Chronic hM3Dq-DREADD-mediated chemogenetic activation of parvalbumin-positive inhibitory interneurons in postnatal life alters anxiety and despair-like behavior in adulthood in a task- and sex-dependent manner.

Animal models of early adversity or neurodevelopmental disorders are associated with altered parvalbumin (PV)-positive inhibitory interneuron number and function, correlated with a dysregulated excitation-inhibition (E/I) balance that is implicated in the pathophysiology of neuropsychiatric disorders. We sought to address whether altering neuronal activity of PV-positive interneurons during the postnatal developmental window influences the emergence of anxio-depressive behaviors in adulthood, which are known to be perturbed in models of early adversity and neurodevelopmental disorders. We used a PV-Cre::hM3Dq-DREADD bigenic mouse line that selectively expresses the hM3Dq-DREADD receptor in PV-positive interneurons, and chemogenetically enhanced Gq signaling in PV-positive interneurons during the postnatal window via administration of the DREADD agonist, clozapine-N-oxide. Immunofluorescence studies have indicated the selective expression of hM3Dq-DREADD in PV-positive interneurons in limbic circuits, and have revealed a reduction in expression of the neuronal activity marker, c-Fos, in these circuits, following chemogenetic hM3Dq-DREADD-mediated activation of PV-positive inhibitory interneurons. We noted no change in either growth or sensorimotor reflex milestones following chronic hM3Dq-DREADD-mediated chemogenetic activation of PV-positive inhibitory interneurons in postnatal life. Adult male and female PV-Cre::hM3DqDREADD bigenic mice with a history of postnatal chemogenetic activation of PV-positive interneurons exhibited a reduction in anxiety and despair-like behavior in adulthood, which was noted in both a behavioral task- and sex-dependent manner. These results indicate that altering neuronal activity within PV-positive interneurons during the critical postnatal developmental window can shape the emergence of anxio-depressive behaviors in adulthood, with sex as a variable playing a key role in determining behavioral outcomes.

Acute immobilization stress evokes sexually dimorphic peripheral and hippocampal neuroimmune responses in adult rats.

Stress perception and response vary across sexes and may contribute to the sex differences in susceptibility to psychopathology. Stress also engages the immune system and baseline immune system markers are known to be sexually dimorphic. Here, we investigated if the neuroimmune consequences following a single episode of acute immobilization stress (AIS) are sexually dimorphic in male and female Sprague-Dawley rats. We analyzed immune parameters in the periphery, and markers of neuroinflammation in the hippocampus, a key target of stress effects in the brain. We observed sexual dimorphism in the pattern of regulation of peripheral cytokines following stress, with males showing a significant increase in the levels of specific cytokines compared to females. Hippocampal cytokine and neuroinflammation-associated gene expression level analysis did not reveal any sexually dimorphic effects of AIS. However, we noted lower baseline expression levels for specific cytokines and many of the genes analyzed in the hippocampus of control females compared to control males. Finally, we assessed the levels of components of the NLRP3 inflammasome in the hippocampus and observed increased NLRP3 protein levels at baseline in females. We further noted that while males showed an increase in NLRP3 levels following AIS, females failed to show a similar change. Together, our results highlight a sexual dimorphism in neuroimmune consequences following AIS, both in the periphery and within the hippocampus, with males displaying robust proinflammatory changes and similar changes not observed in females. Our study underlines the importance of investigating the effect of sex on neuroimmune consequences following acute stress.

Chronic hM4Di-DREADD-Mediated Chemogenetic Inhibition of Forebrain Excitatory Neurons in Postnatal or Juvenile Life Does Not Alter Adult Mood-Related Behavior.

G-protein-coupled receptors (GPCRs) coupled to Gi signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile) in shaping the emergence of adult anxiodepressive behaviors and sensorimotor gating. To address the role of Gi signaling in these developmental windows, we used a CaMKIIα-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD (designer receptor exclusively activated by designer drugs) in forebrain excitatory neurons and enhanced Gi signaling via chronic administration of the DREADD agonist, clozapine-N-oxide (CNO) in the postnatal window (postnatal days 2-14) or the juvenile window (postnatal days 28-40). We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CaMKIIα-positive neurons in the forebrain, and that the administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker c-Fos. hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons did not alter anxiety- or despair-like behaviors in adulthood and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi signaling in CaMKIIα-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxiodepressive behaviors in adulthood.

Postnatal Fluoxetine Treatment Alters Perineuronal Net Formation and Maintenance in the Hippocampus.

Elevation of serotonin via postnatal fluoxetine (PNFlx) treatment during critical temporal windows is hypothesized to perturb the development of limbic circuits thus establishing a substratum for persistent disruption of mood-related behavior. We examined the impact of PNFlx treatment on the formation and maintenance of perineuronal nets (PNNs), extracellular matrix (ECM) structures that deposit primarily around inhibitory interneurons, and mark the closure of critical period plasticity. PNFlx treatment evoked a significant decline in PNN number, with a robust reduction in PNNs deposited around parvalbumin (PV) interneurons, within the CA1 and CA3 hippocampal subfields at postnatal day (P)21 in Sprague Dawley rat pups. While the reduction in CA1 subfield PNN number was still observed in adulthood, we observed no change in colocalization of PV-positive interneurons with PNNs in the hippocampi of adult PNFlx animals. PNFlx treatment did not alter hippocampal PV, calretinin (CalR), or Reelin-positive neuron numbers in PNFlx animals at P21 or in adulthood. We did observe a small, but significant increase in somatostatin (SST)-positive interneurons in the DG subfield of PNFlx-treated animals in adulthood. This was accompanied by altered GABA-A receptor subunit composition, increased dendritic complexity of apical dendrites of CA1 pyramidal neurons, and enhanced neuronal activation revealed by increased c-Fos-positive cell numbers within hippocampi of PNFlx-treated animals in adulthood. These results indicate that PNFlx treatment alters the formation of PNNs within the hippocampus, raising the possibility of a disruption of excitation-inhibition (E/I) balance within this key limbic brain region.

GPCR signaling: role in mediating the effects of early adversity in psychiatric disorders.

Early adversity is a key risk factor for the development of several psychiatric disorders, including anxiety and depression. During early life, neurocircuits that regulate emotionality undergo substantial structural remodeling and functional maturation, and are thus particularly susceptible to modification by environmental experience. Preclinical evidence indicates that early stress enhances adult anxio-depressive behaviors. A commonality noted across diverse early stress models is life-long alterations in neuroendocrine stress responses and monoaminergic neurotransmission in key limbic circuits. Dysregulation of G protein-coupled receptor (GPCR) signaling is noted across multiple early stress models and is hypothesized to be an important player in the programming of aberrant emotionality. This raises the possibility that disruption of GPCR signaling in key limbic regions during critical temporal windows could establish a substrate for enhanced risk of adult psychopathology. Here, we review literature, predominantly from preclinical models, which supports the building hypothesis that a disruption of GPCR signaling could play a central role in programming persistent molecular, cellular, functional, and behavioral changes as a consequence of early adversity.

Chronic postnatal chemogenetic activation of forebrain excitatory neurons evokes persistent changes in mood behavior.

Early adversity is a risk factor for the development of adult psychopathology. Common across multiple rodent models of early adversity is increased signaling via forebrain Gq-coupled neurotransmitter receptors. We addressed whether enhanced Gq-mediated signaling in forebrain excitatory neurons during postnatal life can evoke persistent mood-related behavioral changes. Excitatory hM3Dq DREADD-mediated chemogenetic activation of forebrain excitatory neurons during postnatal life (P2-14), but not in juvenile or adult windows, increased anxiety-, despair-, and schizophrenia-like behavior in adulthood. This was accompanied by an enhanced metabolic rate of cortical and hippocampal glutamatergic and GABAergic neurons. Furthermore, we observed reduced activity and plasticity-associated marker expression, and perturbed excitatory/inhibitory currents in the hippocampus. These results indicate that Gq-signaling-mediated activation of forebrain excitatory neurons during the critical postnatal window is sufficient to program altered mood-related behavior, as well as functional changes in forebrain glutamate and GABA systems, recapitulating aspects of the consequences of early adversity.

Stress and adversity in early childhood can have long-lasting effects, predisposing people to mental illness and mood disorders in adult life. The weeks immediately before and after birth are critical for establishing key networks of neurons in the brain. Therefore, any disruption to these neural circuits during this time can be detrimental to emotional development. However, it is still unclear which cellular mechanisms cause these lasting changes in behavior. Studies in animals suggest that these long-term effects could result from abnormalities in a few signaling pathways in the brain. For example, it has been proposed that overstimulating the cells that activate circuits in the forebrain ­ also known as excitatory neurons ­ may contribute to the behavioral changes that persist into adulthood. To test this theory, Pati et al. used genetic engineering to modulate a signaling pathway in male mice, which is known to stimulate excitatory neurons in the forebrain. The experiments showed that prolonged activation of excitatory neurons in the first two weeks after birth resulted in anxious and despair-like behaviors as the animals aged. The mice also displayed discrepancies in how they responded to certain external sensory information, which is a hallmark of schizophrenia-like behavior. However, engineering the same changes in adolescent and adult mice had no effect on their mood-related behaviors. This animal study reinforces just how critical the first few weeks of life are for optimal brain development. It provides an insight into a possible mechanism of how disruption during this time could alter emotional behavior. The findings are also relevant to psychiatrists interested in the underlying causes of mental illness after early childhood adversity.

Acute Chemogenetic Activation of CamKIIα-Positive Forebrain Excitatory Neurons Regulates Anxiety-Like Behaviour in Mice.

Anxiety disorders are amongst the most prevalent mental health disorders. Several lines of evidence have implicated cortical regions such as the medial prefrontal cortex, orbitofrontal cortex, and insular cortex along with the hippocampus in the top-down modulation of anxiety-like behaviour in animal models. Both rodent models of anxiety, as well as treatment with anxiolytic drugs, result in the concomitant activation of multiple forebrain regions. Here, we sought to examine the effects of chemogenetic activation or inhibition of forebrain principal neurons on anxiety and despair-like behaviour. We acutely activated or inhibited Ca2+/calmodulin-dependent protein kinase II α (CamKIIα)-positive forebrain excitatory neurons using the hM3Dq or the hM4Di Designer Receptor Exclusively Activated by Designer Drug (DREADD) respectively. Circuit activation was confirmed via an increase in expression of the immediate early gene, c-Fos, within both the hippocampus and the neocortex. We then examined the influence of DREADD-mediated activation of forebrain excitatory neurons on behavioural tests for anxiety and despair-like behaviour. Our results indicate that acute hM3Dq DREADD activation of forebrain excitatory neurons resulted in a significant decline in anxiety-like behaviour on the open field, light-dark avoidance, and the elevated plus maze test. In contrast, hM3Dq DREADD activation of forebrain excitatory neurons did not alter despair-like behaviour on either the tail suspension or forced swim tests. Acute hM4Di DREADD inhibition of CamKIIα-positive forebrain excitatory neurons did not modify either anxiety or despair-like behaviour. Taken together, our results demonstrate that chemogenetic activation of excitatory neurons in the forebrain decreases anxiety-like behaviour in mice.

The Ministry of Fear: 'The Conjuring' of Fright in the Amygdala by the Raphe.

In this issue of Neuron, Sengupta and Holmes (2019) characterize a distinct serotonergic circuit from the dorsal raphe nucleus to the basal amygdala that facilitates fear conditioning and memory.

Serotonin regulates mitochondrial biogenesis and function in rodent cortical neurons via the 5-HT2A receptor and SIRT1-PGC-1α axis.

Mitochondria in neurons, in addition to their primary role in bioenergetics, also contribute to specialized functions, including regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. However, the factors that influence mitochondrial biogenesis and function in neurons remain poorly elucidated. Here, we identify an important role for serotonin (5-HT) as a regulator of mitochondrial biogenesis and function in rodent cortical neurons, via a 5-HT2A receptor-mediated recruitment of the SIRT1-PGC-1α axis, which is relevant to the neuroprotective action of 5-HT. We found that 5-HT increased mitochondrial biogenesis, reflected through enhanced mtDNA levels, mitotracker staining, and expression of mitochondrial components. This resulted in higher mitochondrial respiratory capacity, oxidative phosphorylation (OXPHOS) efficiency, and a consequential increase in cellular ATP levels. Mechanistically, the effects of 5-HT were mediated via the 5-HT2A receptor and master modulators of mitochondrial biogenesis, SIRT1 and PGC-1α. SIRT1 was required to mediate the effects of 5-HT on mitochondrial biogenesis and function in cortical neurons. In vivo studies revealed that 5-HT2A receptor stimulation increased cortical mtDNA and ATP levels in a SIRT1-dependent manner. Direct infusion of 5-HT into the neocortex and chemogenetic activation of 5-HT neurons also resulted in enhanced mitochondrial biogenesis and function in vivo. In cortical neurons, 5-HT enhanced expression of antioxidant enzymes, decreased cellular reactive oxygen species, and exhibited neuroprotection against excitotoxic and oxidative stress, an effect that required SIRT1. These findings identify 5-HT as an upstream regulator of mitochondrial biogenesis and function in cortical neurons and implicate the mitochondrial effects of 5-HT in its neuroprotective action.

Pi inhibits intracellular accumulation of methylglyoxal in promastigote form of L. donovani.

Similar to their mammalian counterpart, protozoan parasites including Leishmania donovani detoxify methylglyoxal (MG),(1) a toxic ubiquitous product generated in glycolysis pathway. However, it differs in one or more way(s) from the humans. It is known that MG is eliminated either through glyoxalase (GLO)(2) pathway and/or excreted across the cell membrane. This toxic metabolic by-product is known to be detoxified predominantly by the GLO pathway and excretion across the cell membrane is never considered to be a significant pathway for its detoxification. We have tried to modulate these pathways under various physiological conditions to find ways that may lead to accumulation of MG in L. donovani. Besides targeting the GLO pathway, we intend to understand the mechanism of MG release across the cell membrane and possible ways to inhibit its exclusion from parasites. In our study, it was found that inorganic phosphate (Pi)(3) in the presence of glucose inhibits the production of MG as well as promotes the expulsion of MG from the cell. Moreover, the trivalent form of antimony (Sb(III)) inhibits GLO pathway and thus detoxification of MG. Inhibition of Pi transport, which is a Na(+)/H(+) dependent process, restores the Pi mediated abrogation of MG production. Thus, Sb(III) along with inhibitors of Pi transporter may be a therapeutic advancement for treatment of antimony resistant cases of human visceral leishmaniasis. However, it requires further validation using specific inhibitor(s) of Pi transporter.