Sex Differences in Response-Contingent Cannabis Vapor Administration During Adolescence Mediate Enduring Effects on Behavioral Flexibility and Prefrontal Microglia Activation in Rats.

Introduction: Cannabis is the most used illicit drug in the United States. With many states passing legislation to permit its recreational use, there is concern that cannabis use among adolescents could increase dramatically in the coming years. Historically, it has been difficult to model real-world cannabis use to investigate the causal relationship between cannabis use in adolescence and behavioral and neurobiological effects in adulthood. Materials and Methods: We used a response-contingent vapor administration model to investigate long-term effects of cannabis use during adolescence on the medial prefrontal cortex (mPFC) and mPFC-dependent behaviors in male and female rats. Results: Adolescent (35- to 55-day-old) female rats had significantly higher rates of responding for vaporized Δ9-tetrahydrocannabinol (THC)-dominant cannabis extract (CANTHC) compared with adolescent males. In adulthood (70-110 days old), female, but not male, CANTHC rats also took more trials to reach criterion and made more regressive errors in an automated attentional set-shifting task compared with vehicle rats, thereby indicating sex differences in behavioral flexibility impairments. Notably, sex-treatment interactions were not observed when rats of each sex were exposed to a noncontingent CANTHC vapor dosing regimen that approximated CANTHC vapor deliveries earned by females. No differences were observed in effort-based decision making in either sex. In the mPFC, female (but not male) CANTHC rats displayed more reactive microglia with no changes in myelin basic protein expression or dendritic spine density. Conclusion: Altogether, these data reveal important sex differences in rates of responding for CANTHC vapor in adolescence that may confer enduring alterations to mPFC structure and function and suggest that there may be subtle differences in the effects of response-contingent versus noncontingent cannabis exposure that should be systematically examined in future studies.

Impact of continuous testosterone exposure on reproductive physiology, activity, and pain-related behavior in young adult female rats.

Testosterone may reduce pain in cisgender women and transgender men. Rodents can provide a useful model for investigating physiological effects of hormone therapy. To this end, continuous-release testosterone or blank (placebo) capsules were implanted s.c. into young adult female rats, and three weeks later rats were either ovariectomized or sham-ovariectomized. Testosterone treatment that mimicked previously reported endogenous levels in males eliminated estrous cycling and decreased uterine weight. Testosterone also significantly increased body weight and suppressed the increases in daily wheel running observed in placebo controls over time. Subsequent ovariectomy or sham-ovariectomy decreased wheel running in all groups, but testosterone-treated rats recovered significantly more quickly than did placebo-treated rats. Neither testosterone nor ovariectomy significantly altered hindpaw mechanical threshold. Two weeks after sham/ovariectomy surgery, injection of Complete Freund Adjuvant (CFA) into one hindpaw reduced wheel running and mechanical threshold in all groups; running significantly decreased from the first to second day after CFA in testosterone- but not in placebo-treated rats. Morphine 1.0 but not 3.2 mg/kg increased CFA-suppressed wheel running similarly in all groups, whereas both doses of morphine increased CFA-suppressed mechanical threshold. These data suggest that weeks-long testosterone treatment with or without ovariectomy may provide a useful physiological model of testosterone therapy as used in human gender transition. Although testosterone administered at levels similar to those in gonadally intact males tended to hasten female rats' recovery from surgery, it did not decrease maximal pain-related behaviors after surgery or hindpaw inflammatory insult, nor did it alter opioid antinociception.

Sex and timing of gonadectomy relative to puberty interact to influence weight, body composition, and feeding behaviors in mice.

Gonadal sex steroids are important regulators of energy balance in adult rodents, and gonadectomy (GDX) has opposing effects on weight gain in sexually mature males and females. Puberty is associated with the emergence of sex differences in weight, body composition, and feeding behaviors, yet the role of gonadal hormones at puberty remains unclear. To address this, we performed GDX or sham surgery in male and female C57Bl/6 mice at postnatal day (P)25 (prepubertal) or P60 (postpubertal) timepoints and measured weight and body composition for 35 days, after which ad libitum and operant food intake was measured using Feeding Experimentation Device 3 (FED3s) in the home cage. Consistent with previous studies, postpubertal GDX caused weight gain in females and weight loss in males and increased adiposity in both sexes. However, prepubertal GDX decreased weight gain and altered body composition across the adolescent transition (P25 to P60) in males but had no effect in females. Despite the varied effects on weight, GDX decreased food intake and motivation for food as assessed in operant tasks regardless of sex or timing of surgery relative to puberty. Our findings indicate that GDX interacts with both sex and age at surgery to influence weight, body composition, and feeding behavior.

Sex differences in adolescent cannabis vapor self-administration mediate enduring effects on behavioral flexibility and prefrontal microglia activation in rats.

Cannabis is the most used illicit drug in the United States. With many states passing legislation to permit its recreational use, there is concern that cannabis use among adolescents could increase dramatically in the coming years. Historically, it has been difficult to model real-world cannabis use to investigate the causal relationship between cannabis use in adolescence and behavioral and neurobiological effects in adulthood. To this end, we used a novel volitional vapor administration model to investigate long-term effects of cannabis use during adolescence on the medial prefrontal cortex (mPFC) and mPFC-dependent behaviors in male and female rats. Adolescent (35-55 day old) female rats had significantly higher rates of responding for vaporized Δ9-tetrahydrocannabinol (THC)-dominant cannabis extract (CANTHC) compared to adolescent males. In adulthood (70-110 day old), female, but not male, CANTHC rats also took more trials to reach criterion and made more regressive errors in an automated attentional set-shifting task compared to vehicle rats. Similar set-shifting deficits were observed in males when they were exposed to a non-contingent CANTHC vapor dosing regimen that approximated CANTHC self-administration rates in females. No differences were observed in effort-based decision making in either sex. In the mPFC, female (but not male) CANTHC rats displayed more reactive microglia with no significant changes in myelin basic protein expression or dendritic spine density. Together, these data reveal important sex differences in rates of cannabis vapor self-administration in adolescence that confer enduring alterations to mPFC structure and function. Importantly, female-specific deficits in behavioral flexibility appear to be driven by elevated rates of CANTHC self-administration as opposed to a sex difference in the effects of CANTHC vapor per se.

Activation, but not inhibition, of the indirect pathway disrupts choice rejection in a freely moving, multiple-choice foraging task.

The dorsomedial striatum (DMS) plays a key role in action selection, but less is known about how direct and indirect pathway spiny projection neurons (dSPNs and iSPNs, respectively) contribute to choice rejection in freely moving animals. Here, we use pathway-specific chemogenetic manipulation during a serial choice foraging task to test the role of dSPNs and iSPNs in learned choice rejection. We find that chemogenetic activation, but not inhibition, of iSPNs disrupts rejection of nonrewarded choices, contrary to predictions of a simple "select/suppress" heuristic. Our findings suggest that iSPNs' role in stopping and freezing does not extend in a simple fashion to choice rejection in an ethological, freely moving context. These data may provide insights critical for the successful design of interventions for addiction or other conditions in which it is desirable to strengthen choice rejection.

Synthetic nanosensors for imaging neuromodulators.

Neuromodulation plays a critical role in regulating brain function and its dysregulation is implicated in the pathogenesis of numerous neurological and psychiatric disorders. However, only in the last few years have optical tools become available to probe the spatial and temporal profiles of neuromodulator signaling, including dopamine, with the requisite resolution to uncover mechanisms of neuromodulation. In this review, we summarize the current state of synthetic nanomaterial-based optical nanosensors for monitoring neurotransmission with high spatial and temporal resolution. Specifically, we highlight how synthetic nanosensors can elucidate the spatial distribution of neuromodulator release sites and report the temporal dynamics and spatial diffusion of neuromodulator release. Next, we outline advantages and limitations of currently available nanosensors and their recent application to imaging endogenous dopamine release in brain tissue. Finally, we discuss strategies to improve nanosensors for in vivo use, with implications for translational applications.

Coming of age in the frontal cortex: The role of puberty in cortical maturation.

Across species, adolescence is a period of growing independence that is associated with the maturation of cognitive, social, and affective processing. Reorganization of neural circuits within the frontal cortex is believed to contribute to the emergence of adolescent changes in cognition and behavior. While puberty coincides with adolescence, relatively little is known about which aspects of frontal cortex maturation are driven by pubertal development and gonadal hormones. In this review, we highlight existing work that suggests puberty plays a role in the maturation of specific cell types in the medial prefrontal cortex (mPFC) of rodents, and highlight possible routes by which gonadal hormones influence frontal cortical circuit development.

Sex differences in pubertal associations with fronto-accumbal white matter morphometry: Implications for understanding sensitivity to reward and punishment.

Researchers have reported sex-differentiated maturation of white matter (WM) during puberty. It is not clear, however, whether such distinctions contribute to documented sex differences in sensitivity to reward and punishment during adolescence. Given the role of the orbitofrontal cortex (OFC) and nucleus accumbens (NAcc) in reward and punishment-related behaviors, we tested in a cross-sectional study whether males and females (N = 156, 89 females; ages 9-14 years) differ in the association between pubertal stage and fixel-based morphometry of WM fibers connecting the OFC and NAcc (i.e., the fronto-accumbal tract). Further, we examined whether males and females differ in associations between fronto-accumbal WM measures and self-reported sensitivity to reward and punishment. Pubertal stage was positively associated with fronto-accumbal fiber density and cross-section (FDC) in males, but not in females. Consistent with previous reports, males reported higher reward sensitivity than did females, although fronto-accumbal combined FDC was not related to reward sensitivity in either sex. Meanwhile, only males showed a negative association between fronto-accumbal tract FDC and sensitivity to punishment. Follow-up analyses revealed that fiber cross-section, but not density, was related to pubertal stage and punishment sensitivity in males, as well as to reward sensitivity in all participants. Our findings suggest there are sex differences in puberty-related maturation of the fronto-accumbal tract, and this tract is related to lower punishment sensitivity in adolescent males compared to females.

Sex and Pubertal Status Influence Dendritic Spine Density on Frontal Corticostriatal Projection Neurons in Mice.

In humans, nonhuman primates, and rodents, the frontal cortices exhibit grey matter thinning and dendritic spine pruning that extends into adolescence. This maturation is believed to support higher cognition but may also confer psychiatric vulnerability during adolescence. Currently, little is known about how specific cell types in the frontal cortex mature or whether puberty plays a role in the maturation of some cell types but not others. Here, we used mice to characterize the spatial topography and adolescent development of cross-corticostriatal (cSTR) neurons that project through the corpus collosum to the dorsomedial striatum. We found that apical spine density on cSTR neurons in the medial prefrontal cortex decreased significantly between late juvenile (P29) and young adult time points (P60), with females exhibiting higher spine density than males at both ages. Adult males castrated prior to puberty onset had higher spine density compared to sham controls. Adult females ovariectomized before puberty onset showed greater variance in spine density measures on cSTR cells compared to controls, but their mean spine density did not significantly differ from sham controls. Our findings reveal that these cSTR neurons, a subtype of the broader class of intratelencephalic-type neurons, exhibit significant sex differences and suggest that spine pruning on cSTR neurons is regulated by puberty in male mice.

Parvalbumin Interneuron Dysfunction in a Thalamo-Prefrontal Cortical Circuit in Disc1 Locus Impairment Mice.

Altered cortical excitation-inhibition (E-I) balance resulting from abnormal parvalbumin interneuron (PV IN) function is a proposed pathophysiological mechanism of schizophrenia and other major psychiatric disorders. Preclinical studies have indicated that disrupted-in-schizophrenia-1 (Disc1) is a useful molecular lead to address the biology of prefrontal cortex (PFC)-dependent cognition and PV IN function. To date, PFC inhibitory circuit function has not been investigated in depth in Disc1 locus impairment (LI) mouse models. Therefore, we used a Disc1 LI mouse model to investigate E-I balance in medial PFC (mPFC) circuits. We found that inhibition onto layer 2/3 excitatory pyramidal neurons in the mPFC was significantly reduced in Disc1 LI mice. This reduced inhibition was accompanied by decreased GABA release from local PV, but not somatostatin (SOM) INs, and by impaired feedforward inhibition (FFI) in the mediodorsal thalamus (MD) to mPFC circuit. Our mechanistic findings of abnormal PV IN function in a Disc1 LI model provide insight into biology that may be relevant to neuropsychiatric disorders including schizophrenia.

A role for adaptive developmental plasticity in learning and decision making.

From both a medical and educational perspective, there is enormous value to understanding the environmental factors that sculpt learning and decision making. These questions are often approached from proximate levels of analysis, but may be further informed by the adaptive developmental plasticity framework used in evolutionary biology. The basic adaptive developmental plasticity framework posits that biological sensitive periods evolved to use information from the environment to sculpt emerging phenotypes. Here we lay out how we can apply this framework to learning and decision making in the mammalian brain and propose a working model in which dopamine neurons and their activity may serve to inform downstream circuits about environmental statistics. More widespread use of this evolutionary framework and its associated models can help inform and guide basic research and intervention science.

Variation in early life maternal care predicts later long range frontal cortex synapse development in mice.

Empirical and theoretical work suggests that early postnatal experience may inform later developing synaptic connectivity to adapt the brain to its environment. We hypothesized that early maternal experience may program the development of synaptic density on long range frontal cortex projections. To test this idea, we used maternal separation (MS) to generate environmental variability and examined how MS affected 1) maternal care and 2) synapse density on virally-labeled long range axons of offspring reared in MS or control conditions. We found that MS and variation in maternal care predicted bouton density on dorsal frontal cortex axons that terminated in the basolateral amygdala (BLA) and dorsomedial striatum (DMS) with more, fragmented care associated with higher density. The effects of maternal care on these distinct axonal projections of the frontal cortex were manifest at different ages. Maternal care measures were correlated with frontal cortex → BLA bouton density at mid-adolescence postnatal (P) day 35 and frontal cortex → DMS bouton density in adulthood (P85). Meanwhile, we found no evidence that MS or maternal care affected bouton density on ascending orbitofrontal cortex (OFC) or BLA axons that terminated in the dorsal frontal cortices. Our data show that variation in early experience can alter development in a circuit-specific and age-dependent manner that may be relevant to understanding the effects of early life adversity.

Prepubertal gonadectomy reveals sex differences in approach-avoidance behavior in adult mice.

Adolescence is a developmental period that is associated with physical, cognitive, and affective maturation and a time when sex biases in multiple psychiatric diseases emerge. While puberty onset marks the initiation of adolescence, it is unclear whether the pubertal rise in gonadal hormones generates sex differences in approach-avoidance behaviors that may impact psychiatric vulnerability. To examine the influence of pubertal development on adult behavior, we removed the gonads or performed sham surgery in male and female mice just prior to puberty onset and assessed performance in an odor-guided foraging task and anxiety-related behaviors in adulthood. We observed no significant sex differences in foraging or anxiety-related behaviors between intact adult male and female mice but found significant differences between adult male and female mice that had been gonadectomized (GDX) prior to puberty onset. GDX males failed to acquire the odor-guided foraging task, showed reduced locomotion, and exhibited increased anxiety-like behavior, while GDX females showed the opposite pattern of behavior. These data suggest that puberty may minimize rather than drive differences in approach-avoidance phenotypes in male and female mice.

Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor.

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000-1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

Neuroscience: Sex Hormones at Work in the Neocortex.

Sex hormones have easy access to the brain and their receptors are expressed by cortical neurons. Until recently, little was known about what impact, if any, they have on cortical processing. New data reveal that estradiol potently alters inhibitory neurotransmission in the neocortex.

Adolescence and "Late Blooming" Synapses of the Prefrontal Cortex.

The maturation of the prefrontal cortex (PFC) during adolescence is thought to be important for cognitive and affective development and mental health risk. Whereas many summaries of adolescent development have focused on dendritic spine pruning and gray matter thinning in the PFC during adolescence, we highlight recent rodent data from our laboratory and others to call attention to continued synapse formation and plasticity in the adolescent period in specific cell types and circuits. In particular, we highlight changes in inhibitory neurotransmission onto intratelencephalic (IT-type) projecting cortical neurons and late expansion of connectivity between the amygdala and PFC and the ventral tegmental area and PFC. Continued work on these "late blooming" synapses in specific cell types and circuits, and their interrelationships, will illuminate new opportunities for understanding and shaping the biology of adolescent development. We also address which aspects of adolescent PFC development are dependent on pubertal processes.

Does puberty mark a transition in sensitive periods for plasticity in the associative neocortex?

Postnatal brain development is studded with sensitive periods during which experience dependent plasticity is enhanced. This enables rapid learning from environmental inputs and reorganization of cortical circuits that matches behavior with environmental contingencies. Significant headway has been achieved in characterizing and understanding sensitive period biology in primary sensory cortices, but relatively little is known about sensitive period biology in associative neocortex. One possible mediator is the onset of puberty, which marks the transition to adolescence, when animals shift their behavior toward gaining independence and exploring their social world. Puberty onset correlates with reduced behavioral plasticity in some domains and enhanced plasticity in others, and therefore may drive the transition from juvenile to adolescent brain function. Pubertal onset is also occurring earlier in developed nations, particularly in unserved populations, and earlier puberty is associated with vulnerability for substance use, depression and anxiety. In the present article we review the evidence that supports a causal role for puberty in developmental changes in the function and neurobiology of the associative neocortex. We also propose a model for how pubertal hormones may regulate sensitive period plasticity in associative neocortex. We conclude that the evidence suggests puberty onset may play a causal role in some aspects of associative neocortical development, but that further research that manipulates puberty and measures gonadal hormones is required. We argue that further work of this kind is urgently needed to determine how earlier puberty may negatively impact human health and learning potential. This article is part of a Special Issue entitled SI: Adolescent plasticity.