Early life adversity shapes social subordination and cell type-specific transcriptomic patterning in the ventral hippocampus.

Adverse events in early life can modulate the response to additional stressors later in life and increase the risk of developing psychiatric disorders. The underlying molecular mechanisms responsible for these effects remain unclear. Here, we uncover that early life adversity (ELA) in mice leads to social subordination. Using single-cell RNA sequencing (scRNA-seq), we identified cell type-specific changes in the transcriptional state of glutamatergic and GABAergic neurons in the ventral hippocampus of ELA mice after exposure to acute social stress in adulthood. These findings were reflected by an alteration in excitatory and inhibitory synaptic transmission induced by ELA in response to acute social stress. Finally, enhancing the inhibitory network function through transient diazepam treatment during an early developmental sensitive period reversed the ELA-induced social subordination. Collectively, this study significantly advances our understanding of the molecular, physiological, and behavioral alterations induced by ELA, uncovering a previously unknown cell type-specific vulnerability to ELA.

An optimized protocol for isolation of hepatic leukocytes retrieved from murine and NASH liver biopsies.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

Ketamine exerts its sustained antidepressant effects via cell-type-specific regulation of Kcnq2.

A single sub-anesthetic dose of ketamine produces a rapid and sustained antidepressant response, yet the molecular mechanisms responsible for this remain unclear. Here, we identified cell-type-specific transcriptional signatures associated with a sustained ketamine response in mice. Most interestingly, we identified the Kcnq2 gene as an important downstream regulator of ketamine action in glutamatergic neurons of the ventral hippocampus. We validated these findings through a series of complementary molecular, electrophysiological, cellular, pharmacological, behavioral, and functional experiments. We demonstrated that adjunctive treatment with retigabine, a KCNQ activator, augments ketamine's antidepressant-like effects in mice. Intriguingly, these effects are ketamine specific, as they do not modulate a response to classical antidepressants, such as escitalopram. These findings significantly advance our understanding of the mechanisms underlying the sustained antidepressant effects of ketamine, with important clinical implications.

The role of TET proteins in stress-induced neuroepigenetic and behavioural adaptations.

Over the past decade, critical, non-redundant roles of the ten-eleven translocation (TET) family of dioxygenase enzymes have been identified in the brain during developmental and postnatal stages. Specifically, TET-mediated active demethylation, involving the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and subsequent oxidative derivatives, is dynamically regulated in response to environmental stimuli such as neuronal activity, learning and memory processes, and stressor exposure. Such changes may therefore perpetuate stable and dynamic transcriptional patterns within neuronal populations required for neuroplasticity and behavioural adaptation. In this review, we will highlight recent evidence supporting a role of TET protein function and active demethylation in stress-induced neuroepigenetic and behavioural adaptations. We further explore potential mechanisms by which TET proteins may mediate both the basal and pathological embedding of stressful life experiences within the brain of relevance to stress-related psychiatric disorders.

Adolescent chronic intermittent toluene inhalation dynamically regulates the transcriptome and neuronal methylome within the rat medial prefrontal cortex.

Inhalants containing the volatile solvent toluene are misused to induce euphoria or intoxication. Inhalant abuse is most common during adolescence and can result in cognitive impairments during an important maturational period. Despite evidence suggesting that epigenetic modifications may underpin the cognitive effects of inhalants, no studies to date have thoroughly investigated toluene-induced regulation of the transcriptome or discrete epigenetic modifications within the brain. To address this, we investigated effects of adolescent chronic intermittent toluene (CIT) inhalation on gene expression and DNA methylation profiles within the rat medial prefrontal cortex (mPFC), which undergoes maturation throughout adolescence and has been implicated in toluene-induced cognitive deficits. Employing both RNA-seq and genome-wide Methyl CpG Binding Domain (MBD) Ultra-seq analysis, we demonstrate that adolescent CIT inhalation (10 000 ppm for 1 h/day, 3 days/week for 4 weeks) induces both transient and persistent changes to the transcriptome and DNA methylome within the rat mPFC for at least 2 weeks following toluene exposure. We demonstrate for the first time that adolescent CIT exposure results in dynamic regulation of the mPFC transcriptome likely relating to acute inflammatory responses and persistent deficits in synaptic plasticity. These adaptations may contribute to the cognitive deficits associated with chronic toluene exposure and provide novel molecular targets for preventing long-term neurophysiological abnormalities following chronic toluene inhalation.

Adenosine-to-Inosine RNA Editing Within Corticolimbic Brain Regions Is Regulated in Response to Chronic Social Defeat Stress in Mice.

Adenosine-to-inosine (A-to-I) RNA editing is a co-/posttranscriptional modification of double-stranded RNA, catalyzed by the adenosine deaminase acting on RNA (ADAR) family of enzymes, which results in recognition of inosine as guanosine by translational and splicing machinery causing potential recoding events in amino acid sequences. A-to-I editing is prominent within brain-specific transcripts, and dysregulation of editing at several well-studied loci (e.g., Gria2, Htr2c) has been implicated in acute and chronic stress in rodents as well as neurological (e.g., Alzheimer's) and psychopathological disorders such as schizophrenia and major depressive disorder. However, only a small fraction of recoding sites has been investigated within the brain following stress, and our understanding of the role of RNA editing in transcriptome regulation following environmental stimuli remains poorly understood. Thus, we aimed to investigate A-to-I editing at hundreds of loci following chronic social defeat stress (CSDS) in mice within corticolimbic regions responsive to chronic stress regulation. Adult male mice were subjected to CSDS or control conditions for 21 days and dynamic regulation of A-to-I editing was investigated 2 and 8 days following the final defeat within both the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). Employing a targeted resequencing approach, which utilizes microfluidics-based multiplex polymerase chain reaction (PCR) coupled with next-generation sequencing, we analyzed A-to-I editing at ∼100 high-confidence editing sites within the mouse brain. CSDS resulted in acute regulation of transcripts encoding several ADAR enzymes, which normalized 8 days following the final defeat and was specific for susceptible mice. In contrast, sequencing analysis revealed modest and dynamic regulation of A-to-I editing within numerous transcripts in both the mPFC and BLA of resilient and susceptible mice at both 2 and 8 days following CSDS with minimal overlap between regions and time points. Editing within the Htr2c transcript and relative abundance of Htr2c messenger RNA (mRNA)variants were also observed within the BLA of susceptible mice 2 days following CSDS. These results indicate dynamic RNA editing within discrete brain regions following CSDS in mice, further implicating A-to-I editing as a stress-sensitive molecular mechanism within the brain of potential relevance to resiliency and susceptibility to CSDS.

Central Neuroepigenetic Regulation of the Hypothalamic-Pituitary-Adrenal Axis.

Dynamic adaptation to stressful life events requires the co-ordinated action of the central stress response, which is mediated by the hypothalamic-pituitary-adrenal (HPA) axis, to restore and maintain homeostasis. Excessive exposure to stress or traumatic life events, such as childhood maltreatment, has been linked to HPA axis dysfunction increasing the risk of developing stress-related psychopathologies such as major depressive disorder and post-traumatic-stress-disorder. Mounting evidence supports the notion that stressors throughout pre- and postnatal development as well as adulthood can induce neuroepigenetic regulation of gene expression within key nodes of the brain, which may in part mediate such HPA axis dysfunction. Neuroepigenetic mechanisms, particularly DNA methylation and small non-coding RNAs, are therefore considered to be molecular mechanisms by which stressful life events may perpetuate aberrant behavioral phenotypes associated with psychiatric disorders throughout one's life and even across generations. In this chapter we outline the progress made toward understanding the effects of stress-induced neuroepigenetic changes upon HPA axis function and highlight the need for novel research strategies to deepen our understanding of the establishment, maintenance and reversibility of neuroepigenetic regulation following stress to enable realization of potential novel therapeutic and preventative strategies for stress-related psychiatric disorders.

Epigenetic mechanisms involved in the effects of stress exposure: focus on 5-hydroxymethylcytosine.

5-hydroxymethylcytosine (5hmC) is a recently re-discovered transient intermediate in the active demethylation pathway that also appears to play an independent role in modulating gene function. Epigenetic marks, particularly 5-methylcytosine, have been widely studied in relation to stress-related disorders given the long-lasting effect that stress has on these marks. 5hmC is a good candidate for involvement in the etiology of these disorders given its elevated concentration in mammalian neurons, its dynamic regulation during development of the central nervous system, and its high variability among individuals. Although we are unaware of any studies published to date examining 5 hmC profiles in human subjects who have developed a psychiatric disorder after a life stressor, there is emerging evidence from the animal literature that 5hmC profiles are altered in the context of fear-conditioning paradigms and stress exposure, suggesting a possible role for 5hmC in the biological underpinnings of stress-related disorders. In this review, the authors examine the available approaches for profiling 5hmC and describe their advantages and disadvantages as well as discuss the studies published thus far investigating 5hmC in the context of fear-related learning and stress exposure in animals. The authors also highlight the global versus locus-specific regulation of 5hmC in these studies. Finally, the limitations of the current studies and their implications are discussed.

NMDA receptor binding is reduced within mesocorticolimbic regions following chronic inhalation of toluene in adolescent rats.

The purposeful inhalation of volatile solvents, such as toluene, to induce self-intoxication is prevalent, particularly within adolescent populations. Chronic misuse results in cognitive and neurobiological impairments, as well as an increased risk for addictive behaviours in adulthood. Toluene-induced neuroadaptations within mesocorticolimbic circuitry are thought, in part, to mediate some of the adverse outcomes of toluene misuse, however our understanding of the neuroadaptive processes remains equivocal. An understanding of these processes is particularly important relative to exposure that occurs during adolescence and at concentrations that reflect various patterns of use. Therefore, we exposed male adolescent Wistar rats (postnatal day [PN] 27) to either air or low or high concentrations of inhaled toluene in a chronic and intermittent fashion (CIT, 3,000 or 10,000ppm) for 1 h/day, 3-5 times per week for 4 weeks to model different patterns of human inhalant abuse. Brains were subsequently analysed using autoradiography, qPCR and immunohistochemistry 3 days following the exposure period to investigate toluene-induced neuroadaptations within mesocorticolimbic circuitry. In CIT-exposed rats binding to N-methyl-D-aspartate (NMDA) receptors containing the GluN2B subunit, as determined using [(3)H]-ifenprodil, was decreased in a concentration-related manner in the caudal cingulate cortex, dorsal striatum and accumbens; however, this was not associated with changes in GluN2B protein expression. There were no differences in [(3)H]-epibatidine binding to heteromeric neuronal nicotinic acetylcholine (nACh) receptors. Relative expression of mRNA transcripts encoding NMDA, nACh, γ-aminobutyric acid type-A (GABAA) and dopamine receptor subunits was unchanged in all regions assessed following CIT. Our data suggest that adolescent CIT exposure impacts NMDA receptors within regions of corticostriatal circuitry, possibly via post-translational mechanisms. Dysfunctional glutamatergic signalling within corticostriatal regions may contribute to the adverse outcomes observed following adolescent toluene abuse.

Spatial Learning Requires mGlu5 Signalling in the Dorsal Hippocampus.

We examined the role of hippocampal metabotropic glutamate receptor 5 (mGlu5) in spatial learning and memory. Although it has been shown that mGlu5 signalling is required for certain forms of learning and memory, its role in spatial learning is unclear since studies using pharmacological or knockout mice models provide inconsistent findings. Additionally, the location in the brain where mGlu5 signalling may modulate such learning is yet to be precisely delineated. We stereotaxically injected rAAV-Cre into the dorsal hippocampus of mGlu5(loxP/loxP) mice to knockdown mGlu5 in that region. We show for the first time that knockdown of mGlu5 in the dorsal hippocampus is sufficient to impair spatial learning in Morris Water Maze. Locomotor activity and memory retrieval were unaffected by the mGlu5 knockdown. Taken together, these findings support a key role for dorsal hippocampal mGlu5 signalling in spatial learning.

Chronic intermittent toluene inhalation initiated during adolescence in rats does not alter voluntary consumption of ethanol in adulthood.

Voluntary inhalation of organic solvents, such as toluene, is particularly prevalent in adolescent populations and is considered to be a contributing factor to substance use and dependence later in life. While inhalants are often the initial "drug" experienced during this period, alcohol is another substance readily abused by adolescent populations. Although both substances are thought to have similar actions within the brain, our understanding of the implications of adolescent inhalant abuse upon subsequent exposure to alcohol remains to be investigated. Thus, this study aimed to assess locomotor responses to acute ethanol and voluntary ethanol consumption following a period of toluene inhalation throughout adolescence/early adulthood. Adolescent male Wistar rats (postnatal day [PN] 27) inhaled air or toluene (3000 ppm) for 1 h/day, 3 days/week for 4 (PN 27-52) or 8 weeks (PN 27-80) to mimic the patterns observed in human inhalant abusers. Following the exposure period, cross-sensitization to acute ethanol challenge (0.5 g/kg, intra-peritoneally [i.p.]), and voluntary consumption of 20% ethanol in a chronic intermittent 2-bottle choice paradigm, were assessed. Hepatic ethanol and acetaldehyde metabolism and liver histopathology were also investigated. Chronic intermittent toluene (CIT) exposure throughout adolescence for up to 8 weeks did not alter the behavioral response to acute ethanol or voluntary consumption of ethanol in adulthood, although an age-dependent effect on ethanol consumption was observed (p<0.05). Both liver function and pathology did not differ between treatment groups. Thus, in the paradigm employed, CIT exposure throughout adolescence and early adulthood did not predispose rats to subsequent locomotor sensitivity or voluntary consumption of ethanol in adulthood.

Specific impairments in instrumental learning following chronic intermittent toluene inhalation in adolescent rats.

RATIONALE: Inhalant abuse is prevalent in adolescent populations, with chronic use resulting in neurobiological and cognitive abnormalities in adulthood. However, the nature and persistence of cognitive dysfunction, particularly following adolescent inhalant abuse, remain equivocal. OBJECTIVE: The present study assessed specific cognitive processes beginning in late adolescence and adulthood following adolescent inhalation of toluene, a main component of many compounds readily abused. METHODS: Adolescent male Wistar rats (postnatal day (PN) 27) were exposed to chronic intermittent inhaled toluene (10,000 ppm) for 1 h/day, 3 days/week for 4 weeks (PN 27-52) to mimic the patterns observed in human adolescent inhalant abusers. Following toluene exposure, motor and cognitive function was assessed. RESULTS: Adolescent toluene exposure did not alter motor learning in the Rotarod task (PN 58) or acquisition, reversal, or retention of spatial learning in the Morris water maze (PN 55-64). In contrast, it delayed acquisition of instrumental responding for sucrose (5 % w/v) and impaired operant reversal learning and cue-induced reinstatement of sucrose seeking in adulthood (PN 57-100). CONCLUSION: This study demonstrates that exposure to toluene at an abuse concentration during adolescence results in specific impairments in aspects of instrumental learning, without altering motor function and spatial learning in late adolescence/early adulthood. Our data imply that persistent alterations in reward processing may occur following adolescent inhalant misuse.

Adolescent toluene inhalation in rats affects white matter maturation with the potential for recovery following abstinence.

Inhalant misuse is common during adolescence, with ongoing chronic misuse associated with neurobiological and cognitive abnormalities. While human imaging studies consistently report white matter abnormalities among long-term inhalant users, longitudinal studies have been lacking with limited data available regarding the progressive nature of such abnormalities, including the potential for recovery following periods of sustained abstinence. We exposed adolescent male Wistar rats (postnatal day 27) to chronic intermittent inhaled toluene (3,000 ppm) for 1 hour/day, 3 times/week for 8 weeks to model abuse patterns observed in adolescent and young adult human users. This dosing regimen resulted in a significant retardation in weight gain during the exposure period (p<0.05). In parallel, we performed longitudinal magnetic resonance imaging (T2-weighted) and diffusion tensor imaging prior to exposure, and after 4 and 8 weeks, to examine the integrity of white matter tracts, including the anterior commissure and corpus callosum. We also conducted imaging after 8 weeks of abstinence to assess for potential recovery. Chronic intermittent toluene exposure during adolescence and early adulthood resulted in white matter abnormalities, including a decrease in axial (p<0.05) and radial (p<0.05) diffusivity. These abnormalities appeared region-specific, occurring in the anterior commissure but not the corpus callosum and were not present until after at least 4 weeks of exposure. Toluene-induced effects on both body weight and white matter parameters recovered following abstinence. Behaviourally, we observed a progressive decrease in rearing activity following toluene exposure but no difference in motor function, suggesting cognitive function may be more sensitive to the effects of toluene. Furthermore, deficits in rearing were present by 4 weeks suggesting that toluene may affect behaviour prior to detectable white matter abnormalities. Consequently, exposure to inhalants that contain toluene during adolescence and early adulthood appear to differentially affect white matter maturation and behavioural outcomes, although recovery can occur following abstinence.