Early life adversity accelerates hypothalamic drive of pubertal timing in female rats with associated enhanced acoustic startle.

Early life adversity in the form of childhood maltreatment in humans or as modeled by maternal separation (MS) in rodents is often associated with an earlier emergence of puberty in females. Earlier pubertal initiation is an example of accelerated biological aging and predicts later risk for anxiety in women, especially in populations exposed to early life trauma. Here we investigated external pubertal markers as well as hypothalamic gene expression of pubertal regulators kisspeptin and gonadotropin-releasing hormone, to determine a biological substrate for MS-induced accelerated puberty. We further investigated a mechanism by which developmental stress might regulate pubertal timing. As kisspeptin and gonadotropin-releasing hormone secretion are typically inhibited by corticotropin releasing hormone at its receptor CRH-R1, we hypothesized that MS induces a downregulation of Crhr1 gene transcription in a cell-specific manner. Finally, we explored the association between pubertal timing and anxiety-like behavior in an acoustic startle paradigm, to drive future preclinical research linking accelerated puberty and anxiety. We replicated previous findings that MS leads to earlier puberty in females but not males, and found expression of kisspeptin and gonadotropin-releasing hormone mRNA to be prematurely increased in MS females. RNAscope confirmed increased expression of these genes, and further revealed that kisspeptin-expressing neurons in females were less likely to express Crhr1 after MS. Early puberty was associated with higher acoustic startle magnitude in females. Taken together, these findings indicate precocial maturation of central pubertal timing mechanisms after MS, as well as a potential role of CRH-R1 in these effects and an association with a translational measure of anxiety.

Effects of early life stress on cocaine conditioning and AMPA receptor composition are sex-specific and driven by TNF.

Exposure to early life adversity can predispose adolescents to the formation of substance abuse disorders. In rodents, early stressors such as repeated maternal separation (MS) impact AMPAR activity in the prefrontal cortex (PFC) and nucleus accumbens (NAc), regions involved in drug-cue association after cocaine-induced conditioned place preference (CPP). Notably, previous reports suggest that the pro-inflammatory cytokine tumor necrosis factor (TNF) regulates AMPAR subunit composition; increased TNF levels are reported to reduce GluA2-positive AMPARs. Since MS can elevate adolescent TNF levels, the stressor may therefore alter AMPAR subunit composition via neuroimmune signaling, thereby affecting cocaine-induced CPP. We tested the specific role of soluble TNF in MS-induced GluA2 loss and cocaine-induced CPP with biologic disruption of TNF signaling. TNF gene and protein expression were elevated in both PFC and NAc of MS males, but not females. GluA2 expression was reduced in both regions in only male MS rats, and systemic treatment with either ibudilast - a phosphodiesterase inhibitor, or XPro1595 - a blood-brain barrier-permeable blocker of soluble TNF - reversed such loss. MS males also formed greater preference for a cocaine-paired environment, the expression of which returned to control levels after XPro1595 administration. These data suggest a sex-specific mechanistic link between TNF signaling and changes in GluA2 expression and drug-cue conditioning, thereby providing further evidence for a role of MS and neuro-immune activity in cortical and striatal AMPAR changes. Moreover, manipulation of the TNF signaling pathway represents a novel approach for influencing response to reinforcing effects of drug use.

22 kHz and 55 kHz ultrasonic vocalizations differentially influence neural and behavioral outcomes: Implications for modeling anxiety via auditory stimuli in the rat.

The communicative role of ultrasonic vocalizations (USVs) in rats is well established, with distinct USVs indicative of different affective states. USVs in the 22 kHz range are typically emitted by adult rats when in anxiety- or fear-provoking situations (e.g. predator odor, social defeat), while 55 kHz range USVs are typically emitted in appetitive situations (e.g., play, anticipation of reward). Previous work indicates that USVs (real-time and playback) can effectively communicate these affective states and influence changes in behavior and neural activity of the receiver. Changes in cFos activation following 22 kHz USVs have been seen in cortical and limbic regions involved in anxiety, including the basolateral amygdala (BLA). However, it is unclear how USV playback influences cFos activity within the bed nucleus of the stria terminalis (BNST), a region also thought to be critical in processing anxiety-related information, and the nucleus accumbens, a region associated with reward. The present work sought to characterize distinct behavioral, physiological, and neural responses in rats presented with aversive (22 kHz) compared to appetitive (55 kHz) USVs or silence. Our findings show that rats exposed to 22 kHz USVs: 1) engage in anxiety-like behaviors in the elevated zero maze, and 2) show distinct patterns of cFos activation within the BLA and BNST that contrast those seen in 55 kHz playback and silence. Specifically, 22 kHz USVs increased cFos density in the anterodorsal nuclei, while 55 kHz playback increased cFos in the oval nucleus of the BNST, without significant changes within the nucleus accumbens. These results provide important groundwork for leveraging ethologically-relevant stimuli in the rat to improve our understanding of anxiety-related responses in both typical and pathological populations.

Effects of early adolescent environmental enrichment on cognitive dysfunction, prefrontal cortex development, and inflammatory cytokines after early life stress.

Early postnatal stress such as maternal separation causes cognitive dysfunction later in life, including working memory deficits that are largely mediated by the prefrontal cortex. Maternal separation in male rats also yields a loss of parvalbumin-containing prefrontal cortex interneurons in adolescence, which may occur via inflammatory or oxidative stress mechanisms. Environmental enrichment can prevent several effects of maternal separation; however, effects of enrichment on prefrontal cortex development are not well understood. Here, we report that enrichment prevented cognitive dysfunction in maternally separated males and females, and prevented elevated circulating pro-inflammatory cytokines that was evident in maternally separated males, but not females. However, enrichment did not prevent parvalbumin loss or adolescent measures of oxidative stress. Significant correlations indicated that adolescents with higher oxidative damage and less prefrontal cortex parvalbumin in adolescence committed more errors on the win-shift task; therefore, maternal separation may affect cognitive dysfunction via aberrant interneuron development. © 2015 Wiley Periodicals, Inc. Dev Psychobiol 58: 482-491, 2016.

Functional Uncoupling NMDAR NR2A Subunit from PSD-95 in the Prefrontal Cortex: Effects on Behavioral Dysfunction and Parvalbumin Loss after Early-Life Stress.

Exposure to early-life stress increases vulnerability to psychiatric disorders, including depression, schizophrenia, and anxiety. Growing evidence implicates aberrant development of the prefrontal cortex (PFC) in the effects of early-life stress, which often emerge in adolescence or young adulthood. Specifically, early-life stress in the form of maternal separation (MS) in rodents has been shown to decrease parvalbumin (PVB)-positive interneurons in the adolescent PFC; however, the mechanism underpinning behavioral dysfunction and PVB loss is not yet known. We recently reported that MS causes overexpression of the NMDA subunit NR2A in the PFC of adolescent rats. Elevated PFC NR2A is also found in developmental models of schizophrenia and is correlated with behavioral deficits, acting largely through its association with the postsynaptic protein PSD-95. In addition, adolescent maturation of PVB-positive interneurons relies on NR2A-driven NMDA activity. Therefore, it is possible that the NR2A/PSD-95 signaling complex has a role in adolescent MS effects. Here, we aimed to determine whether a discrete manipulation of PFC NR2A could prevent MS effects on PFC-controlled behaviors, including cognition, anxiety, and novelty-induced hyperlocomotion, as well as PVB loss in adolescence. We intracranially infused the NR2A-specific blocking peptide TAT2A in order to uncouple NR2A from PSD-95 in the early-adolescent PFC, without antagonizing the NMDA receptor. We demonstrated that MS rats treated with TAT2A during early adolescence were protected from MS-induced PVB loss and exhibited less anxious behavior than those infused with control peptide. These data implicate NR2A-related N-methyl-D-aspartate receptor development in adolescent behavioral and neural consequences of early-life stress.

Viral over-expression of D1 dopamine receptors in the prefrontal cortex increase high-risk behaviors in adults: comparison with adolescents.

RATIONALE: Adolescents are often described as "lacking brakes" resulting in an increase in several behaviors associated with risk for addiction. Prefrontal cortex dopamine and cortico-limbic interaction play an important role in addiction, and we have previously shown that the dopamine D1 receptor is elevated on prelimbic prefrontal output neurons in adolescent rats. We hypothesized that a constellation of risk-related behaviors is mediated by prefrontal output neuron expression of D1. OBJECTIVES: We aimed to determine the role of the dopamine D1 receptor in behavioral and neural correlates of risk for addiction that are often observed in adolescents. Therefore, high-risk behaviors as well as subcortical D2 receptor expression were investigated in adult animals with experimentally elevated D1 on prefrontal glutamatergic neurons. METHODS: A lentiviral vector that selectively expressed the D1 receptor within glutamate neurons was injected in the prelimbic prefrontal cortex of adult male rats. Place conditioning to cocaine, alcohol, and nicotine, as well as delay discounting, novelty preferences, anxiety, cocaine self-administration, and sucrose preferences were assessed. RESULTS: Virally mediated D1 over-expression in adults leads to stronger drug-cue associations and greater consumption of sweet solutions, elevates bias towards immediate satisfaction rather than delaying gratification, decreases anxiety, and causes rats to work harder for and take more cocaine. Furthermore, elevated cortical D1 reduces D2 receptors in the accumbens (a putative risk marker). CONCLUSIONS: Together, these data suggest a common mechanism for increased motivational drive to seek and consume substances with hedonic value, consistent with adolescent addictive processes.

Evidence for a neuroinflammatory mechanism in delayed effects of early life adversity in rats: relationship to cortical NMDA receptor expression.

Postnatal maternal separation in rats causes a reduction of GABAergic parvalbumin-containing interneurons in the prefrontal cortex that first occurs in adolescence. This parvalbumin loss can be prevented by pre-adolescent treatment with a non-steroidal anti-inflammatory drug that also protects against excitotoxicity. Therefore, the neuropsychiatric disorders associated with early life adversity and interneuron dysfunction may involve neuroinflammatory processes and/or aberrant glutamatergic activity. Here, we aimed to determine whether delayed parvalbumin loss after maternal separation was due to inflammatory activity, and whether central administration of the anti-inflammatory cytokine interleukin (IL)-10 could protect against such loss. We also investigated the effects of maternal separation and IL-10 treatment on cortical NMDA receptor expression. Male rat pups were isolated for 4h/day between postnatal days 2-20. IL-10 was administered intracerebroventricularly through an indwelling cannula between P30 and 38. Adolescent prefrontal cortices were analyzed using Western blotting and immunohistochemistry for parvalbumin and NMDA NR2A subunit expression. We demonstrate that central IL-10 administration during pre-adolescence protects maternally separated animals from parvalbumin loss in adolescence. Linear regression analyses revealed that increased circulating levels of the pro-inflammatory cytokines IL-1ß and IL-6 predicted lowered parvalbumin levels in maternally separated adolescents. Maternal separation also increases cortical expression of the NR2A NMDA receptor subunit in adolescence, which is prevented by IL-10 treatment. These data suggest that inflammatory damage to parvalbumin interneurons may occur via aberrant glutamatergic activity in the prefrontal cortex. Our findings provide a novel interactive mechanism between inflammation and neural dysfunction that helps explain deleterious effects of early life adversity on prefrontal cortex interneurons.

Differential activation of cAMP response element binding protein in discrete nucleus accumbens subregions during early and late cocaine sensitization.

The present study examined the differential cocaine-induced activation of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) throughout discrete zones of analysis of the nucleus accumbens (NAc) in rats. CREB-dependent gene transcription, which may underlie long-lasting drug-induced changes in behavior and the subjective effects of cocaine, varies depending on the stage of drug exposure or withdrawal and the cell population involved. Using immunohistochemistry, the authors analyzed changes in CREB phosphorylation in the NAc after 5 days of cocaine, a short or long drug-free period, and a subsequent challenge injection. The NAc shell was separated into 5 zones of analysis previously defined by neurochemistry and connectivity. Repeated cocaine resulted in CREB phosphorylation in all analyzed subregions of the NAc excluding the most ventrolateral region of the shell 2 weeks after cessation of repeated cocaine, but rats challenged after 2 drug-free days yielded a more localized activation of CREB in the 3 most dorsomedial zones of the shell. The temporal and anatomical determinants of cocaine-induced CREB activity may indicate functional differences among NAc shell subregions and suggest the involvement of CREB in early and late cocaine effects.

Electrolytic lesions of a discrete area within the nucleus accumbens shell attenuate the long-term expression, but not early phase, of sensitization to cocaine.

Repeated exposure to cocaine leads to behavioral sensitization, which is the augmentation of the locomotor response to a subsequent exposure to the drug. The nucleus accumbens (NAc), a major termination site of dopaminergic neurons, is believed to be involved in behavioral sensitization and studies have demonstrated that the NAc shell can be split into five zones of analysis; the vertex, arch, cone, intermediate and ventrolateral zones [Todtenkopf MS, Stellar JR. Assessment of tyrosine hydroxylase immunoreactive innervation in five subregions of the nucleus accumbens shell in rats treated with repeated cocaine. Synapse 2000;38:261-70]. Several reports show cocaine-induced c-fos expression particularly in the intermediate zone after 14, but not 2, drug-free days following repeated cocaine administration, suggesting that this region may be involved in sensitization and particularly in the later phase of expression, versus the earlier phase of sensitization. Bilateral electrolytic lesions of the intermediate zone were made in two groups of rats, which were then repeatedly exposed to cocaine (15 mg/kg, twice/day for 5 days). One group was subsequently given a single cocaine challenge injection (15 mg/kg) after 14 drug-free days, while the other group was challenged after only 2 drug-free days. Two sham surgery groups in which an electrode was lowered but no current was passed served as controls. Results show that lesioned animals as well as sham controls exhibited behavioral sensitization to the drug. However, following a 14-day drug-free period, the lesioned animals showed significant reduction in sensitization, compared to sham controls. Together these findings suggest that the intermediate zone of the NAc shell is indeed involved in the expression phase of behavioral sensitization to cocaine.