Neuroimmune Activation and Microglia Reactivity in Female Rats Following Alcohol Dependence.

The rates of alcohol use disorder among women are growing, yet little is known about how the female brain is affected by alcohol. The neuroimmune system, and specifically microglia, have been implicated in mediating alcohol neurotoxicity, but most preclinical studies have focused on males. Further, few studies have considered changes to the microglial phenotype when examining the effects of ethanol on brain structure and function. Therefore, we quantified microglial reactivity in female rats using a binge model of alcohol dependence, assessed through morphological and phenotypic marker expression, coupled with regional cytokine levels. In a time- and region-dependent manner, alcohol altered the microglial number and morphology, including the soma and process area, and the overall complexity within the corticolimbic regions examined, but no significant increases in the proinflammatory markers MHCII or CD68 were observed. The majority of cytokine and growth factor levels examined were similarly unchanged. However, the expression of the proinflammatory cytokine TNFα was increased, and the anti-inflammatory IL-10, decreased. Thus, female rats showed subtle differences in neuroimmune reactivity compared to past work in males, consistent with reports of enhanced neuroimmune responses in females across the literature. These data suggest that specific neuroimmune reactions in females may impact their susceptibility to alcohol neurotoxicity and other neurodegenerative events with microglial contributions.

Predator-induced transgenerational plasticity of parental care behaviour in male three-spined stickleback fish across two generations.

Parental care is a critical determinant of offspring fitness, and parents adjust their care in response to ecological challenges, including predation risk. The experiences of both mothers and fathers can influence phenotypes of future generations (transgenerational plasticity). If it is adaptive for parents to alter parental care in response to predation risk, then we expect F1 and F2 offspring who receive transgenerational cues of predation risk to shift their parental care behaviour if these ancestral cues reliably predict a similarly risky environment as their F0 parents. Here, we used three-spined sticklebacks (Gasterosteus aculeatus) to understand how paternal exposure to predation risk prior to mating alters reproductive traits and parental care behaviour in unexposed F1 sons and F2 grandsons. Sons of predator-exposed fathers took more attempts to mate than sons of control fathers. F1 sons and F2 grandsons with two (maternal and paternal) predator-exposed grandfathers shifted their paternal care (fanning) behaviour in strikingly similar ways: they fanned less initially, but fanned more near egg hatching. This shift in fanning behaviour matches shifts observed in response to direct exposure to predation risk, suggesting a highly conserved response to pre-fertilization predator exposure that persists from the F0 to the F1 and F2 generations.

Cold nociception as a measure of hyperalgesia during spontaneous heroin withdrawal in mice.

Opioids are powerful analgesic drugs that are used clinically to treat pain. However, chronic opioid use causes compensatory neuroadaptations that result in greater pain sensitivity during withdrawal, known as opioid withdrawal-induced hyperalgesia (OWIH). Cold nociception tests are commonly used in humans, but preclinical studies often use mechanical and heat stimuli to measure OWIH. Thus, further characterization of cold nociception stimuli is needed in preclinical models. We assessed three cold nociception tests-thermal gradient ring (5-30 °C, 5-50 °C, 15-40 °C, and 25-50 °C), dynamic cold plate (4 °C to -1 °C at -1 °C/min, -1 °C to 4 °C at +1 °C/min), and stable cold plate (10 °C, 6 °C, and 2 °C)-to measure hyperalgesia in a mouse protocol of heroin dependence. On the thermal gradient ring, mice in the heroin withdrawal group preferred warmer temperatures, and the results depended on the ring's temperature range. On the dynamic cold plate, heroin withdrawal increased the number of nociceptive responses, with a temperature ramp from 4 °C to -1 °C yielding the largest response. On the stable cold plate, heroin withdrawal increased the number of nociceptive responses, and a plate temperature of 2 °C yielded the most significant increase in responses. Among the three tests, the stable cold plate elicited the most robust change in behavior between heroin-dependent and nondependent mice and had the highest throughput. To pharmacologically characterize the stable cold plate test, we used µ-opioid and non-opioid receptor-targeting drugs that have been previously shown to reverse OWIH in mechanical and heat nociception assays. The full µ-opioid receptor agonist methadone and µ-opioid receptor partial agonist buprenorphine decreased OWIH, whereas the preferential µ-opioid receptor antagonist naltrexone increased OWIH. Two N-methyl-d-aspartate receptor antagonists (ketamine, MK-801), a corticotropin-releasing factor 1 receptor antagonist (R121919), a ß2-adrenergic receptor antagonist (butoxamine), an α2-adrenergic receptor agonist (lofexidine), and a 5-hydroxytryptamine-3 receptor antagonist (ondansetron) had no effect on OWIH. These data demonstrate that the stable cold plate at 2 °C yields a robust, reliable, and concise measure of OWIH that is sensitive to opioid agonists.

Heroin- and Fentanyl-Induced Respiratory Depression in a Rat Plethysmography Model: Potency, Tolerance, and Sex Differences.

The opioid overdose death toll in the United States is an ongoing public health crisis. We characterized the magnitude and duration of respiratory depression, the leading cause of death in opioid overdose cases, induced by heroin or fentanyl and the development of tolerance in male and female rats. We used whole-body plethysmography to first establish dose-response curves by recording breathing for 60 minutes post-intravenous opioid injection. We then tested the development of respiratory tolerance to acute heroin or fentanyl over several weeks and to chronic fentanyl with acute fentanyl or heroin challenge. Heroin and fentanyl each provoked dose-dependent respiratory depression. Heroin caused prolonged (45-60 minute) respiratory depression in female and male rats, characterized by decreased frequency, tidal volume, and minute ventilation and increased inspiratory time and apneic pause. Fentanyl produced similar changes with a shorter duration (10-15 minutes). High-dose heroin or fentanyl produced robust respiratory depression that was slightly more severe in females and, when given intermittently (acute doses 2 to 3 weeks apart), did not lead to tolerance. In contrast, chronic fentanyl delivered with an osmotic minipump resulted in tolerance to acute fentanyl and heroin, characterized by a shorter duration of respiratory depression. This effect persisted during withdrawal in males only. Our model and experimental design will allow for investigation of the neurobiology of opioid-induced respiratory depression and for testing potential therapeutics to reverse respiratory depression or stimulate breathing. SIGNIFICANCE STATEMENT: Fentanyl was more potent and had shorter duration in producing respiratory depression than heroin in both sexes, whereas female rats were more sensitive than males to heroin-induced respiratory depression. Tolerance/cross-tolerance develops in chronic fentanyl administration but is minimized with long interadministration intervals.

Extended access to fentanyl vapor self-administration leads to addiction-like behaviors in mice: Blood chemokine/cytokine levels as potential biomarkers.

Rodent models are useful for understanding the mechanisms that underlie opioid addiction, but most preclinical studies have focused on rewarding and consummatory aspects of opioids without components of dependence-induced escalation of drug taking or seeking. We characterized several opioid-related behaviors in mice using a model of vaporized fentanyl self-administration. Male and female C57BL/6J mice were assigned to short-access (ShA; 1 h, nondependent) or long-access (LgA; 6 h, dependent) fentanyl vapor self-administration and subsequently tested in a battery of behavioral tests, followed by blood collection during withdrawal. Compared with mice in the ShA group, mice in the LgA group escalated their fentanyl intake, were more motivated to work to obtain the drug, exhibited greater hyperalgesia, and exhibited greater signs of naloxone-precipitated withdrawal. Principal component analysis indicated the emergence of two independent behavioral constructs: "intake/motivation" and "hyperalgesia/punished seeking." In mice in the LgA condition only, "hyperalgesia/punished seeking" was associated with plasma levels of proinflammatory interleukin-17 (IL-17), chemokine (C-C motif) ligand 4 (CCL-4), and tumor necrosis factor α (TNF-α). Overall, the results suggest that extended access to opioids leads to addiction-like behavior, and some constructs that are associated with addiction-like behavior may be associated with levels of the proinflammatory cytokines/chemokines IL-17, TNF-α, and CCL-4 in blood.

The neuroimmune system - Where aging and excess alcohol intersect.

As the percentage of the global population over age 65 grows, and with it a subpopulation of individuals with alcohol use disorder (AUD), understanding the effect of alcohol on the aged brain is of utmost importance. Neuroinflammation is implicated in both natural aging as well as alcohol use, and its role in alterations to brain morphology and function may be exacerbated in aging individuals who drink alcohol to excess. The neuroimmune response to alcohol in aging is complex. The few studies investigating this issue have reported heightened basal activity and either hypo- or hyper-reactivity to an alcohol challenge. This review of preclinical research will first introduce key players of the immune system, then explore changes in neuroimmune function with aging or alcohol alone, with discussion of vulnerable brain regions, changes in cytokines, and varied reactions of microglia and astrocytes. We will then consider different levels of alcohol exposure, relevant animal models of AUD, and neuroimmune activation by alcohol across the lifespan. By identifying key findings, challenges, and targets for future research, we hope to bring more attention and resources to this underexplored area of inquiry.

The interplay between sperm-mediated and care-mediated paternal effects in threespine sticklebacks.

The environment experienced by one generation can influence the phenotypes of future generations. Because parental cues can be conveyed to offspring at multiple points in time, ranging from fertilization to posthatching/parturition, offspring can potentially receive multiple cues from their parents via different mechanisms. We have relatively little information regarding how different mechanisms operate in isolation and in tandem, but it is possible, for example, that offspring phenotypes induced by nongenetic changes to gametes may be amplified by, mitigated by, or depend upon parental care. Here, we manipulated paternal experience with predation risk prior to fertilization in threespine stickleback, Gasterosteus aculeatus, and then examined the potential of paternal care to mitigate and/or amplify sperm-mediated paternal effects. Specifically, we compared (1) offspring of predator-exposed fathers who were reared without paternal care, (2) offspring of predator-exposed fathers who were reared with paternal care, (3) offspring of control (unexposed) fathers who were reared without paternal care and (4) offspring of control fathers who were reared with paternal care. We found that offspring of predator-exposed fathers were less active and had higher cortisol following a simulated predator attack. Although predator-exposed males shifted their paternal care behaviours - reduced fanning early in egg development and increased fanning right before egg hatching compared to control males - this shift in paternal behavior did not appear to affect offspring traits. This suggests that paternal care neither amplifies nor compensates for these phenotypic effects induced by sperm and that nongenetic changes induced by sperm may occur independently of nongenetic changes induced by paternal care. Overall, these results underscore the importance of considering how parents may have multiple nongenetic mechanisms by which they can influence offspring.

κ-Opioid receptor antagonism reverses heroin withdrawal-induced hyperalgesia in male and female rats.

Although opioids are potent analgesics, a consequence of chronic opioid use is hyperalgesia during withdrawal, which may contribute to opioid misuse. Dynorphin, the endogenous ligand of κ-opioid receptors (KORs), is upregulated in opioid-dependent rats and in animal models of chronic pain. However, the role of KORs in opioid withdrawal-induced hyperalgesia remains to be determined. We hypothesized that KOR antagonism would reverse opioid withdrawal-induced hyperalgesia in opioid-dependent rats. Male and female Wistar rats received daily injections of heroin (2-6 mg/kg, SC) and were tested for mechanical sensitivity in the electronic von Frey test 4-6 h into withdrawal. Female rats required significantly more heroin than male rats to reach comparable levels of both heroin-induced analgesia and hyperalgesia (6 mg/kg vs. 2 mg/kg). Once hyperalgesia was established, we tested the effects of the KOR antagonists nor-binaltorphimine (norBNI; 30 mg/kg, SC) and 5'-guanidinonaltrindole (5'GNTI; 30 mg/kg, SC). When the animals continued to receive their daily heroin treatment (or saline treatment in the repeated saline group) five times per week throughout the experiment, both KOR antagonists reversed heroin withdrawal-induced hyperalgesia. The anti-hyperalgesia effect of norBNI was more prolonged in males than in females (14 days vs. 7 days), whereas 5'GNTI had more prolonged effects in females than in males (14 days vs. 4 days). The behavioral effects of 5'GNTI coincided with higher 5'GNTI levels in the brain than in plasma when measured at 24 h, whereas 5'GNTI did not reverse hyperalgesia at 30 min posttreatment when 5'GNTI levels were higher in plasma than in the brain. Finally, we tested the effects of 5'GNTI on naloxone-induced and spontaneous signs of opioid withdrawal and found no effect in either male or female rats. These findings indicate a functional role for KORs in heroin withdrawal-induced hyperalgesia that is observed in rats of both sexes.

Drug addiction co-morbidity with alcohol: Neurobiological insights.

Addiction is a chronic disorder that consists of a three-stage cycle of binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. These stages involve, respectively, neuroadaptations in brain circuits involved in incentive salience and habit formation, stress surfeit and reward deficit, and executive function. Much research on addiction focuses on the neurobiology underlying single drug use. However, alcohol use disorder (AUD) can be co-morbid with substance use disorder (SUD), called dual dependence. The limited epidemiological data on dual dependence indicates that there is a large population of individuals suffering from addiction who are dependent on more than one drug and/or alcohol, yet dual dependence remains understudied in addiction research. Here, we review neurobiological data on neurotransmitter and neuropeptide systems that are known to contribute to addiction pathology and how the involvement of these systems is consistent or divergent across drug classes. In particular, we highlight the dopamine, opioid, corticotropin-releasing factor, norepinephrine, hypocretin/orexin, glucocorticoid, neuroimmune signaling, endocannabinoid, glutamate, and GABA systems. We also discuss the limited research on these systems in dual dependence. Collectively, these studies demonstrate that the use of multiple drugs can produce neuroadaptations that are distinct from single drug use. Further investigation into the neurobiology of dual dependence is necessary to develop effective treatments for addiction to multiple drugs.

Sex-specific plasticity across generations II: Grandpaternal effects are lineage specific and sex specific.

Transgenerational plasticity (TGP) occurs when the environment encountered by one generation (F0) alters the phenotypes of one or more future generations (e.g. F1 and F2). Sex selective TGP, via specific lineages or to only male or female descendants, has been underexplored in natural systems, and may be adaptive if it allows past generations to fine-tune the phenotypes of future generations in response to sex-specific life-history strategies. We sought to understand if exposing males to predation risk can influence grandoffspring via sperm in three-spined stickleback Gasterosteus aculeatus. We specifically tested the hypothesis that grandparental effects are transmitted in a sex-specific way down the male lineage, from paternal grandfathers to F2 males. We reared F1 offspring of unexposed and predator-exposed F0 males under 'control' conditions and used them to generate F2s with control grandfathers, a predator-exposed maternal grandfather (i.e. predator-exposed F0 males to F1 daughters to F2s), a predator-exposed paternal grandfather (i.e. predator-exposed F0 males to F1 sons to F2s) or two predator-exposed grandfathers. We then assayed male and female F2s for a variety of traits related to antipredator defence. We found little evidence that transgenerational effects were mediated to only male descendants via the paternal lineage. Instead, grandpaternal effects depended on lineage and were mediated largely across sexes, from F1 males to F2 females and from F1 females to F2 males. When their paternal grandfather was exposed to predation risk, female F2s were heavier and showed a reduced change in behaviour in response to a simulated predator attack relative to grandoffspring of control, unexposed grandparents. In contrast, male F2s showed reduced antipredator behaviour when their maternal grandfather was exposed to predation risk. However, these patterns were only evident when one grandfather, but not both grandfathers, was exposed to predation risk, suggesting the potential for non-additive interactions across lineages. If sex-specific and lineage effects are common, then grandparental effects are likely underestimated in the literature. These results draw attention to the importance of sex-selective inheritance of environmental effects and raise new questions about the proximate and ultimate causes of selective transmission across generations.