Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

All-optical approaches to studying psychiatric disease.

Improvements in all-optical means of monitoring and manipulating neural activity have generated new ways of studying psychiatric disease. The combination of calcium imaging techniques with optogenetics to concurrently record and manipulate neural activity has been used to create new disease models that link distinct circuit abnormalities to specific disease dimensions. These approaches represent a new path towards the development of more effective treatments, as they allow researchers to identify circuit manipulations that normalize pathological network activity. In this review we highlight the utility of all-optical approaches to generate new psychiatric disease models where the specific circuit abnormalities associated with disease symptomology can be assessed in vivo and in response to manipulations designed to normalize disease states. We then outline the principles underlying all-optical interrogations of neural circuits and discuss practical considerations for experimental design.

Lysergic acid diethylamide (LSD) promotes social behavior through mTORC1 in the excitatory neurotransmission.

Clinical studies have reported that the psychedelic lysergic acid diethylamide (LSD) enhances empathy and social behavior (SB) in humans, but its mechanism of action remains elusive. Using a multidisciplinary approach including in vivo electrophysiology, optogenetics, behavioral paradigms, and molecular biology, the effects of LSD on SB and glutamatergic neurotransmission in the medial prefrontal cortex (mPFC) were studied in male mice. Acute LSD (30 µg/kg) injection failed to increase SB. However, repeated LSD (30 µg/kg, once a day, for 7 days) administration promotes SB, without eliciting antidepressant/anxiolytic-like effects. Optogenetic inhibition of mPFC excitatory neurons dramatically inhibits social interaction and nullifies the prosocial effect of LSD. LSD potentiates the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and 5-HT2A, but not N-methyl-D-aspartate (NMDA) and 5-HT1A, synaptic responses in the mPFC and increases the phosphorylation of the serine-threonine protein kinases Akt and mTOR. In conditional knockout mice lacking Raptor (one of the structural components of the mTORC1 complex) in excitatory glutamatergic neurons (Raptorf/f:Camk2alpha-Cre), the prosocial effects of LSD and the potentiation of 5-HT2A/AMPA synaptic responses were nullified, demonstrating that LSD requires the integrity of mTORC1 in excitatory neurons to promote SB. Conversely, in knockout mice lacking Raptor in GABAergic neurons of the mPFC (Raptorf/f:Gad2-Cre), LSD promotes SB. These results indicate that LSD selectively enhances SB by potentiating mPFC excitatory transmission through 5-HT2A/AMPA receptors and mTOR signaling. The activation of 5-HT2A/AMPA/mTORC1 in the mPFC by psychedelic drugs should be explored for the treatment of mental diseases with SB impairments such as autism spectrum disorder and social anxiety disorder.

Off-Target Influences of Arch-Mediated Axon Terminal Inhibition on Network Activity and Behavior.

Archaerhodopsin (ArchT)-mediated photoinhibition of axon terminals is commonly used to test the involvement of specific long-range neural projections in behavior. Although sustained activation of this opsin in axon terminals has the unintended consequence of enhancing spontaneous vesicle release, it is unclear whether this desynchronized signaling is consequential for ArchT's behavioral effects. Here, we compare axon terminal and cell body photoinhibition of nucleus accumbens (NAc) afferents to test the utility of these approaches for uncovering pathway-specific contributions of neural circuits to behavior. First, in brain slice recordings we confirmed that ArchT photoinhibition of glutamatergic axons reduces evoked synaptic currents and increases spontaneous transmitter release. A further consequence was increased interneuron activity, which served to broadly suppress glutamate input via presynaptic GABAB receptors. In vivo, axon terminal photoinhibition increased feeding and reward-seeking behavior irrespective of the afferent pathway targeted. These behavioral effects are comparable to those obtained with broad inhibition of NAc neurons. In contrast, cell body inhibition of excitatory NAc afferents revealed a pathway-specific contribution of thalamic input to feeding behavior and amygdala input to reward-seeking under extinction conditions. These findings underscore the off-target behavioral consequences of ArchT-mediated axon terminal inhibition while highlighting cell body inhibition as a valuable alternative for pathway-specific optogenetic silencing.

Nucleus Accumbens Cell Type- and Input-Specific Suppression of Unproductive Reward Seeking.

The nucleus accumbens (NAc) contributes to behavioral inhibition and compulsions, but circuit mechanisms are unclear. Recent evidence suggests that amygdala and thalamic inputs exert opposing control over behavior, much like direct and indirect pathway output neurons. Accordingly, opponent processes between these NAc inputs or cell types may underlie efficient reward seeking. We assess the contributions of these circuit elements to mouse operant behavior during recurring conditions when reward is and is not available. Although direct pathway stimulation is rewarding and indirect pathway stimulation aversive, the activity of both cell types is elevated during periods of behavioral suppression, and the inhibition of either cell-type selectively increases unproductive reward seeking. Amygdala and thalamic inputs are also necessary for behavioral suppression, even though they both support self-stimulation and innervate different NAc subregions. These data suggest that efficient reward seeking relies on complementary activity across NAc cell types and inputs rather than opponent processes between them.

Hippocampal Input to the Nucleus Accumbens Shell Enhances Food Palatability.

BACKGROUND: Insight into the neural basis of hedonic processing has come from studies of food palatability in rodents. Pharmacological manipulations of the nucleus accumbens shell (NAcSh) have repeatedly been demonstrated to increase hedonic taste reactivity, yet the contribution of specific NAcSh circuit components is unknown. METHODS: Bidirectional optogenetic manipulations were targeted to the principal NAcSh projection neurons and afferent pathways in mice during free feeding assays. Number of licks per bout of consumption was used as a measure of food palatability as it was confirmed to track sucrose concentration and subjective flavor preferences. RESULTS: Photoinhibition of NAcSh neurons, whether general or cell-type specific, was found to alter consumption without affecting its hedonic impact. Among the principal excitatory afferent pathways, we showed that ventral hippocampal (vHipp) input alone enhances palatability upon low-frequency photostimulation time-locked to consumption. This enhancement in palatability was independent of opioid signaling and not recapitulated by NAcSh or dopamine neuron photostimulation. We further demonstrated that vHipp input photostimulation is sufficient to condition a flavor preference, while its inhibition impedes sucrose-driven flavor preference conditioning. CONCLUSIONS: These results demonstrate a novel contribution of vHipp-NAcSh pathway activity to palatability that may relate to its innervation of a particular region or neuronal ensemble in the NAcSh. These findings are consistent with the evidence that vHipp-NAcSh activity is relevant to the pathophysiology of anhedonia and depression as well as the increasing appreciation of hippocampal involvement in people's food pleasantness ratings, hunger, and weight.

Cue-Evoked Dopamine Neuron Activity Helps Maintain but Does Not Encode Expected Value.

Cue-evoked midbrain dopamine (DA) neuron activity reflects expected value, but its influence on reward assessment is unclear. In mice performing a trial-based operant task, we test if bidirectional manipulations of cue or operant-associated DA neuron activity drive learning as a result of under- or overexpectation of reward value. We target optogenetic manipulations to different components of forced trials, when only one lever is presented, and assess lever biases on choice trials in the absence of photomanipulation. Although lever biases are demonstrated to be flexible and sensitive to changes in expected value, augmentation of cue or operant-associated DA signaling does not significantly alter choice behavior, and blunting DA signaling during any component of the forced trials reduces choice trial responses on the associated lever. These data suggest cue-evoked DA helps maintain cue-value associations but does not encode expected value as to set the benchmark against which received reward is judged.

Coordinated Reductions in Excitatory Input to the Nucleus Accumbens Underlie Food Consumption.

Reward-seeking behavior is regulated by a diverse collection of inputs to the nucleus accumbens (NAc). The information encoded in each excitatory afferent to the NAc is unknown, in part because it is unclear when these pathways are active in relation to behavior. Here we compare the activity profiles of amygdala, hippocampal, and thalamic inputs to the NAc shell in mice performing a cued reward-seeking task using GCaMP-based fiber photometry. We find that the rostral and caudal ends of the NAc shell are innervated by distinct but intermingled populations of forebrain neurons that exhibit divergent feeding-related activity. In the rostral NAc shell, a coordinated network-wide reduction in excitatory drive correlates with feeding, and reduced input from individual pathways is sufficient to promote it. Overall, the data suggest that pathway-specific input activity at a population level may vary more across the NAc than between pathways.

Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex.

It is generally assumed that infralimbic cortex (ILC) and prelimbic cortex, two adjacent areas of the medial prefrontal cortex (mPFC) in rodents, provide selective excitatory glutamatergic inputs to the nucleus accumbens (NAc) shell and core, respectively. It is also generally believed that mPFC influences the extracellular levels of dopamine in the NAc primarily by an excitatory collateral to the ventral tegmental area (VTA). In the present study, we first established the existence of a selective functional connection between ILC and the posteromedial portions of the VTA (pmVTA) and the mNAc shell (pmNAc shell), by measuring striatal neuronal activation (immunohistochemical analysis of ERK1/2 phosphorylation) and glutamate release (in vivo microdialysis) upon ILC electrical stimulation. A novel optogenetic-microdialysis approach allowed the measurement of extracellular concentrations of glutamate and dopamine in the pmNAc shell upon local light-induced stimulation of glutamatergic terminals from ILC. Cortical electrical and local optogenetic stimulation produced significant increases in the extracellular concentrations of glutamate and dopamine in the pmNAc shell. Local blockade of glutamate release by perfusion of an adenosine A2A receptor antagonist in the pmNAc shell blocked the dopamine release induced by local optogenetic stimulation but only partially antagonized dopamine release induced by cortical electrical stimulation. The results demonstrate that ILC excitatory afferents directly modulate the extracellular concentration of dopamine in the pmNAc shell, but also support the involvement of an indirect mechanism of dopamine control, through a concomitant ILC-mediated activation of the pmVTA. Significance statement: We established the existence of a functional connection between the infralimbic cortex (ILC) and the posteromedial portions of the ventral tegmental area (pmVTA) and the medial nucleus acumbens shell (pmNAc shell). A novel optogenetic-microdialysis approach allowed us to demonstrate that local glutamate release from glutamatergic terminals from the ILC exert a significant modulation of extracellular concentration of dopamine in the pmNAc shell. This mechanism provides the frame for a selective cortical-mediated tonic dopaminergic modulation of specific striatal compartments.

Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons.

Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.

Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry.

The dorsal raphe nucleus (DRN) contains the largest group of serotonin-producing neurons in the brain and projects to regions controlling reward. Although pharmacological studies suggest that serotonin inhibits reward seeking, electrical stimulation of the DRN strongly reinforces instrumental behavior. Here, we provide a targeted assessment of the behavioral, anatomical, and electrophysiological contributions of serotonergic and nonserotonergic DRN neurons to reward processes. To explore DRN heterogeneity, we used a simultaneous two-vector knockout/optogenetic stimulation strategy, as well as cre-induced and cre-silenced vectors in several cre-expressing transgenic mouse lines. We found that the DRN is capable of reinforcing behavior primarily via nonserotonergic neurons, for which the main projection target is the ventral tegmental area (VTA). Furthermore, these nonserotonergic projections provide glutamatergic excitation of VTA dopamine neurons and account for a large majority of the DRN-VTA pathway. These findings help to resolve apparent discrepancies between the roles of serotonin versus the DRN in behavioral reinforcement.

Optogenetics in preclinical neuroscience and psychiatry research: recent insights and potential applications.

There have been significant advances in the treatment of psychiatric disease in the last half century, but it is still unclear which neural circuits are ultimately responsible for specific disease states. Fortunately, technical limitations that have constrained this research have recently been mitigated by advances in research tools that facilitate circuit-based analyses. The most prominent of these tools is optogenetics, which refers to the use of genetically encoded, light-sensitive proteins that can be used to manipulate discrete neural circuits with temporal precision. Optogenetics has recently been used to examine the neural underpinnings of both psychiatric disease and symptom relief, and this research has rapidly identified novel therapeutic targets for what could be a new generation of rational drug development. As these and related methodologies for controlling neurons ultimately make their way into the clinic, circuit-based strategies for alleviating psychiatric symptoms could become a remarkably refined approach to disease treatment.

Methamphetamine downregulates striatal glutamate receptors via diverse epigenetic mechanisms.

BACKGROUND: Chronic methamphetamine (METH) exposure causes neuroadaptations at glutamatergic synapses. METHODS: To identify the METH-induced epigenetic underpinnings of these neuroadaptations, we injected increasing METH doses to rats for 2 weeks and measured striatal glutamate receptor expression. We then quantified the effects of METH exposure on histone acetylation. We also measured METH-induced changes in DNA methylation and DNA hydroxymethylation. RESULTS: Chronic METH decreased transcript and protein expression of GluA1 and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and GluN1 N-methyl-D-aspartate receptor subunits. These changes were associated with altered electrophysiological glutamatergic responses in striatal neurons. Chromatin immunoprecipitation-polymerase chain reaction revealed that METH decreased enrichment of acetylated histone H4 on GluA1, GluA2, and GluN1 promoters. Methamphetamine exposure also increased repressor element-1 silencing transcription factor (REST) corepressor 1, methylated CpG binding protein 2, and histone deacetylase 2 enrichment, but not of sirtuin 1 or sirtuin 2, onto GluA1 and GluA2 gene sequences. Moreover, METH caused interactions of REST corepressor 1 and methylated CpG binding protein 2 with histone deacetylase 2 and of REST with histone deacetylase 1. Surprisingly, methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation-polymerase chain reaction revealed METH-induced decreased enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at GluA1 and GluA2 promoter sequences. Importantly, the histone deacetylase inhibitor, valproic acid, blocked METH-induced decreased expression of AMPAR and N-methyl-D-aspartate receptor subunits. Finally, valproic acid also attenuated METH-induced decrease H4K16Ac recruitment on AMPAR gene sequences. CONCLUSIONS: These observations suggest that histone H4 hypoacetylation may be the main determinant of METH-induced decreased striatal glutamate receptor expression.

Alcohol and tobacco: how smoking may promote excessive drinking.

Cigarette smokers tend to drink more alcohol than their nonsmoking peers. In this issue of Neuron, Doyon et al. (2013) found that nicotine-induced increases in stress hormones can augment ethanol self-administration in rats, suggesting that a drug interaction may contribute to this phenomenon.

Optogenetic interrogations of the neural circuits underlying addiction.

Exposure to addictive drugs can result in maladaptive alterations in neural circuit function. This review highlights recent progress made in identifying the organization, function, and cellular plasticity of the ventral tegmental area (VTA) and nucleus accumbens (NAc), two brain regions strongly implicated in substance use disorders. Emphasis is given to advances made with new research methodologies, particularly optogenetics, which have provided scientists with an unprecedented ability to map neural circuitry and pinpoint drug-induced synaptic modifications. A better understanding of these adaptive events will aid the development of pharmacological treatments for drug addiction and, more generally, further our understanding of motivated behaviors.

Synaptic and behavioral profile of multiple glutamatergic inputs to the nucleus accumbens.

Excitatory afferents to the nucleus accumbens (NAc) are thought to facilitate reward seeking by encoding reward-associated cues. Selective activation of different glutamatergic inputs to the NAc can produce divergent physiological and behavioral responses, but mechanistic explanations for these pathway-specific effects are lacking. Here, we compared the innervation patterns and synaptic properties of ventral hippocampus, basolateral amygdala, and prefrontal cortex input to the NAc. Ventral hippocampal input was found to be uniquely localized to the medial NAc shell, where it was predominant and selectively potentiated after cocaine exposure. In vivo, bidirectional optogenetic manipulations of this pathway attenuated and enhanced cocaine-induced locomotion. Challenging the idea that any of these inputs encode motivationally neutral information, activation of each discrete pathway reinforced instrumental behaviors. Finally, direct optical activation of medium spiny neurons proved to be capable of supporting self-stimulation, demonstrating that behavioral reinforcement is an explicit consequence of strong excitatory drive to the NAc.

Use of channelrhodopsin for activation of CNS neurons.

Optogenetics-the use of optically activated proteins to control cell function-allows for control of neurons with an unprecedented degree of spatial, temporal, and neurochemical precision. Three protocols are presented in this unit describing the use of channelrhodopsin-2 (ChR2), a light-activated cation channel. These protocols emphasize practical issues of working with ChR2, including guidelines for selecting a gene delivery method, light source, and method of tissue implantation, as well as steps for fabricating fiber optic patch cables and chronic implantable optical fibers. The first protocol describes the use of ChR2 in electrophysiological recordings from brain slices. The second and third involve the use of ChR2 in vivo, with light delivered through chronic fiber implants or guide cannula.

Optogenetic modulation of neural circuits that underlie reward seeking.

The manifestation of complex neuropsychiatric disorders, such as drug and alcohol addiction, is thought to result from progressive maladaptive alterations in neural circuit function. Clearly, repeated drug exposure alters a distributed network of neural circuit elements. However, a more precise understanding of addiction has been hampered by an inability to control and, consequently, identify specific circuit components that underlie addictive behaviors. The development of optogenetic strategies for selectively modulating the activity of genetically defined neuronal populations has provided a means for determining the relationship between circuit function and behavior with a level of precision that has been previously unobtainable. Here, we briefly review the main optogenetic studies that have contributed to elucidate neural circuit connectivity within the ventral tegmental area and the nucleus accumbens, two brain nuclei that are essential for the manifestation of addiction-related behaviors. Additional targeted manipulation of genetically defined neural populations in these brain regions, as well as afferent and efferent structures, promises to delineate the cellular mechanisms and circuit components required for the transition from natural goal-directed behavior to compulsive reward seeking despite negative consequences.

Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice.

Treatment of Parkinson disease (PD) with L-3,4-dihydroxyphenylalanine (L-DOPA) dramatically relieves associated motor deficits, but L-DOPA-induced dyskinesias (LID) limit the therapeutic benefit over time. Previous investigations have noted changes in striatal medium spiny neurons, including abnormal activation of extracellular signal-regulated kinase1/2 (ERK). Using two PD models, the traditional 6-hydroxydopamine toxic lesion and a genetic model with nigrostriatal dopaminergic deficits, we found that acute dopamine challenge induces ERK activation in medium spiny neurons in denervated striatum. After repeated L-DOPA treatment, however, ERK activation diminishes in medium spiny neurons and increases in striatal cholinergic interneurons. ERK activation leads to enhanced basal firing rate and stronger excitatory responses to dopamine in striatal cholinergic neurons. Pharmacological blockers of ERK activation inhibit L-DOPA-induced changes in ERK phosphorylation, neuronal excitability, and the behavioral manifestation of LID. In addition, a muscarinic receptor antagonist reduces LID. These data indicate that increased dopamine sensitivity of striatal cholinergic neurons contributes to the expression of LID, which suggests novel therapeutic targets for LID.