanisms involved in regulating protein phosphatase 1 activity include interactions with the structurally related cytoskeletal elements spinophilin and neurabin, synaptic scaffolding proteins that are highly enriched in dendritic spines. The requirement for these proteins in dopamine-related neuromodulation was tested using knockout mice. Dopamine D1-mediated regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor activity was deficient in both striatal and prefrontal cortical neurons from neurabin knockout mice; in spinophilin knockout mice this deficit was manifest only in striatal neurons. At corticostriatal synapses long-term potentiation was deficient in neurabin knockout mice, but not in spinophilin knockout mice, and was rescued by a D1 receptor agonist. In contrast, long-term depression was deficient in spinophilin knockout mice but not in neurabin knockout mice, and was rescued by D2 receptor activation. Spontaneous excitatory post-synaptic current frequency was increased in neurabin knockout mice, but not in spinophilin knockout mice, and this effect was normalized by D2 receptor agonist application. Both knockout strains displayed increased induction of GluR1 Ser(845) phosphorylation in response to D1 receptor stimulation in slices, and also displayed enhanced locomotor activation in response to cocaine administration. These effects could be dissociated from cocaine reward, which was enhanced only in spinophilin knockout mice, and was accompanied by increased immediate early gene induction. These data establish a requirement for synaptic scaffolding in dopamine-mediated responses, and further indicate that spinophilin and neurabin play distinct roles in dopaminergic signal transduction and psychostimulant response.

Cocaine self-administration in mice is inversely related to phosphorylation at Thr34 (protein kinase A site) and Ser130 (kinase CK1 site) of DARPP-32.

The reinforcing effect of cocaine is associated with increases in dopamine in the striatum. The phosphoprotein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein) has been shown to mediate the intracellular events after activation of dopamine receptors. DARPP-32 is phosphorylated at multiple sites by different protein kinases, but little is known about the functional role of these different sites. Cocaine self-administration and striatal levels of dopamine after acute "binge" cocaine administration were measured in separate lines of mice with alanine mutations introduced into DARPP-32 at either Thr34 (protein kinase A site, Thr34A), Thr75, (cyclin-dependent kinase 5 site, Thr75A), Ser97 (kinase CK2 site, Ser97A), or Ser130 (kinase CK1 site, Ser130A). Acquisition of stable cocaine self-administration required significantly more time in Thr34A-/- mice. Both Thr34A- and Ser130A-DARPP-32 mutant mice self-administered more cocaine than their respective wild-type controls. Also, cocaine-induced increases of dopamine in dorsal striatum were attenuated in the Thr34A- and Ser130A-DARPP-32 phosphomutant mice compared with wild-type mice. Notably, levels of P-Thr34- and P-Ser130-DARPP-32 were reduced after self-administration of cocaine in wild-type mice. Thus, phosphorylation states of Thr34- and Ser130-DARPP-32 play important roles in modulating the reinforcing effects of cocaine.

A network of control mediated by regulator of calcium/calmodulin-dependent signaling.

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.

Ovarian function and morphology after deletion of the DARPP-32 gene in mice.

A plethora of systemic and local signaling molecules regulate ovarian function, but how different signaling molecules interact within an ovarian target cell is not known. Here we report that endocrine cells of the ovary express a phosphoprotein, DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein of Mr 32,000), which integrates signaling molecules in neurons. We thus hypothesized that DARPP-32 might act in a similar way in ovarian endocrine cells and therefore studied whether DARPP-32 gene deletion has consequences for ovarian functions in mice. Reproductive performance of adult mutants did not differ from wild-type females, as judged from numbers of litters and pups delivered. Similar steroid levels in mutant and wild-type mice ruled out gross abnormalities in the hypothalamic-pituitary-ovarian axis. However, an analysis of ovarian morphology, using serially sectioned ovaries, revealed several differences. Ovaries of young adult mutant mice at 2 - 3 months contained luteinized follicles, but fewer corpora lutea. At 5 - 6 months, large cysts were found in mutant mice, as well as reduced numbers of preantral follicles and antral follicles. Interstitial cell hypertrophy and degeneration was marked in all mutant ovaries at this age. Thus, while the lack of DARPP-32 does not overtly alter reproductive performance in adult mice, it is associated with progressive alterations and derangements of growth and development of ovarian follicles, suggesting premature ovarian ageing. This implies that ovarian DARPP-32 is involved in follicular development, presumably by integrating effects of signaling molecules, which act together to ensure efficient follicular development.

Estradiol affects spinophilin protein differently in gonadectomized males and females.

Estrogen (E) treatment of ovariectomized animals increases dendritic spines and/or synaptic protein expression in the hippocampus of female rats [J Neurosci 12 (1992) 2549; Endocrinology 142 (2001) 1284; Endocrinol Rev 20 (1999) 279; Annu Rev Pharmacol Toxicol 41 (2001) 569], mice [Proc Natl Acad Sci USA 101 (2004) 2185], rhesus monkeys [Proc Natl Acad Sci USA 98 (2001) 8071; Endocrinology 144 (2003) 4734; J Comp Neurol 465 (2003) 540] and hippocampal cells in vitro [J Neurosci 16 (1996) 4059; Neuroscience 124 (2004) 549]. The role of E in hippocampal synaptic structural plasticity in males is less well understood. In the present study, we have used a recently developed technique to count spinophilin immunogold-reactive (Ir) puncta as well as in situ hybridization to compare E effects on spinophilin-Ir and mRNA in gonadectomized female and male rats 48 h after E treatment. Spinophilin is an established spine marker, which interacts with several proteins (including actin and protein phosphatase 1) that are highly enriched in spines [Proc Natl Acad Sci USA 94 (1997) 9956; Proc Natl Acad Sci USA 97 (2000) 9287]. We report that E exerts sex-specific effects on dendritic spinophilin-labeled spines in the CA1 region: E treatment significantly increased spinophilin-Ir puncta, indicative of spines, in females, but led to a decrease in males. Furthermore, while hippocampal spinophilin mRNA changes could have occurred earlier, spinophilin mRNA levels were unchanged after 48 h of E in both males and females. This suggests the possibility that E regulates spinophilin protein expression and or stability within dendrites via post-transcriptional mechanisms.