A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Deletion of astrocytic BMAL1 results in metabolic imbalance and shorter lifespan in mice.

Disruption of the circadian cycle is strongly associated with metabolic imbalance and reduced longevity in humans. Also, rodent models of circadian arrhythmia, such as the constitutive knockout of the clock gene Bmal1, leads to metabolic disturbances and early death. Although astrocyte clock regulates molecular and behavioral circadian rhythms, its involvement in the regulation of energy balance and lifespan is unknown. Here, we show that astrocyte-specific deletion of Bmal1 is sufficient to alter energy balance, glucose homeostasis, and reduce lifespan. Mutant animals displayed impaired hypothalamic molecular clock, age-dependent astrogliosis, apoptosis of hypothalamic astrocytes, and increased glutamate and GABA levels. Importantly, modulation of GABAA-receptor signaling completely restored glutamate levels, delayed the reactive gliosis as well as the metabolic phenotypes and expanded the lifespan of the mutants. Our results demonstrate that the astrocytic clock can influence many aspects of brain function and neurological disease and suggest astrocytes and GABAA receptor as pharmacological targets to prevent the metabolic dysfunctions and shortened lifespan associated with alterations of circadian rhythms.

Oxytocin, vasopressin and social behavior in the age of genome editing: A comparative perspective.

Behavioral neuroendocrinology has a rich history of using diverse model organisms to elucidate general principles and evolution of hormone-brain-behavior relationships. The oxytocin and vasopressin systems have been studied in many species, revealing their role in regulating social behaviors. Oxytocin and vasopressin receptors show remarkable species and individual differences in distribution in the brain that have been linked to diversity in social behaviors. New technologies allow for unprecedented interrogation of the genes and neural circuitry regulating behaviors, but these approaches often require transgenic models and are most often used in mice. Here we discuss seminal findings relating the oxytocin and vasopressin systems to social behavior with a focus on non-traditional animal models. We then evaluate the potential of using CRISPR/Cas9 genome editing to examine the roles of genes and enable circuit dissection, manipulation and activity monitoring of the oxytocin and vasopressin systems. We believe that it is essential to incorporate these genetic and circuit level techniques in comparative behavioral neuroendocrinology research to ensure that our field remains innovative and attractive for the next generation of investigators and funding agencies.

Diet-induced neuropeptide expression: feasibility of quantifying extended and highly charged endogenous peptide sequences by selected reaction monitoring.

Understanding regulation and action of endogenous peptides, especially neuropeptides, which serve as inter- and intracellular signal transmitters, is key in understanding a variety of functional processes, such as energy balance, memory, circadian rhythm, drug addiction, etc. Therefore, accurate and reproducible quantification of these bioactive endogenous compounds is highly relevant. The biosynthesis of endogenous peptides, involving multiple possible trimming and modification events, hinders the de novo prediction of the active peptide sequences, making MS-based measurements very valuable in determining the actual active compounds. Here, we report an extended selected reaction monitoring (SRM)-based strategy to reproducibly and quantitatively monitor the abundances of a set of 15 endogenously occurring peptides from Rattus norvegicus hypothalamus. We demonstrate that SRM can be extended toward reproducible detection and quantification of peptides, bearing characteristics very different from tryptic peptides. We show that long peptide sequences, producing precursors with up to five and MS2 fragment ions with up to three charges, can be targeted by SRM on a triple quadrupole instrument. Using this approach to quantify endogenous peptide levels in hypothalami of animals subjected to different diets revealed several significant changes, most notably the significant upregulation of VGF-derived signaling peptide AQEE-30 upon high caloric feeding.

Profiling of diet-induced neuropeptide changes in rat brain by quantitative mass spectrometry.

Neuropeptides are intercellular signal transmitters that play key roles in modulation of many behavioral and physiological processes. Neuropeptide signaling in several nuclei in the hypothalamus contributes to the control of food intake. Additionally, food intake regulation involves neuropeptide signaling in the reward circuitry in the striatum. Here, we analyze neuropeptides extracted from hypothalamus and striatum from rats in four differentially treated dietary groups including a high-fat/high-sucrose diet, mimicking diet-induced obesity. We employ high-resolution tandem mass spectrometry using higher-energy collision dissociation and electron transfer dissociation fragmentation for sensitive identification of more than 1700 unique endogenous peptides, including virtually all key neuropeptides known to be involved in food intake regulation. Label-free quantification of differential neuropeptide expression revealed comparable upregulation of orexigenic and anorexigenic neuropeptides in rats that were fed on a high-fat/high-sucrose diet.

Natural variation in oxytocin receptor signaling causes widespread changes in brain transcription: a link to the natural killer gene complex.

Oxytocin (OXT) is a highly conserved neuropeptide that modulates social cognition, and variation in its receptor gene (Oxtr) is associated with divergent social phenotypes. The cellular mechanisms connecting Oxtr genotype to social phenotype remain obscure. We exploit an association between Oxtr polymorphisms and striatal-specific OXTR density in prairie voles to investigate how OXTR signaling influences the brain transcriptome. We discover widespread, OXTR signaling-dependent transcriptomic changes. Interestingly, OXTR signaling robustly modulates gene expression of C-type lectin-like receptors (CTLRs) in the natural killer gene complex, a genomic region associated with immune function. CTLRs are positioned to control microglial synaptic pruning; a process important for shaping neural circuits. Similar relationships between OXTR RNA and CTLR gene expression were found in human striatum. These data suggest a potential molecular mechanism by which variation in OXTR signaling due to genetic background and/or life-long social experiences, including nurturing/neglect, may affect circuit connectivity and social behavior.

An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept.

A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

Genomewide analysis of rat barrel cortex reveals time- and layer-specific mRNA expression changes related to experience-dependent plasticity.

Because of its anatomical organization, the rodent whisker-to-barrel system is an ideal model to study experience-dependent plasticity. Manipulation of sensory input causes changes in the properties of the barrels at the physiological, structural, and functional levels. However, much less is known about the molecular events underlying these changes. To explore such molecular events, we have used a genomewide approach to identify key genes and molecular pathways involved in experience-induced plasticity in the barrel cortex of adult rats. Given the natural tendency of rats to explore novel objects, exposure to an enriched environment (EE) was used to stimulate the activity of the whisker-to-barrel cortex in vivo. Microarray analysis at two different time points after EE revealed differential expression of genes encoding transcription factors, including nuclear receptors, as well as of genes involved in the regulation of synaptic plasticity, cell differentiation, metabolism, and, surprisingly, blood vessel morphogenesis. These expression differences reflect changes in somatosensory information processing because unilateral whisker clipping showed EE-induced differential expression patterns in the spared versus deprived barrel cortex. Finally, in situ hybridization revealed cortical layer patterns specific for each selected gene. Together, the present study offers the first genomewide exploration of the key genes regulated by somatosensory stimulation in the barrel cortex and thus provides a solid experimental framework for future in-depth analysis of the mechanisms underlying experience-dependent plasticity.

Social experience alters oxytocinergic modulation in the nucleus accumbens of female prairie voles.

Social relationships are dynamic and evolve with shared and personal experiences. Whether the functional role of social neuromodulators also evolves with experience to shape the trajectory of relationships is unknown. We utilized pair bonding in the socially monogamous prairie vole as an example of socio-sexual experience that dramatically alters behaviors displayed toward other individuals. We investigated oxytocin-dependent modulation of excitatory synaptic transmission in the nucleus accumbens as a function of pair-bonding status. We found that an oxytocin receptor agonist decreases the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in sexually naive virgin, but not pair-bonded, female voles, while it increases the amplitude of electrically evoked EPSCs in paired voles, but not in virgins. This oxytocin-induced potentiation of synaptic transmission relies on the de novo coupling between oxytocin receptor signaling and endocannabinoid receptor type 1 (CB1) receptor signaling in pair-bonded voles. Blocking CB1 receptors after pair-bond formation increases the occurrence of a specific form of social rejection-defensive upright response-that is displayed toward the partner, but not toward a novel individual. Altogether, our results demonstrate that oxytocin's action in the nucleus accumbens is changed through social experience in a way that regulates the trajectory of social interactions as the relationship with the partner unfolds, potentially promoting the maintenance of a pair bond by inhibiting aggressive responses. These results provide a mechanism by which social experience and context shift oxytocinergic signaling to impact neural and behavioral responses to social cues.

Striatal Astrocytes Shape Behavioral Flexibility via Regulation of the Glutamate Transporter EAAT2.

BACKGROUND: Striatal circuits must be modulated for behavioral flexibility, the ability to adapt to environmental changes. Striatal astrocytes contribute to circuit neuromodulation by controlling the activity of ambient neurotransmitters. In particular, extracellular glutamate levels are tightly controlled by the astrocytic glutamate transporter EAAT2, influencing synaptic functioning and neural network activity. However, it remains unclear if EAAT2 responds to environmental cues to specifically shape action control. METHODS: To investigate the relationship between behavioral flexibility and experience-dependent regulation of EAAT2 expression in the dorsal striatum, mice were trained on an instrumental task. We manipulated EAAT2 expression using chemogenetic activation of astrocytic Gq signaling or in vivo morpholinos and determined the ability to adapt to novel environmental contingencies. RESULTS: The loss of behavioral flexibility with task overtraining is associated with the upregulation of EAAT2, which results in enhanced glutamate clearance and altered modulation of glutamatergic neurotransmission in the lateral part of the dorsal striatum. Interfering with EAAT2 upregulation in this striatal area preserves behavioral flexibility. CONCLUSIONS: Astrocytes are emerging as critical regulators of striatal functions. This work demonstrates that plasticity of EAAT2 expression in the lateral part of the dorsal striatum shapes behavior, thus providing novel mechanistic insights into how flexibility in action control is regulated.

Young, active and well-connected: adult-born neurons in the zebra finch are activated during singing.

Neuronal replacement in the pallial song control nucleus HVC of adult zebra finches constitutes an interesting case of homeostatic plasticity; in spite of continuous addition and attrition of neurons in ensembles that code song elements, adult song remains remarkably invariant. New neurons migrate into HVC and later synapse with their target, arcopallial song nucleus RA (HVCRA). New HVCRA neurons respond to auditory stimuli (in anaesthetised animals), but whether and when they become functionally active during singing is unknown. We studied this, using 5-bromo-2'-deoxyuridine to birth-date neurons, combined with immunohistochemical detection of immediate-early gene (IEG) expression and retrograde tracer injections into RA to track connectivity. Interestingly, singing was followed by IEG expression in a substantial fraction of new neurons that were not retrogradely labelled from RA, suggesting a possible role in HVC-intrinsic network function. As new HVC neurons matured, the proportion of HVCRA neurons that expressed IEGs after singing increased significantly. Since it was previously shown that singing induces IEG expression in HVC also in deaf birds and that hearing song does not induce IEG expression in HVC, our data provide the first direct evidence that new HVC neurons are engaged in song motor behaviour.

Cannabinoid, melanocortin and opioid receptor expression on DRD1 and DRD2 subpopulations in rat striatum.

The striatum harbors two neuronal populations that enable action selection. One population represents the striatonigral pathway, expresses the dopamine receptor D1 (DRD1) and promotes the execution of motor programs, while the other population represents the striatopallidal pathway, expresses the dopamine receptor D2 (DRD2) and suppresses voluntary activity. The two populations integrate distinct sensorimotor, cognitive, and emotional information streams and their combined activity enables the selection of adaptive behaviors. Characterization of these populations is critical to the understanding of their role in action selection, because it aids the identification of the molecular mechanisms that separate them. To that end, we used fluorescent in situ hybridization to quantify the percentage of striatal cells that (co)express dopaminergic receptors and receptors of the cannabinoid, melanocortin or opioid neurotransmitters systems. Our main findings are that the cannabinoid 1 receptor is equally expressed on both populations with a gradient from dorsal to ventral striatum, that the opioid receptors have a preference for expression with either the DRD1 or DRD2 and that the melanocortin 4 receptor (MC4R) is predominantly expressed in ventral parts of the striatum. In addition, we find that the level of MC4R expression determines its localization to either the DRD1 or the DRD2 population. Thereby, we provide insight into the sensitivity of the two dopaminoceptive populations to these neurotransmitters and progress the understanding of the mechanisms that enable action selection.

The obesity-associated gene Negr1 regulates aspects of energy balance in rat hypothalamic areas.

Neural growth regulator 1 (Negr1) is among the first common variants that have been associated with the regulation of body mass index. Using AAV technology directed to manipulate Negr1 expression in vivo, we find that decreased expression of Negr1 in periventricular hypothalamic areas leads to increases in body weight, presumably via increased food intake. Moreover, we observed that both increased and decreased levels of Negr1 lead to reduced locomotor activity and body temperature. In sum, our results provide further support for a role of hypothalamic expressed Negr1 in the regulation of energy balance.

Recombinant adeno-associated virus: efficient transduction of the rat VMH and clearance from blood.

To promote the efficient and safe application of adeno-associated virus (AAV) vectors as a gene transfer tool in the central nervous system (CNS), transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH). No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×109 genomic copies of AAV would be fully cleared from blood after 2 days.

Combined use of the canine adenovirus-2 and DREADD-technology to activate specific neural pathways in vivo.

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM3D(Gq)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM3D(Gq)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM3D(Gq)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM3D(Gq)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.

AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.

Nutritional state affects the expression of the obesity-associated genes Etv5, Faim2, Fto, and Negr1.

Obesity is a risk factor for type II diabetes, atherosclerosis, and some forms of cancer. Variation in common measures of obesity (e.g., BMI, waist/hip ratio) is largely explained by heritability. The advent of genome-wide association studies (GWAS) has made it possible to identify several genetic variants that associate with measures of obesity, but how exactly these genetic variants contribute to overweight has remained largely unresolved. One first hint is given by the fact that many of the associated variants reside in or near genes that act in the central nervous system, which implicates neuronal signaling in the etiology of obesity. Although the brain controls both energy intake and expenditure, it has more capacity to regulate energy intake rather than energy expenditure. In environments where food is abundant, this renders the body prone to weight increases. To gain more insight into the neurobiological mechanisms involved, we set out to investigate the effect of dietary exposure on the expression levels of obesity-associated genes in the ventro-medial hypothalamus (VMH)/arcuate nucleus (ARC) and the substantia nigra (SN)/ventral tegmental area (VTA), two brain regions that are implicated in feeding behavior. We show that the expression of Etv5, Faim2, Fto, Negr1 but not Sh2b1 is affected by nutritional state in these two areas, thereby providing insight into the relationship between nutritional state and expression levels of obesity-associated genes in two brain areas relevant to feeding.

Together or alone?: foraging strategies in Caenorhabditis elegans.

A central goal in Life Sciences is to understand how genes encode behaviour and how environmental factors influence the expression of the genes concerned. To reach this goal a combined ecological, molecular biological and physiological approach is required in combination with a suitable model organism. Such an approach allows the elucidation of all parts of the complicated chain of events that lead from induction of gene expression to behaviour, i.e. from environmental stimulus, sensory organs and extracellular and intracellular neuronal signal processing to activation of effector organs. A particularly good model species with which to take this approach is the nematode Caenorhabditis elegans, as it has been described in great detail at the genomic, cellular and behavioural levels. Different strains of C. elegans display prominent behavioural variation in foraging behaviour. Some strains will form social feeding groups when subjected to certain environmental stimuli, while others do not. This variation is due to the existence of just two isoforms of the gene npr-1, namely 215F and 215V. Here, we describe these behavioural variations at the molecular and cellular levels to attempt to determine the environmental inputs that cause aggregation of these small nematodes. As many different stimuli affect aggregation either positively or negatively, aggregation behaviour seems to be displayed when it improves survival chances. However, not much is known about the ecological context in which C. elegans lives. Investigation of the habitats of different strains of C. elegans would help us to understand why and how a specific foraging strategy enhances survival. The relatively well-understood molecular pathways that direct its social feeding behaviour make C. elegans a highly suitable model organism to test ecological and behavioural hypotheses about the mechanisms that differentiate between aggregation and solitary behaviours.