Chronic chemogenetic activation of hippocampal progenitors enhances adult neurogenesis and modulates anxiety-like behavior and fear extinction learning.

Adult hippocampal neurogenesis is a lifelong process that involves the integration of newborn neurons into the hippocampal network, and plays a role in cognitive function and the modulation of mood-related behavior. Here, we sought to address the impact of chemogenetic activation of adult hippocampal progenitors on distinct stages of progenitor development, including quiescent stem cell activation, progenitor turnover, differentiation and morphological maturation. We find that hM3Dq-DREADD-mediated activation of nestin-positive adult hippocampal progenitors recruits quiescent stem cells, enhances progenitor proliferation, increases doublecortin-positive newborn neuron number, accompanied by an acceleration of differentiation and morphological maturation, associated with increased dendritic complexity. Behavioral analysis indicated anxiolytic behavioral responses in transgenic mice subjected to chemogenetic activation of adult hippocampal progenitors at timepoints when newborn neurons are predicted to integrate into the mature hippocampal network. Furthermore, we noted an enhanced fear memory extinction on a contextual fear memory learning task in transgenic mice subjected to chemogenetic activation of adult hippocampal progenitors. Our findings indicate that hM3Dq-DREAD-mediated chemogenetic activation of adult hippocampal progenitors impacts distinct aspects of hippocampal neurogenesis, associated with the regulation of anxiety-like behavior and fear memory extinction.

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.

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.

Chemogenetic Activation of Excitatory Neurons Alters Hippocampal Neurotransmission in a Dose-Dependent Manner.

Designer receptors exclusively activated by designer drugs (DREADD)-based chemogenetic tools are extensively used to manipulate neuronal activity in a cell type-specific manner. Whole-cell patch-clamp recordings indicate membrane depolarization, coupled with increased neuronal firing rate, following administration of the DREADD ligand, clozapine-N-oxide (CNO) to activate the Gq-coupled DREADD, hM3Dq. Although hM3Dq has been used to enhance neuronal firing in order to manipulate diverse behaviors, often within 30 min to 1 h after CNO administration, the physiological effects on excitatory neurotransmission remain poorly understood. We investigated the influence of CNO-mediated hM3Dq DREADD activation on distinct aspects of hippocampal excitatory neurotransmission at the Schaffer collateral-CA1 synapse in hippocampal slices derived from mice expressing hM3Dq in Ca2+/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons. Our results indicate a clear dose-dependent effect on field EPSP (fEPSP) slope, with no change noted at the lower dose of CNO (1 µM) and a significant, long-term decline in fEPSP slope observed at higher doses (5-20 µM). Further, we noted a robust θ burst stimulus (TBS) induced long-term potentiation (LTP) in the presence of the lower CNO (1 µM) dose, which was significantly attenuated at the higher CNO (20 µM) dose. Whole-cell patch-clamp recording revealed both complex dose-dependent regulation of excitability, and spontaneous and evoked activity of CA1 pyramidal neurons in response to hM3Dq activation across CNO concentrations. Our data indicate that CNO-mediated activation of the hM3Dq DREADD results in dose-dependent regulation of excitatory hippocampal neurotransmission and highlight the importance of careful interpretation of behavioral experiments involving chemogenetic manipulation.

Acute pharmacogenetic activation of medial prefrontal cortex excitatory neurons regulates anxiety-like behaviour.

The medial prefrontal cortex (mPFC) is implicated in anxiety-like behaviour. In rodent models, perturbations of mPFC neuronal activity through pharmacological manipulations, optogenetic activation of mPFC neurons or cell-type specific pharmacogenetic inhibition of somatostatin interneurons indicate conflicting effects on anxiety-like behaviour. In the present study we examined the effects of pharmacogenetic activation of Ca 2+/calmodulin-dependent protein kinase alpha (CamKII alpha)-positive excitatory neurons on anxiety-like behaviour. We used clozapine-N-oxide (CNO) to pharmacogenetically activate virally delivered CamKII alpha-hM3Dq-DREADD in mPFC excitatory neurons. The effects of acute CNO or vehicle treatment on anxiety-like behaviour in the open field and elevated plus maze tests were examined in rats virally infected with either CamKII alpha-hM3Dq-DREADD or CamKII alpha-GFP. In addition, the effects of acute CNO treatment on the expression of the neuronal activity marker c-Fos were examined in the mPFC as well as downstream target neuronal circuits using immunohistochemistry. Acute pharmacogenetic activation of mPFC excitatory neurons evoked a significant decrease in anxiety-like behaviour selectively on the elevated plus maze task, but not the open field test. Acute CNO treatment resulted in enhanced c-Fos-immunopositive cell number in the infralimbic, prelimbic and cingulate subdivisions of the mPFC. This was also accompanied by enhanced c-Fos-immunopositive cell number in multiple downstream circuits of the mPFC in CNO-treated hM3Dq animals. Acute pharmacogenetic activation of mPFC excitatory neurons reduces anxietylike behaviour in a task-specific fashion accompanied by enhanced c-Fos expression in the mPFC and multiple target circuits implicated in the regulation of anxiety-like behaviour.

Early emergence of altered 5-HT2A receptor-evoked behavior, neural activation and gene expression following maternal separation.

The early stress of Maternal Separation (MS) contributes to the establishment of adult psychopathology. The serotonergic (5-HT) system is implicated during this temporal window in mediating the development of mood-related behaviors. MS is reported to evoke altered 5-HT2A receptor function in adulthood. However, the ontogeny of altered 5-HT2A receptor responsivity following MS remains unknown. Here, we examined 5-HT2A receptor agonist, DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (2mg/kg) evoked responses, namely stereotypical head-twitch behaviors in control and MS Sprague-Dawley rat pups at postnatal day 21 (P21). MS involved a separation of pups from the dam for 3h daily from postnatal day 2-14. MS pups at P21 exhibited significantly enhanced head-twitch behaviors compared to controls. Using c-Fos cell counting we examined neural activation in control and MS pups following DOI treatment. MS pups exhibited altered DOI-evoked c-Fos expression within all mPFC subdivisions, but not in the hippocampus, lateral septum and hypothalamus, suggesting differential prefrontal neural activation upon 5-HT2A receptor stimulation following early stress. Gene profiling of 5-HT2A receptor-regulated immediate early genes (IEGs) indicated a decline in the expression of Fos, Fra1 and Egr1 mRNA under baseline conditions in the mPFC of MS pups. MS pups also showed an altered pattern in the regulation of several 5-HT2A receptor-regulated IEGs (Fos, Fra1, Bdnf, Egr1, Egr3) following DOI treatment. Collectively, these results highlight an early emergence of altered 5-HT2A receptor-evoked behavioral responses and neural activation patterns in multiple brain regions in animals with a history of MS.