Spinal projecting neurons in rostral ventromedial medulla co-regulate motor and sympathetic tone.

Many behaviors require the coordinated actions of somatic and autonomic functions. However, the underlying mechanisms remain elusive. By opto-stimulating different populations of descending spinal projecting neurons (SPNs) in anesthetized mice, we show that stimulation of excitatory SPNs in the rostral ventromedial medulla (rVMM) resulted in a simultaneous increase in somatomotor and sympathetic activities. Conversely, opto-stimulation of rVMM inhibitory SPNs decreased both activities. Anatomically, these SPNs innervate both sympathetic preganglionic neurons and motor-related regions in the spinal cord. Fiber-photometry recording indicated that the activities of rVMM SPNs correlate with different levels of muscle and sympathetic tone during distinct arousal states. Inhibiting rVMM excitatory SPNs reduced basal muscle and sympathetic tone, impairing locomotion initiation and high-speed performance. In contrast, silencing the inhibitory population abolished muscle atonia and sympathetic hypoactivity during rapid eye movement (REM) sleep. Together, these results identify rVMM SPNs as descending spinal projecting pathways controlling the tone of both the somatomotor and sympathetic systems.

A transcriptomic taxonomy of mouse brain-wide spinal projecting neurons.

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.

The whisking oscillator circuit.

Central oscillators are primordial neural circuits that generate and control rhythmic movements1,2. Mechanistic understanding of these circuits requires genetic identification of the oscillator neurons and their synaptic connections to enable targeted electrophysiological recording and causal manipulation during behaviours. However, such targeting remains a challenge with mammalian systems. Here we delimit the oscillator circuit that drives rhythmic whisking-a motor action that is central to foraging and active sensing in rodents3,4. We found that the whisking oscillator consists of parvalbumin-expressing inhibitory neurons located in the vibrissa intermediate reticular nucleus (vIRtPV) in the brainstem. vIRtPV neurons receive descending excitatory inputs and form recurrent inhibitory connections among themselves. Silencing vIRtPV neurons eliminated rhythmic whisking and resulted in sustained vibrissae protraction. In vivo recording of opto-tagged vIRtPV neurons in awake mice showed that these cells spike tonically when animals are at rest, and transition to rhythmic bursting at the onset of whisking, suggesting that rhythm generation is probably the result of network dynamics, as opposed to intrinsic cellular properties. Notably, ablating inhibitory synaptic inputs to vIRtPV neurons quenched their rhythmic bursting, impaired the tonic-to-bursting transition and abolished regular whisking. Thus, the whisking oscillator is an all-inhibitory network and recurrent synaptic inhibition has a key role in its rhythmogenesis.

Elevated TNF-α Leads to Neural Circuit Instability in the Absence of Interferon Regulatory Factor 8.

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.

Lovastatin suppresses hyperexcitability and seizure in Angelman syndrome model.

Epilepsy is prevalent and often medically intractable in Angelman syndrome (AS). AS mouse model (Ube3am-/p+) shows reduced excitatory neurotransmission but lower seizure threshold. The neural mechanism linking the synaptic dysfunction to the seizure remains elusive. We show that the local circuits of Ube3am-/p+in vitro are hyperexcitable and display a unique epileptiform activity, a phenomenon that is reminiscent of the finding in fragile X syndrome (FXS) mouse model. Similar to the FXS model, lovastatin suppressed the epileptiform activity and audiogenic seizures in Ube3am-/p+. The in vitro model of Ube3am-/p+ is valuable for dissection of neural mechanism and epilepsy drug screening in vivo.

Mechanisms of increased hippocampal excitability in the Mashl+/- mouse model of Na+ /K+ -ATPase dysfunction.

OBJECTIVE: Na+ /K+ -ATPase dysfunction, primary (mutation) or secondary (energy crisis, neurodegenerative disease) increases neuronal excitability in the brain. To evaluate the mechanisms underlying such increased excitability we studied mice carrying the D801N mutation, the most common mutation causing human disease, specifically alternating hemiplegia of childhood (AHC) including epilepsy. Because the gene is expressed in all neurons, particularly γ-aminobutyric acid (GABA)ergic interneurons, we hypothesized that the pathophysiology would involve both pyramidal cells and interneurons and that fast-spiking interneurons, which have increased firing rates, would be most vulnerable. METHODS: We performed extracellular recordings, as well as whole-cell patch clamp recordings from pyramidal cells and interneurons, in the CA1 region on hippocampal slices. We also performed immunohistochemistry from hippocampal sections to count CA1 pyramidal cells as well as parvalbumin-positive interneurons. In addition, we performed video-electroencephalography (EEG) recordings from the dorsal hippocampal CA1 region. RESULTS: We observed that juvenile knock-in mice carrying the above mutation reproduce the human phenotype of AHC. We then demonstrated in the CA1 region of these mice the following findings as compared to wild type: (1) Increased number of spikes evoked by electrical stimulation of Schaffer collaterals; (2) equalization by bicuculline of the number of spikes induced by Schaffer collateral stimulation; (3) reduced miniature, spontaneous, and evoked inhibitory postsynaptic currents, but no change in excitatory postsynaptic currents; (4) robust action potential frequency adaptation in response to depolarizing current injection in CA1 fast-spiking interneurons; and (5) no change in the number of pyramidal cells, but reduced number of parvalbumin positive interneurons. SIGNIFICANCE: Our data indicate that, in our genetic model of Atp1α3 mutation, there is increased excitability and marked dysfunction in GABAergic inhibition. This supports the performance of further investigations to determine if selective expression of the mutation in GABAergic and or glutamatergic neurons is necessary and sufficient to result in the behavioral phenotype.

Pupil Response to Threat in Trauma-Exposed Individuals With or Without PTSD.

An infrequently studied and potentially promising physiological marker for posttraumatic stress disorder (PTSD) is pupil response. This study tested the hypothesis that pupil responses to threat would be significantly larger in trauma-exposed individuals with PTSD compared to those without PTSD. Eye-tracking technology was used to evaluate pupil response to threatening and neutral images. Recruited for participation were 40 trauma-exposed individuals; 40.0% (n = 16) met diagnostic criteria for PTSD. Individuals with PTSD showed significantly more pupil dilation to threat-relevant stimuli compared to the neutral elements (Cohen's d = 0.76), and to trauma-exposed controls (Cohen's d = 0.75). Pupil dilation significantly accounted for 12% of variability in PTSD after time elapsed since most recent trauma, cumulative violence exposure, and trait anxiety were statistically adjusted. The final logistic regression model was associated with 85% of variability in PTSD status and correctly classified 93.8% of individuals with PTSD and 95.8% of those without. Pupil reactivity showed promise as a physiological marker for PTSD.

Feed-forward Activation of Habenula Cholinergic Neurons by Local Acetylcholine.

While the functional and behavioral role of the medial habenula (MHb) is still emerging, recent data indicate an involvement of this nuclei in regulating mood, aversion, and addiction. Unique to the MHb is a large cluster of cholinergic neurons that project to the interpeduncular nucleus and densely express acetylcholine receptors (AChRs) suggesting that the activity of these cholinergic neurons may be regulated by ACh itself. Whether endogenous ACh from within the habenula regulates cholinergic neuron activity has not been demonstrated. Supporting a role for ACh in modulating MHb activity, acetylcholinesterase inhibitors increased the firing rate of MHb cholinergic neurons in mouse habenula slices, an effect blocked by AChR antagonists and mediated by ACh which was detected via expressing fluorescent ACh sensors in MHb in vivo. To test if cholinergic afferents innervate MHb cholinergic neurons, we used anterograde and retrograde viral tracing to identify cholinergic inputs. Surprisingly, tracing experiments failed to detect cholinergic inputs into the MHb, including from the septum, suggesting that MHb cholinergic neurons may release ACh within the MHb to drive cholinergic activity. To test this hypothesis, we expressed channelrhodopsin in a portion of MHb cholinergic neurons while recording from non-opsin-expressing neurons. Light pulses progressively increased activity of MHb cholinergic neurons indicating feed-forward activation driven by MHb ACh release. These data indicate MHb cholinergic neurons may utilize a unique feed-forward mechanism to synchronize and increase activity by releasing local ACh.

Synaptic plasticity in mouse models of autism spectrum disorders.

Analysis of synaptic plasticity together with behavioral and molecular studies have become a popular approach to model autism spectrum disorders in order to gain insight into the pathosphysiological mechanisms and to find therapeutic targets. Abnormalities of specific types of synaptic plasticity have been revealed in numerous genetically modified mice that have molecular construct validity to human autism spectrum disorders. Constrained by the feasibility of technique, the common regions analyzed in most studies are hippocampus and visual cortex. The relevance of the synaptic defects in these regions to the behavioral abnormalities of autistic like behaviors is still a subject of debate. Because the exact regions or circuits responsible for the core features of autistic behaviors in humans are still poorly understood, investigation using region-specific conditional mutant mice may help to provide the insight into the neuroanatomical basis of autism in the future.

Midbrain Dopamine Controls Anxiety-like Behavior by Engaging Unique Interpeduncular Nucleus Microcircuitry.

BACKGROUND: Dopamine (DA) is hypothesized to modulate anxiety-like behavior, although the precise role of DA in anxiety behaviors and the complete anxiety network in the brain have yet to be elucidated. Recent data indicate that dopaminergic projections from the ventral tegmental area (VTA) innervate the interpeduncular nucleus (IPN), but how the IPN responds to DA and what role this circuit plays in anxiety-like behavior are unknown. METHODS: We expressed a genetically encoded G protein-coupled receptor activation-based DA sensor in mouse midbrain to detect DA in IPN slices using fluorescence imaging combined with pharmacology. Next, we selectively inhibited or activated VTA→IPN DAergic inputs via optogenetics during anxiety-like behavior. We used a biophysical approach to characterize DA effects on neural IPN circuits. Site-directed pharmacology was used to test if DA receptors in the IPN can regulate anxiety-like behavior. RESULTS: DA was detected in mouse IPN slices. Silencing/activating VTA→IPN DAergic inputs oppositely modulated anxiety-like behavior. Two neuronal populations in the ventral IPN (vIPN) responded to DA via D1 receptors (D1Rs). vIPN neurons were controlled by a small population of D1R neurons in the caudal IPN that directly respond to VTA DAergic terminal stimulation and innervate the vIPN. IPN infusion of a D1R agonist and antagonist bidirectionally controlled anxiety-like behavior. CONCLUSIONS: VTA DA engages D1R-expressing neurons in the caudal IPN that innervate vIPN, thereby amplifying the VTA DA signal to modulate anxiety-like behavior. These data identify a DAergic circuit that mediates anxiety-like behavior through unique IPN microcircuitry.

Group I metabotropic glutamate receptors generate two types of intrinsic membrane oscillations in hippocampal oriens/alveus interneurons.

GABAergic interneurons in the hippocampus are critically involved in almost all hippocampal circuit functions including coordinated network activity. Somatostatin-expressing oriens-lacunosum moleculare (O-LM) interneurons are a major subtype of dendritically projecting interneurons in hippocampal subregions (e.g., CA1), and express group I metabotropic glutamate receptors (mGluRs), specifically mGluR1 and mGluR5. Group I mGluRs are thought to regulate hippocampal circuit functions partially through GABAergic interneurons. Previous studies suggest that a group I/II mGluR agonist produces slow supra-threshold membrane oscillations (<0.1 Hz), which are associated with high-frequency action potential (AP) discharges in O-LM interneurons. However, the properties and underlying mechanisms of these slow oscillations remain largely unknown. We performed whole-cell patch-clamp recordings from mouse interneurons in the stratum oriens/alveus (O/A interneurons) including CA1 O-LM interneurons. Our study revealed that the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced slow membrane oscillations (<0.1 Hz), which were associated with gamma frequency APs followed by AP-free perithreshold gamma oscillations. The selective mGluR1 antagonist (S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) reduced the slow oscillations, and the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) partially blocked them. Blockade of nonselective cation-conducting transient receptor potential channels, L-type Ca2+ channels, or ryanodine receptors all abolished the slow oscillations, suggesting the involvement of multiple mechanisms. Our findings suggest that group I mGluR activation in O/A interneurons may play an important role in coordinated network activity, and O/A interneuron vulnerability to excitotoxicity, in disease states like seizures, is at least in part due to an excessive rise in intracellular Ca2+.

Epigenetic dysregulation of Oxtr in Tet1-deficient mice has implications for neuropsychiatric disorders.

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice - by deleting the largest coding exon 4 (Tet1Δe4) - we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4-/- mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4-/- mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.

Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism.

Human neuroimaging studies suggest that aberrant neural connectivity underlies behavioural deficits in autism spectrum disorders (ASDs), but the molecular and neural circuit mechanisms underlying ASDs remain elusive. Here, we describe a complete knockout mouse model of the autism-associated Shank3 gene, with a deletion of exons 4-22 (Δe4-22). Both mGluR5-Homer scaffolds and mGluR5-mediated signalling are selectively altered in striatal neurons. These changes are associated with perturbed function at striatal synapses, abnormal brain morphology, aberrant structural connectivity and ASD-like behaviour. In vivo recording reveals that the cortico-striatal-thalamic circuit is tonically hyperactive in mutants, but becomes hypoactive during social behaviour. Manipulation of mGluR5 activity attenuates excessive grooming and instrumental learning differentially, and rescues impaired striatal synaptic plasticity in Δe4-22(-/-) mice. These findings show that deficiency of Shank3 can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities that underlie deficits in learning and ASD-like behaviours. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs.

A Brief Introduction to the Transduction of Neural Activity into Fos Signal.

The immediate early gene c-fos has long been known as a molecular marker of neural activity. The neuron's activity is transformed into intracellular calcium influx through NMDA receptors and L-type voltage sensitive calcium channels. For the transcription of c-fos, neural activity should be strong enough to activate mitogen-activated protein kinase (MAPK) signaling pathway which shows low calcium sensitivity. Upon translation, the auto-inhibition by Fos protein regulates basal Fos expression. The pattern of external stimuli and the valence of the stimulus to the animal change Fos signal, thus the signal reflects learning and memory aspects. Understanding the features of multiple components regulating Fos signaling is necessary for the optimal generation and interpretation of Fos signal.

Parental origin impairment of synaptic functions and behaviors in cytoplasmic FMRP interacting protein 1 (Cyfip1) deficient mice.

CYFIP1 maps to the interval between proximal breakpoint 1 (BP1) and breakpoint 2 (BP2) of chromosomal 15q11-q13 deletions that are implicated in the Angelman (AS) and Prader-Willi syndrome (PWS). There is only one breakpoint (BP3) at the distal end of deletion. CYFIP1 is deleted in AS patients with the larger class I deletion (BP1 to BP3) and the neurological presentations in these patients are more severe than that of patients with class II (BP2 to BP3) deletion. The haploinsufficiency of CYFIP1 is hypothesized to contribute to more severe clinical presentations in class I AS patients. The expression of CYFIP1 is suggested to be bi-allelic in literature but the possibility of parental origin of expression is not completely excluded. We generated and characterized Cyfip1 mutant mice. Homozygous Cyfip1 mice were early embryonic lethal. However, there was a parental origin specific effect between paternal Cyfip1 deficiency (m+/p-) and maternal deficiency (m-/p+) on both synaptic transmissions and behaviors in hippocampal CA1 synapses despite no evidence supporting the parental origin difference for the expression. Both m-/p+ and m+/p- showed the impaired input-output response and paired-pulse facilitation. While the long term-potentiation and group I mGluR mediated long term depression induced by DHPG was not different between Cyfip1 m-/p+ and m+/p- mice, the initial DHPG induced response was significantly enhanced in m-/p+ but not in m+/p- mice. m+/p- but not m-/p+ mice displayed increased freezing in cued fear conditioning and abnormal transitions in zero-maze test. The impaired synaptic transmission and behaviors in haploinsufficiency of Cyfip1 mice provide the evidence supporting the role of CYFIP1 modifying the clinical presentation of class I AS patients and in human neuropsychiatric disorders.

Therapeutic approaches for shankopathies.

Despite recent advances in understanding the molecular mechanisms of autism spectrum disorders (ASD), the current treatments for these disorders are mostly focused on behavioral and educational approaches. The considerable clinical and molecular heterogeneity of ASD present a significant challenge to the development of an effective treatment targeting underlying molecular defects. Deficiency of SHANK family genes causing ASD represent an exciting opportunity for developing molecular therapies because of strong genetic evidence for SHANK as causative genes in ASD and the availability of a panel of Shank mutant mouse models. In this article, we review the literature suggesting the potential for developing therapies based on molecular characteristics and discuss several exciting themes that are emerging from studying Shank mutant mice at the molecular level and in terms of synaptic function.

Ginsenoside Rg3 regulates GABAA receptor channel activity: involvement of interaction with the γ2 subunit.

Ginseng exhibits beneficial effects on GABAA receptor-related anxiety and sleep disorders. However, little is known regarding the cellular and molecular bases of the ginseng action on GABAA receptor. The present study was performed to elucidate the molecular mechanism of the ginseng effect on GABAA receptor. The effect of ginsenoside Rg3 (Rg3), one of the active ingredients of ginseng, on γ-aminobutyric acid (GABA)A receptor channel activity was examined in Xenopus oocytes using two-electrode voltage-clamp technique. Rg3 itself evoked an inward current in Xenopus oocytes expressing GABAA receptor subunits (α1ß1γ2) and the Rg3 itself-elicited inward current was only selective to γ2 subunit expression ratio, since Rg3 alone had no effects in oocytes expressing other subunits such as γ1, γ3, δ, or ε. Co-treatment of Rg3 with GABA enhanced GABA receptor (α1ß1γ2)-mediated inward currents (IGABA) but Rg3-mediated IGABA enhancement was independent on γ2. Rg3 itself-elicited inward current was blocked by GABAA receptor antagonist. The present results indicate that Rg3-induced GABAA receptor activation via the γ2 subunit and IGABA enhancement by Rg3 might be one of the molecular bases of ginseng effects on GABAA receptor.

Recent progress in GABAergic excitation from mature brain.

The excitatory effect of γ-Aminobutyric acid (GABA) has been recognized in very young animals and in seizure generation, but not so much in animals after weaning age or in adults. The existence of this phenomenon in mature brain is still controversial. In the course of debate, creative studies have identified and characterized the phenomenon in suprachiasmatic nucleus, cortex, hippocampus and basolateral amygdala, albeit mostly in single neurons. In neural circuit activity, presumed GABAergic excitation was observed in basolateral amygdala during the study of a neuropeptide, cholecystokinin. Though the functional meaning of this phenomenon in vivo remains to be uncovered, it may be implicated in epilepsy or anxiety in the adult brain.

Modulation of neural circuit actvity by ethanol in basolateral amygdala.

Ethanol actions in the amygdala formation may underlie in part the reinforcing effects of ethanol consumption. Previously a physiological phenomenon in the basolateral amygdala (BLA) that is dependent on neuronal network activity, compound postsynaptic potentials (cPSPs) were characterized. Effects of acute ethanol application on the frequency of cPSPs were subsequently investigated. Whole cell patch clamp recordings were performed from identified projection neurons in a rat brain slice preparation containing the amygdala formation. Acute ethanol exposure had complex effects on cPSP frequency, with both increases and decreases dependent on concentration, duration of exposure and age of the animal. Ethanol produces complex biphasic effects on synaptically-driven network activity in the BLA. These findings may relate to subjective effects of ethanol on arousal and anxiolysis in humans.

Impact of retrotransposons in pluripotent stem cells.

Retrotransposons, which constitute approximately 40% of the human genome, have the capacity to 'jump' across the genome. Their mobility contributes to oncogenesis, evolution, and genomic plasticity of the host genome. Induced pluripotent stem cells as well as embryonic stem cells are more susceptible than differentiated cells to genomic aberrations including insertion, deletion and duplication. Recent studies have revealed specific behaviors of retrotransposons in pluripotent cells. Here, we review recent progress in understanding retrotransposons and provide a perspective on the relationship between retrotransposons and genomic variation in pluripotent stem cells.