Persistent enhancement of basolateral amygdala-dorsomedial striatum synapses causes compulsive-like behaviors in mice.

Compulsive behaviors are observed in a range of psychiatric disorders, however the neural substrates underlying the behaviors are not clearly defined. Here we show that the basolateral amygdala-dorsomedial striatum (BLA-DMS) circuit activation leads to the manifestation of compulsive-like behaviors. We revealed that the BLA neurons projecting to the DMS, mainly onto dopamine D1 receptor-expressing neurons, largely overlap with the neuronal population that responds to aversive predator stress, a widely used anxiogenic stressor. Specific optogenetic activation of the BLA-DMS circuit induced a strong anxiety response followed by compulsive grooming. Furthermore, we developed a mouse model for compulsivity displaying a wide spectrum of compulsive-like behaviors by chronically activating the BLA-DMS circuit. In these mice, persistent molecular changes at the BLA-DMS synapses observed were causally related to the compulsive-like phenotypes. Together, our study demonstrates the involvement of the BLA-DMS circuit in the emergence of enduring compulsive-like behaviors via its persistent synaptic changes.

Orexinergic neurons modulate stress coping responses in mice.

Stress is a critical precipitating factor for major depression. However, individual responses to the same stressor vary widely, possibly owing to individual variations in stress resilience. Nevertheless, the factors that determine stress susceptibility and resilience remain poorly understood. Orexin neurons have been implicated in the control of stress-induced arousal. Therefore, we investigated whether orexin-expressing neurons are involved in the regulation of stress resilience in male mice. We found that the level of c-fos expression was significantly different in susceptible versus resilient mice in the learned helplessness test (LHT). Furthermore, activating orexinergic neurons induced resilience in the susceptible group, and this resilience was also consistently observed in other behavioral tests. However, activating orexinergic neurons during the induction period (during inescapable stress exposure) did not affect stress resilience in the escape test. In addition, analyses using pathway-specific optic stimulation revealed that activating orexinergic projections to the medial part of the nucleus accumbens (NAc) alone mediated a decrease in anxiety but was not sufficient to induce resilience in the LHT. Collectively, our data suggest that orexinergic projections to multiple targets control diverse and flexible stress-related behaviors in response to various stressors.

Myristoylation-dependent palmitoylation of cyclin Y modulates long-term potentiation and spatial learning.

Many psychiatric disorders accompany deficits in cognitive functions and synaptic plasticity, and abnormal lipid modifications of neuronal proteins are associated with their pathophysiology. Lipid modifications, including palmitoylation and myristoylation, play crucial roles in the subcellular localization and trafficking of proteins. Cyclin Y (CCNY), enriched in the postsynaptic compartment, acts as an inhibitory modulator of functional and structural long-term potentiation (LTP) in the hippocampal neurons. However, cellular and molecular mechanisms underlying CCNY-mediated inhibitory functions in the synapse remain largely unknown. Here, we report that myristoylation located CCNY to the trans-Golgi network (TGN), and subsequent palmitoylation directed the myristoylated CCNY from the TGN to the synaptic cell surface. This myristoylation-dependent palmitoylation of CCNY was required for the inhibitory role of CCNY in excitatory synaptic transmission, activity-induced dynamics of AMPA receptors and PSD-95, LTP, and spatial learning. Furthermore, spatial learning significantly reduced palmitoyl- and myristoyl-CCNY levels, indicating that spatial learning lowers the synaptic abundance of CCNY. Our findings provide mechanistic insight into how CCNY is clustered adjacent to postsynaptic sites where it could play its inhibitory roles in synaptic plasticity and spatial learning.

Dynamic Changes in the Bridging Collaterals of the Basal Ganglia Circuitry Control Stress-Related Behaviors in Mice.

The basal ganglia network has been implicated in the control of adaptive behavior, possibly by integrating motor learning and motivational processes. Both positive and negative reinforcement appear to shape our behavioral adaptation by modulating the function of the basal ganglia. Here, we examined a transgenic mouse line (G2CT) in which synaptic transmissions onto the medium spiny neurons (MSNs) of the basal ganglia are depressed. We found that the level of collaterals from direct pathway MSNs in the external segment of the globus pallidus (GPe) ('bridging collaterals') was decreased in these mice, and this was accompanied by behavioral inhibition under stress. Furthermore, additional manipulations that could further decrease or restore the level of the bridging collaterals resulted in an increase in behavioral inhibition or active behavior in the G2CT mice, respectively. Collectively, our data indicate that the striatum of the basal ganglia network integrates negative emotions and controls appropriate coping responses in which the bridging collateral connections in the GPe play a critical regulatory role.

CRMP2 mediates GSK3ß actions in the striatum on regulating neuronal structure and mania-like behavior.

BACKGROUND: Genetic and physiological studies have implicated the striatum in bipolar disorder (BD). Although Glycogen synthase kinase 3 beta (GSK3ß) has been suggested to play a role in the pathophysiology of BD since it is inhibited by lithium, it remains unknown how GSK3ß activity might be involved. Therefore we examined the functional roles of GSK3ß and one of its substrates, CRMP2, within the striatum. METHODS: Using CRISPR-Cas9 system, we specifically ablated GSK3ß in the striatal neurons in vivo and in vitro. Sholl analysis was performed for the structural studies of medium spiny neurons (MSNs) and amphetamine-induced hyperlocomotion was measured to investigate the effects of gene ablations on the mania-like symptom of BD. RESULTS: GSK3ß deficiency in cultured neurons and in neurons of adult mouse brain caused opposite patterns of neurite changes. Furthermore, specific knockout of GSK3ß in the MSNs of the indirect pathway significantly suppressed amphetamine-induced hyperlocomotion. We demonstrated that these phenotypes of GSK3ß ablation were mediated by CRMP2, a major substrate of GSK3ß. LIMITATIONS: Amphetamine-induced hyperlocomotion only partially recapitulate the symptoms of BD. It requires further study to examine whether abnormality in GSK3ß or CRMP2 is also involved in depression phase of BD. Additionally, we could not confirm whether the behavioral changes observed in GSK3ß-ablated mice were indeed caused by the cellular structural changes observed in the striatal neurons. CONCLUSION: Our results demonstrate that GSK3ß and its substrate CRMP2 critically regulate the neurite structure of MSNs and their functions specifically within the indirect pathway of the basal ganglia network play a critical role in manifesting mania-like behavior of BD. Moreover, our data also suggest lithium may exert its effect on BD through a GSK3ß-independent mechanism, in addition to the GSK3ß inhibition-mediated mechanism.

Dopamine D2 receptor-mediated circuit from the central amygdala to the bed nucleus of the stria terminalis regulates impulsive behavior.

Impulsivity is closely associated with addictive disorders, and changes in the brain dopamine system have been proposed to affect impulse control in reward-related behaviors. However, the central neural pathways through which the dopamine system controls impulsive behavior are still unclear. We found that the absence of the D2 dopamine receptor (D2R) increased impulsive behavior in mice, whereas restoration of D2R expression specifically in the central amygdala (CeA) of D2R knockout mice (Drd2-/-) normalized their enhanced impulsivity. Inhibitory synaptic output from D2R-expressing neurons in the CeA underlies modulation of impulsive behavior because optogenetic activation of D2R-positive inhibitory neurons that project from the CeA to the bed nucleus of the stria terminalis (BNST) attenuate such behavior. Our identification of the key contribution of D2R-expressing neurons in the CeA → BNST circuit to the control of impulsive behavior reveals a pathway that could serve as a target for approaches to the management of neuropsychiatric disorders associated with impulsivity.

Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator.

Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%. Surprisingly, the range of V220D was similar to that of V220K with shifted optical responses towards negative potentials. In contrast, the V220E mutant mirrored the responses of the V220R mutation suggesting that the length of the side chain plays in role in determining the voltage range of the GEVI. Charged mutations at the 219 position all behaved similarly slightly shifting the optical response to more negative potentials. Charged mutations to the 221 position behaved erratically suggesting interactions with the plasma membrane and/or other amino acids in the VSD. Introduction of bulky amino acids at the V220 position increased the range of the optical response to include hyperpolarizing signals. Combining The V220W mutant with the R217Q mutation resulted in a probe that reduced the depolarizing signal and enhanced the hyperpolarizing signal which may lead to GEVIs that only report neuronal inhibition.

Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators.

The age of genetically encoded voltage indicators (GEVIs) has matured to the point that changes in membrane potential can now be observed optically in vivo. Improving the signal size and speed of these voltage sensors has been the primary driving forces during this maturation process. As a result, there is a wide range of probes using different voltage detecting mechanisms and fluorescent reporters. As the use of these probes transitions from optically reporting membrane potential in single, cultured cells to imaging populations of cells in slice and/or in vivo, a new challenge emerges-optically resolving the different types of neuronal activity. While improvements in speed and signal size are still needed, optimizing the voltage range and the subcellular expression (i.e., soma only) of the probe are becoming more important. In this review, we will examine the ability of recently developed probes to report synaptic activity in slice and in vivo. The voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes are all good at reporting changes in membrane potential, but all have difficulty distinguishing subthreshold depolarizations from action potentials and detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few possible ways to improve the optical resolution of the various types of neuronal activities.

Early life stress increases stress vulnerability through BDNF gene epigenetic changes in the rat hippocampus.

Early life stress (ELS) exerts long-lasting epigenetic influences on the brain and makes an individual susceptible to later depression. It is poorly understood whether ELS and subsequent adult chronic stress modulate epigenetic mechanisms. We examined the epigenetic mechanisms of the BDNF gene in the hippocampus, which may underlie stress vulnerability to postnatal maternal separation (MS) and adult restraint stress (RS). Rat pups were separated from their dams (3 h/day from P1-P21). When the pups reached adulthood (8 weeks old), we introduced RS (2 h/day for 3 weeks) followed by escitalopram treatment. We showed that both the MS and RS groups expressed reduced levels of total and exon IV BDNF mRNA. Furthermore, RS potentiated MS-induced decreases in these expression levels. Similarly, both the MS and RS groups showed decreased levels of acetylated histone H3 and H4 at BDNF promoter IV, and RS exacerbated MS-induced decreases of H3 and H4 acetylation. Both the MS and RS groups had increased MeCP2 levels at BDNF promoter IV, as well as increased HDAC5 mRNA, and the combination of MS and RS exerted a greater effect on these parameters than did RS alone. In the forced swimming test, the immobility time of the MS + RS group was significantly higher than that of the RS group. Additionally, chronic escitalopram treatment recovered these alterations. Our results suggest that postnatal MS and subsequent adult RS modulate epigenetic changes in the BDNF gene, and that these changes may be related to behavioral phenotype. These epigenetic mechanisms are involved in escitalopram action.

Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors.

Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein.

Association of brain-derived neurotrophic factor DNA methylation and reduced white matter integrity in the anterior corona radiata in major depression.

Considerable evidence suggests a crucial role for the epigenetic regulation of brain-derived neurotrophic factor (BDNF) in the pathophysiology of major depressive disorder (MDD). However, the relationship between BDNF DNA methylation and white matter (WM) integrity in MDD has not yet been investigated. In the current study, we examined the association between the DNA methylation status of the BDNF promoter region and WM integrity in MDD. Sixty patients with MDD and 53 healthy controls underwent T1-weighted structural magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), to assess their WM integrity. BDNF DNA methylation at 4 CpG sites of the promoter region was also measured. As compared to healthy controls, the MDD group demonstrated reduced fractional anisotropy (FA) in the bilateral anterior and posterior corona radiata (ACR and PCR), genu of the corpus callosum, and the bilateral posterior thalamic radiations. We observed a significant inverse correlation between the DNA methylation of the BDNF promoter region and the FA of the right ACR in MDD patients. Our findings demonstrate a relationship between methylation of the BDNF promoter region and the integrity of the ACR, a key structural component of the emotional and cognitive control network involved in the pathophysiology of MDD. This correlation suggests that BDNF DNA methylation may contribute to structural WM changes in MDD patients.

Altered trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) in the striatum leads to behavioral changes in emotional responses.

The striatum receives and integrates multiple inputs from diverse areas in the brain and plays a critical role in the regulation of motor activity. However, whether the striatum is involved in the alteration of behavior in the presence of emotional challenges is unknown. Here, we examined whether alterations in the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) in the dorsal striatum would affect anxiety-related behaviors. We found that the transient expression of G1CT or G2CT, AMPAR-derived peptides, in the dorsomedial striatum led to decreased mobility in high-anxiety circumstances; however, the expression of these peptides in the dorsolateral striatum did not affect anxiety-related behavior. These data suggest that excitatory connections within the dorsomedial striatum play important roles in the control of motor actions in the presence of emotional challenges.

Orexin administration to mice that underwent chronic stress produces bimodal effects on emotion-related behaviors.

Orexin plays diverse roles in regulating behaviors, such as sleep and wake, reward processing, arousal, and stress and anxiety. The orexin system may accomplish these multiple tasks through its complex innervations throughout the brain. The emerging evidence indicates a role of orexin in emotional behaviors; however, most of the previous studies have investigated the function of orexin in naïve animals. Here, we examined a functional role of orexin in mice that had been exposed to repeated stress. Chronic social defeat stress produced differential social interaction behaviors in mice (susceptible versus resilient) and these two groups of mice displayed different levels of prepro-orexin in the hypothalamus. Exogenously added orexin A to the brain induced an antidepressant-like effect in only the susceptible mice but not in the resilient mice. In contrast, orexin A and orexin B infused together produced an anxiogenic effect in only the resilient mice and not in the susceptible mice. Furthermore, we found that the antidepressant-like effect of orexin A is mediated by the bed nucleus of the stria terminalis (BNST) after exposure to chronic restraint stress. These findings reveal a bimodal effect of the orexin system in regulating emotional behavior that depends on stress susceptibility.

Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization.

Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

Snx14 regulates neuronal excitability, promotes synaptic transmission, and is imprinted in the brain of mice.

Genomic imprinting describes an epigenetic process through which genes can be expressed in a parent-of-origin-specific manner. The monoallelic expression of imprinted genes renders them particularly susceptible to disease causing mutations. A large proportion of imprinted genes are expressed in the brain, but little is known about their functions. Indeed, it has proven difficult to identify cell type-specific imprinted genes due to the heterogeneity of cell types within the brain. Here we used laser capture microdissection of visual cortical neurons and found evidence that sorting nexin 14 (Snx14) is a neuronally imprinted gene in mice. SNX14 protein levels are high in the brain and progressively increase during neuronal development and maturation. Snx14 knockdown reduces intrinsic excitability and severely impairs both excitatory and inhibitory synaptic transmission. These data reveal a role for monoallelic Snx14 expression in maintaining normal neuronal excitability and synaptic transmission.

The expression of PHO92 is regulated by Gcr1, and Pho92 is involved in glucose metabolism in Saccharomyces cerevisiae.

Ydr374c (Pho92) contains a YTH domain in its C-terminal region and is a human YTHDF2 homologue. Previously, we reported that Pho92 regulates phosphate metabolism by regulating PHO4 mRNA stability. In this study, we found that growth of the ∆pho92 strain on SG media was slower than that of the wild type and that PHO92 expression was up-regulated by non-fermentable carbon sources, such as ethanol and glycerol, but not by fermentable carbon sources. Furthermore, two conserved Gcr1-binding regions were identified in the upstream, untranslated region of PHO92. Gcr1 is an important factor involved in the coordinated regulation of glycolytic gene expression. Mutation of two Gcr1-binding sites of the PHO92 upstream region resulted in a growth defect on SD media. Finally, mutagenesis of the Gcr1-binding sites of the PHO92 upstream region and deletion of GCR1 resulted in up-regulation of PHO92, and this resulted from inhibition of PHO4 mRNA degradation. Based on these results, we suggest that Gcr1 regulates the expression of PHO92, and Pho92 is involved in glucose metabolism.

Development of a novel screening system for allatostatin receptor agonists in search of new candidate insect growth regulators.

Allatostatins (ASTs) are insect neuropeptide hormones that regulate diverse physiological functions, including feeding, growth and development, and reproduction. Therefore, regulation of allatostatin receptor (AstR) activity can be an effective tool for controlling insect growth and proliferation. Here, we describe a novel screening system using a mammalian cell line in which AstR is ectopically expressed, combined with fluorescence-based measurements of the membrane potential. HEK293T cells that do not express cognate receptors for AST became responsive to AST upon transfection with AstR. The response of the membrane potential to AST could be reliably detected by measuring the fluorescence of DiBAC4(3), a voltage-sensitive dye. We also discovered that overexpressing GIRK1/2 in this cell line could augment the magnitude of hyperpolarization by AST. Our screening system produces a fast and reliable readout for the efficient screening of AstR agonists.

Remodeling of the dendritic structure of the striatal medium spiny neurons accompanies behavioral recovery in a mouse model of Parkinson's disease.

Medium spiny neurons (MSNs) are the major type of neurons found in the striatum. The dendritic spines on these cells contain glutamatergic synaptic contacts between the cortex (or the thalamus) and the striatum. The complexity of the dendritic structure of MSNs may therefore reflect the functional status of the basal ganglia because the striatum is the major input structure in which signals from different regions are integrated. We examined the structural alterations in the dendrites of striatal MSNs in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease (PD). Acute MPTP treatment rapidly damaged dopaminergic neurons and their terminals within the striatum and caused behavioral impairments. However, mice injected with MPTP spontaneously recovered from these behavioral impairments within one week. This recovery was accompanied by the restoration of dendritic structures on MSNs, but the damage to dopaminergic neurons remained extensive. Furthermore, we demonstrated that rasagiline, a monoamine oxidase-B (MAO-B) inhibitor that has been shown to be efficacious for PD, could enhance the dendritic complexity of cultured MSNs. The effect of rasagiline on the spine-like structures of dendrites, however, appears not to require DA availability because the small protrusions of dendrites in cultured MSNs without major source of DA input was similarly changed by rasagiline. Our data suggest that the dendritic structures of striatal MSNs change dynamically, reflecting the progression of motor-related symptoms in PD, and the restoration of functional synapses in the MSNs of PD patients may constitute a clinical target for symptomatic alleviation.

Different locomotor sensitization responses to repeated cocaine injections are associated with differential phosphorylation of GluA1 in the dorsomedial striatum of adult rats.

Behavioral sensitization to psychostimulants reflects neural adaptation, which might share a common mechanism with drug addiction. Outbred male rats show different locomotor sensitization responses to cocaine, and cocaine also produces varied addictive progress in humans. We investigated whether differences in the induction of sensitization would affect the long-term persistence of sensitized locomotor activity, and we sought to determine the molecular basis for the variability in sensitization. Male Sprague-Dawley rats that showed sensitized locomotor responses over 5 consecutive daily cocaine injections (SENS) had significantly lower initial locomotor responses to the 1st cocaine exposure than did rats that did not show locomotor sensitization (NONS). Furthermore, rats that underwent 1 month of cocaine withdrawal after 5 repeated cocaine injections also exhibited sensitized or non-sensitized locomotor responses to a challenge injection of cocaine (SENS-C or NONS-C, respectively). This variability was also related to the initial responsiveness to cocaine. We examined the level of phosphorylation of the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropioniate receptor (AMPAR) in the dorsal striatum and found that there were significant differences between the sensitized rats and the non-sensitized rats. pGluA1-Ser831 was increased in the SENS rats during the induction of locomotor sensitization, and pGluA1-Ser845 was increased in the SENS-C rats during the expression of locomotor sensitization. These phosphorylation changes were observed in the dorsomedial striatum (DMS) of adult rats but not in the dorsolateral striatum (DLS) of adults. Our findings suggest that differential phosphorylation of AMPAR might be an important mechanism that contributes to the development of locomotor sensitization to cocaine in adult rats.

Basal blood corticosterone level is correlated with susceptibility to chronic restraint stress in mice.

Corticosterone is released in response to stress and manifests as various bodily stress responses in rodents. While corticosterone reflects acute adaptive responses, how the basal steady-state corticosterone level relates to the subsequent stress response is largely unknown. Here, we investigated how basal corticosterone levels can affect the susceptibility to chronic restraint stress in mice. We designed a longitudinal experiment, enabling us to compare the basal corticosterone level and the subsequent response to repeated restraint stress within the same animal. We found that the mice had differential changes in plasma corticosterone levels, which either increased or decreased, with exposure to chronic stress. These differential changes reflected the differential stress susceptibility of the mice, as evaluated by changes in body weight. The extent of the changes in corticosterone level during chronic stress exposure was predicted by the basal corticosterone level. In addition, the behavioral consequence of chronic stress was also correlated with the basal corticosterone level prior to chronic stress experience. These data reveal that the basal steady-state corticosterone level is a predictor of stress susceptibility or resilience to subsequent stress exposures.