RESUMEN
Brain-wide fluctuations in local field potential oscillations reflect emergent network-level signals that mediate behavior. Cracking the code whereby these oscillations coordinate in time and space (spatiotemporal dynamics) to represent complex behaviors would provide fundamental insights into how the brain signals emotional pathology. Using machine learning, we discover a spatiotemporal dynamic network that predicts the emergence of major depressive disorder (MDD)-related behavioral dysfunction in mice subjected to chronic social defeat stress. Activity patterns in this network originate in prefrontal cortex and ventral striatum, relay through amygdala and ventral tegmental area, and converge in ventral hippocampus. This network is increased by acute threat, and it is also enhanced in three independent models of MDD vulnerability. Finally, we demonstrate that this vulnerability network is biologically distinct from the networks that encode dysfunction after stress. Thus, these findings reveal a convergent mechanism through which MDD vulnerability is mediated in the brain.
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Encéfalo/fisiología , Depresión/patología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Depresión/fisiopatología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Electrodos Implantados , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ketamina/farmacología , Aprendizaje Automático , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenómenos Fisiológicos/efectos de los fármacos , Corteza Prefrontal/fisiología , Estrés PsicológicoRESUMEN
Schizophrenia is an idiopathic psychiatric disorder with a high degree of polygenicity. Evidence from genetics, single-cell transcriptomics, and pharmacological studies suggest an important, but untested, overlap between genes involved in the etiology of schizophrenia and the cellular mechanisms of action of antipsychotics. To directly compare genes with antipsychotic-induced differential expression to genes involved in schizophrenia, we applied single-cell RNA-sequencing to striatal samples from male C57BL/6 J mice chronically exposed to a typical antipsychotic (haloperidol), an atypical antipsychotic (olanzapine), or placebo. We identified differentially expressed genes in three cell populations identified from the single-cell RNA-sequencing (medium spiny neurons [MSNs], microglia, and astrocytes) and applied multiple analysis pipelines to contextualize these findings, including comparison to GWAS results for schizophrenia. In MSNs in particular, differential expression analysis showed that there was a larger share of differentially expressed genes (DEGs) from mice treated with olanzapine compared with haloperidol. DEGs were enriched in loci implicated by genetic studies of schizophrenia, and we highlighted nine genes with convergent evidence. Pathway analyses of gene expression in MSNs highlighted neuron/synapse development, alternative splicing, and mitochondrial function as particularly engaged by antipsychotics. In microglia, we identified pathways involved in microglial activation and inflammation as part of the antipsychotic response. In conclusion, single-cell RNA sequencing may provide important insights into antipsychotic mechanisms of action and links to findings from psychiatric genomic studies.
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Antipsicóticos , Animales , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Expresión Génica , Haloperidol/farmacología , Haloperidol/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Olanzapina , ARNRESUMEN
The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles--predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation--suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.
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Axones/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Conos de Crecimiento/metabolismo , Animales , Axones/efectos de los fármacos , Axones/fisiología , Procesos de Crecimiento Celular , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Conos de Crecimiento/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-DawleyRESUMEN
Transcranial magnetic stimulation and deep brain stimulation have emerged as therapeutic modalities for treatment refractory depression; however, little remains known regarding the circuitry that mediates the therapeutic effect of these approaches. Here we show that direct optogenetic stimulation of prefrontal cortex (PFC) descending projection neurons in mice engineered to express Chr2 in layer V pyramidal neurons (Thy1-Chr2 mice) models an antidepressant-like effect in mice subjected to a forced-swim test. Furthermore, we show that this PFC stimulation induces a long-lasting suppression of anxiety-like behavior (but not conditioned social avoidance) in socially stressed Thy1-Chr2 mice: an effect that is observed >10 d after the last stimulation. Finally, we use optogenetic stimulation and multicircuit recording techniques concurrently in Thy1-Chr2 mice to demonstrate that activation of cortical projection neurons entrains neural oscillatory activity and drives synchrony across limbic brain areas that regulate affect. Importantly, these neural oscillatory changes directly correlate with the temporally precise activation and suppression of limbic unit activity. Together, our findings show that the direct activation of cortical projection systems is sufficient to modulate activity across networks underlying affective regulation. They also suggest that optogenetic stimulation of cortical projection systems may serve as a viable therapeutic strategy for treating affective disorders.
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Afecto/fisiología , Ansiedad/fisiopatología , Conducta Animal/fisiología , Corteza Cerebral/fisiología , Red Nerviosa/fisiología , Animales , Masculino , Ratones , Neuronas/fisiologíaRESUMEN
BACKGROUND: Increased vulnerability to stress is a major risk factor for several mood disorders, including major depressive disorder. Although cellular and molecular mechanisms associated with depressive behaviors following stress have been identified, little is known about the mechanisms that confer the vulnerability that predisposes individuals to future damage from chronic stress. METHODS: We used multisite in vivo neurophysiology in freely behaving male and female C57BL/6 mice (n = 12) to measure electrical brain network activity previously identified as indicating a latent stress vulnerability brain state. We combined this neurophysiological approach with single-cell RNA sequencing of the prefrontal cortex to identify distinct transcriptomic differences between groups of mice with inherent high and low stress vulnerability. RESULTS: We identified hundreds of differentially expressed genes (padjusted < .05) across 5 major cell types in animals with high and low stress vulnerability brain network activity. This unique analysis revealed that GABAergic (gamma-aminobutyric acidergic) neuron gene expression contributed most to the network activity of the stress vulnerability brain state. Upregulation of mitochondrial and metabolic pathways also distinguished high and low vulnerability brain states, especially in inhibitory neurons. Importantly, genes that were differentially regulated with vulnerability network activity significantly overlapped (above chance) with those identified by genome-wide association studies as having single nucleotide polymorphisms significantly associated with depression as well as genes more highly expressed in postmortem prefrontal cortex of patients with major depressive disorder. CONCLUSIONS: This is the first study to identify cell types and genes involved in a latent stress vulnerability state in the brain.
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In rodents, anxiety is charactered by heightened vigilance during low-threat and uncertain situations. Though activity in the frontal cortex and limbic system are fundamental to supporting this internal state, the underlying network architecture that integrates activity across brain regions to encode anxiety across animals and paradigms remains unclear. Here, we utilize parallel electrical recordings in freely behaving mice, translational paradigms known to induce anxiety, and machine learning to discover a multi-region network that encodes the anxious brain-state. The network is composed of circuits widely implicated in anxiety behavior, it generalizes across many behavioral contexts that induce anxiety, and it fails to encode multiple behavioral contexts that do not. Strikingly, the activity of this network is also principally altered in two mouse models of depression. Thus, we establish a network-level process whereby the brain encodes anxiety in health and disease.
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Increased vulnerability to stress is a major risk factor for the manifestation of several mood disorders, including major depressive disorder (MDD). Despite the status of MDD as a significant donor to global disability, the complex integration of genetic and environmental factors that contribute to the behavioral display of such disorders has made a thorough understanding of related etiology elusive. Recent developments suggest that a brain-wide network approach is needed, taking into account the complex interplay of cell types spanning multiple brain regions. Single cell RNA-sequencing technologies can provide transcriptomic profiling at the single-cell level across heterogenous samples. Furthermore, we have previously used local field potential oscillations and machine learning to identify an electrical brain network that is indicative of a predisposed vulnerability state. Thus, this study combined single cell RNA-sequencing (scRNA-Seq) with electrical brain network measures of the stress-vulnerable state, providing a unique opportunity to access the relationship between stress network activity and transcriptomic changes within individual cell types. We found especially high numbers of differentially expressed genes between animals with high and low stress vulnerability brain network activity in astrocytes and glutamatergic neurons but we estimated that vulnerability network activity depends most on GABAergic neurons. High vulnerability network activity included upregulation of microglia and mitochondrial and metabolic pathways, while lower vulnerability involved synaptic regulation. Genes that were differentially regulated with vulnerability network activity significantly overlapped with genes identified as having significant SNPs by human GWAS for depression. Taken together, these data provide the gene expression architecture of a previously uncharacterized stress vulnerability brain state, enabling new understanding and intervention of predisposition to stress susceptibility.
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The architecture whereby activity across many brain regions integrates to encode individual appetitive social behavior remains unknown. Here we measure electrical activity from eight brain regions as mice engage in a social preference assay. We then use machine learning to discover a network that encodes the extent to which individual mice engage another mouse. This network is organized by theta oscillations leading from prelimbic cortex and amygdala that converge on the ventral tegmental area. Network activity is synchronized with cellular firing, and frequency-specific activation of a circuit within this network increases social behavior. Finally, the network generalizes, on a mouse-by-mouse basis, to encode individual differences in social behavior in healthy animals but fails to encode individual behavior in a 'high confidence' genetic model of autism. Thus, our findings reveal the architecture whereby the brain integrates distributed activity across timescales to encode an appetitive brain state underlying individual differences in social behavior.
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Conducta Apetitiva , Encéfalo , Amígdala del Cerebelo , Animales , Encéfalo/fisiología , Ratones , Conducta Social , Área Tegmental VentralRESUMEN
Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.
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Cisteína/metabolismo , Lipoilación , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Acilación , Electroforesis en Gel de Poliacrilamida/métodos , Sefarosa/análogos & derivados , Sefarosa/química , Proteínas ras/análisisRESUMEN
While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness.
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Microagresión , Racismo/prevención & control , Ciencia/organización & administración , Ingeniería , Humanos , Matemática , Estudiantes , Tecnología , UniversidadesRESUMEN
Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic and gender identity groups in Science, Technology, Engineering, Mathematics and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community.
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Comunicación , Relaciones Interprofesionales , Tutoría , Mentores , Humanos , ConfianzaRESUMEN
BACKGROUND: The prefrontal cortex plays a critical role in regulating emotional behaviors, and dysfunction of prefrontal cortex-dependent networks has been broadly implicated in mediating stress-induced behavioral disorders including major depressive disorder. METHODS: Here we acquired multicircuit in vivo activity from eight cortical and limbic brain regions as mice were subjected to the tail suspension test (TST) and an open field test. We used a linear decoder to determine whether cellular responses across each of the cortical and limbic areas signal movement during the TST and open field test. We then performed repeat behavioral testing to identify which brain areas show cellular adaptations that signal the increase in immobility induced by repeat TST exposure. RESULTS: The increase in immobility observed during repeat TST exposure is linked to a selective functional upregulation of cellular activity in infralimbic cortex and medial dorsal thalamus, and to an increase in the spatiotemporal dynamic interaction between these structures. Inducing this spatiotemporal dynamic using closed-loop optogenetic stimulation is sufficient to increase movement in the TST in stress-naive mice, while stimulating above the carrier frequency of this circuit suppressed movement. This demonstrates that the adaptations in infralimbic cortex-medial dorsal thalamus circuitry observed after stress reflect a compensatory mechanism whereby the brain drives neural systems to counterbalance stress effects. CONCLUSIONS: Our findings provide evidence that targeting endogenous spatiotemporal dynamics is a potential therapeutic approach for treating stress-induced behavioral disorders, and that dynamics are a critical axis of manipulation for causal optogenetic studies.
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Corteza Cerebral/fisiopatología , Sistema Límbico/fisiopatología , Estrés Psicológico/fisiopatología , Potenciales de Acción , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Trastorno Depresivo Mayor/fisiopatología , Modelos Animales de Enfermedad , Reacción de Fuga/fisiología , Suspensión Trasera , Masculino , Ratones Endogámicos BALB C , Ratones Transgénicos , Microelectrodos , Actividad Motora , Vías Nerviosas/fisiopatología , Neuronas/fisiología , Optogenética , Estimulación Luminosa , Factores de TiempoRESUMEN
Circuits distributed across cortico-limbic brain regions compose the networks that mediate emotional behavior. The prefrontal cortex (PFC) regulates ultraslow (<1 Hz) dynamics across these networks, and PFC dysfunction is implicated in stress-related illnesses including major depressive disorder (MDD). To uncover the mechanism whereby stress-induced changes in PFC circuitry alter emotional networks to yield pathology, we used a multi-disciplinary approach including in vivo recordings in mice and chronic social defeat stress. Our network model, inferred using machine learning, linked stress-induced behavioral pathology to the capacity of PFC to synchronize amygdala and VTA activity. Direct stimulation of PFC-amygdala circuitry with DREADDs normalized PFC-dependent limbic synchrony in stress-susceptible animals and restored normal behavior. In addition to providing insights into MDD mechanisms, our findings demonstrate an interdisciplinary approach that can be used to identify the large-scale network changes that underlie complex emotional pathologies and the specific network nodes that can be used to develop targeted interventions.
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Amígdala del Cerebelo/fisiopatología , Conducta Animal/fisiología , Emociones/fisiología , Corteza Prefrontal/fisiopatología , Estrés Psicológico/patología , Animales , Trastorno Depresivo Mayor/fisiopatología , Ratones Endogámicos C57BL , Corteza Prefrontal/patologíaRESUMEN
Psychological stress contributes to the onset and exacerbation of nearly all neuropsychiatric disorders. Individual differences in stress-regulatory circuits can therefore dramatically affect vulnerability to these illnesses. Here we identify neural circuit mechanisms underlying individual differences in vulnerability to stress using a murine model of chronic social defeat stress. In chronically stressed mice, we find that the degree of prefrontal cortex (PFC) control of amygdala activity predicts stress susceptibility in individual mice. Critically, we also find that individual differences in PFC activation (that is, reactivity) during exposure to an aggressor mouse predict the emergence stress-induced behavioural deficits in stress-naïve mice. Finally, we show that naturally occurring differences in PFC reactivity directly correspond to the intrinsic firing rate of PFC neurons. This demonstrates that naturally occurring differences in PFC function underlie individual differences in vulnerability to stress, raising the hypothesis that PFC modulation may prevent stress-induced psychiatric disorders.
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Agresión/psicología , Amígdala del Cerebelo/fisiopatología , Corteza Prefrontal/fisiopatología , Conducta Social , Estrés Psicológico/fisiopatología , Animales , Ratones , Ratones Endogámicos C57BL , Monitorización NeurofisiológicaRESUMEN
Calmodulin (CaM) is a small (148 amino acid), ubiquitously expressed eukaryotic protein essential for Ca(2+) regulation and signaling. This highly acidic polypeptide (pI<4) has two homologous domains (N and C), each consisting of two EF-hand Ca(2+)-binding sites. Despite significant homology, the domains have intrinsic differences in their Ca(2+)-binding properties and separable roles in regulating physiological targets such as kinases and ion channels. In mammalian full-length CaM, sites III and IV in the C-domain bind Ca(2+) cooperatively with ~10-fold higher affinity than sites I and II in the N-domain. However, the difference is only twofold when CaM is severed at residue 75, indicating that anticooperative interactions occur in full-length CaM. The Ca(2+)-binding properties of sites I and II are regulated by several factors including the interplay of interdomain linker residues far from the binding sites. Our prior thermodynamic studies showed that these residues inhibit thermal denaturation and decrease calcium affinity. Based on high-resolution structures and NMR spectra, there appear to be interactions between charged residues in the sequence 75-80 and those near the amino terminus of CaM. To explore electrostatic contributions to interdomain interactions in CaM, KCl was used to perturb the Ca(2+)-binding affinity, thermal stability, and hydrodynamic size of a nested set of recombinant mammalian CaM (rCaM) fragments terminating at residues 75, 80, 85, or 90. Potassium chloride is known to decrease Ca(2+)-binding affinity of full-length CaM. It may act directly by competition with acidic side chains that chelate Ca(2+) in the binding sites, and indirectly elsewhere in the molecule by changing tertiary constraints and conformation. In all proteins studied, KCl decreased Ca(2+)-affinity, decreased Stokes radius, and increased thermal stability, but not monotonically. Crystallographic structures of Ca(2+)-saturated rCaM(1-75) (3B32.pdb) and rCaM(1-90) (3IFK.pdb) were determined, offering cautionary notes about the effect of packing interactions on flexible linkers. This chapter describes an array of methods for characterizing system-specific thermodynamic properties that in concert govern structure and function.
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Calmodulina/química , Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Calmodulina/genética , Cristalografía por Rayos X/métodos , Fluorometría/métodos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
A hydrophobic core is a widely accepted determinant of protein stability. However, regulatory proteins undergoing ligand-induced conformational switching may expose interior residues to solvent and cannot afford to be extremely rigid. Optimizing the energetic balance between stability and binding is challenging. The addition of five interdomain residues to rat and Paramecium calmodulin N-domain fragments (residues 1-75) increased their thermostability by 9 degrees C and lowered their calcium affinity by a factor of 4. This demonstrates that the flexible linker regulates functional properties as well as tethering the neighboring domains and that protein stability may be increased markedly by minor modifications of the C-terminus. The sensitivity of this domain to few and conservative variations in helices A and D (D2E, S17A, T70S and M71L) is demonstrated by the rat CaM fragments having lower stability and higher calcium affinity than fragments of the same length derived from Paramecium CaM.