Neuroscience in addiction research.

The prevention and treatment of addiction (moderate to severe substance use disorder-SUD) have remained challenging because of the dynamic and complex interactions between multiple biological and social determinants that shape SUD. The pharmacological landscape is ever changing and the use of multiple drugs is increasingly common, requiring an unraveling of pharmacological interactions to understand the effects. There are different stages in the trajectory from drug use to addiction that are characterized by distinct cognitive and emotional features. These are directed by different neurobiological processes that require identification and characterization including those that underlie the high co-morbidity with other disorders. Finally, there is substantial individual variability in the susceptibility to develop SUD because there are multiple determinants, including genetics, sex, developmental trajectories and times of drug exposures, and psychosocial and environmental factors including commercial determinants that influence drug availability. Elucidating how these factors interact to determine risk is essential for identifying the biobehavioral basis of addiction and developing prevention and treatment strategies. Basic research is tasked with addressing each of these challenges. The recent proliferation of technological advances that allow for genetic manipulation, visualization of molecular reactions and cellular activity in vivo, multiscale whole brain mapping across the life span, and the mining of massive data sets including multimodality human brain imaging are accelerating our ability to understand how the brain functions and how drugs influence it. Here, we highlight how the application of these tools to the study of addiction promises to illuminate its neurobiological basis and guide strategies for prevention and treatment.

Sex-specific alterations in corticotropin-releasing factor regulation of coerulear-cortical network activity.

The locus coeruleus (LC)-norepinephrine system is a stress responsive system that regulates arousal and cognitive functions through extensive projections, including to the prefrontal cortex. LC-cortical circuits are activated by stressors, and this activation is thought to contribute to stress-induced impairments in executive function. Because corticotropin-releasing factor (CRF) is a mediator of stress-induced LC activation, we examined the effects of CRF administered into the LC of male and female rats on network activity of two functionally distinct regions of the PFC, the medial PFC (mPFC) and the orbitofrontal cortex (OFC). Network activity, measured as local field potentials, was recorded in awake animals before and after intra-LC infusion of aCSF or CRF (2 or 20 ng). CRF had qualitatively distinct effects on network activity in males and females with respect to dose, region and timecourse. CRF (20 ng) produced a prominent theta oscillation (7-9 Hz) selectively in female rats shortly after LC infusion and 20 min later. In contrast, in male rats, CRF (2 and 20 ng) decreased the amplitude of power in the 4-6 Hz range in the mPFC 10 min after injection. Lastly, CRF (20 ng) increased mPFC-OFC coherence in females and decreased mPFC-OFC coherence in males. In sum, these results show sex differences in CRF modulation of the LC-norepinephrine system that regulates prefrontal cortical networks, which may underlie sex differences in cognitive and behavioral responses to stress.

Somatostatin Neurons in the Mouse Pontine Nucleus Activate GABAA Receptor Mediated Synaptic Currents in Locus Coeruleus Neurons.

The pontine nuclei comprising the locus coeruleus (LC) and Barrington's nucleus (BRN) amongst others form the neural circuitry(s) that coordinates arousal and voiding behaviors. However, little is known about the synaptic connectivity of neurons within or across these nuclei. These include corticotropin-releasing factor (CRF+) expressing neurons in the BRN that control bladder contraction and somatostatin expressing (SST+) neurons whose role in this region has not been discerned. To determine the synaptic connectivity of these neurons, we employed optogenetic stimulation with recordings from BRN and LC neurons in brain stem slices of channelrhodopsin-2 expressing SST or CRF neurons. Optogenetic stimulation of CRF+ BRN neurons of Crf Cre ;chr2-yfp mice had little effect on either CRF+ BRN neurons, CRF- BRN neurons, or LC neurons. In contrast, in Sst Cre ;chr2-yfp mice light-activated inhibitory postsynaptic currents (IPSCs) were reliably observed in a majority of LC but not BRN neurons. The GABAA receptor antagonist, bicuculline, completely abolished the light-induced IPSCs. To ascertain if these neurons were part of the neural circuitry that controls the bladder, the trans-synaptic tracer, pseudorabies virus (PRV) was injected into the bladder wall of Crf Cre ;tdTomato or Sst Cre ;tdTomato mice. At 68-72 h post-viral infection, PRV labeled neurons were present only in the BRN, being preponderant in CRF+ neurons with few SST+ BRN neurons labeled from the bladder. At 76 and 96 h post-virus injection, increased labeling was observed in both BRN and LC neurons. Our results suggest SST+ neurons rather than CRF+ neurons in BRN can regulate the activity of LC neurons.

Corticotropin-Releasing Hormone from the Pontine Micturition Center Plays an Inhibitory Role in Micturition.

Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.

Presynaptic Inhibitory Effects of Acetylcholine in the Hippocampus: A 40-Year Evolution of a Serendipitous Finding.

Cholinergic regulation of hippocampal circuit activity has been an active area of neurophysiological research for decades. The prominent cholinergic innervation of intrinsic hippocampal circuitry, potent effects of cholinomimetic drugs, and behavioral responses to cholinergic modulation of hippocampal circuitry have driven investigators to discover diverse cellular actions of acetylcholine in distinct sites within hippocampal circuitry. Further research has illuminated how these actions organize circuit activity to optimize encoding of new information, promote consolidation, and coordinate this with recall of prior memories. The development of the hippocampal slice preparation was a major advance that accelerated knowledge of how hippocampal circuits functioned and how acetylcholine modulated these circuits. Using this preparation in the early 1980s, we made a serendipitous finding of a novel presynaptic inhibitory effect of acetylcholine on Schaffer collaterals, the projections from CA3 pyramidal neurons to dendrites of CA1 pyramidal cells. We characterized this effect at cellular and pharmacological levels, published the findings in the first volume of the Journal of Neuroscience, and proceeded to pursue other scientific directions. We were surprised and thrilled to see that, nearly 40 years later, the paper is still being cited and downloaded because the data became an integral piece of the foundation of the science of cholinergic regulation of hippocampal function in learning and memory. This Progressions article is a story of how single laboratory findings evolve through time to be confirmed, challenged, and reinterpreted by other laboratories to eventually become part of the basis of fundamental concepts related to important brain functions.

Sex differences in µ-opioid regulation of coerulear-cortical transmission.

Stress-induced activation of locus coeruleus (LC)-norepinephrine (NE) projections to the prefrontal cortex are thought to promote cognitive responses to stressors. LC activation by stressors is modulated by endogenous opioids that restrain LC activation and facilitate a return to baseline activity upon stress termination. Sex differences in this opioid influence could be a basis for sex differences in stress vulnerability. Consistent with this, we recently demonstrated that µ-opioid receptor (MOR) expression is decreased in the female rat LC compared to the male LC, and this was associated with sexually distinct consequences of activating MOR in the LC on cognitive flexibility. Given that the LC-NE system affects cognitive flexibility through its projections to the medial prefrontal cortex (mPFC), the present study quantified and compared the effects of LC-MOR activation on mPFC neural activity in male and female rats. Local field potential (LFPs) were recorded from the mPFC of freely behaving male and female rats before and following local LC microinjection of the MOR agonist, DAMGO, or vehicle. Intra-LC DAMGO altered the LFP power spectrum selectively in male but not female rats, resulting in a time-dependent increase in the power in delta and alpha frequency bands. LC microinfusion of ACSF had no effect on either sex. Together, the results are consistent with previous evidence for decreased MOR function in the female rat LC and demonstrate that this translates to a diminished effect on cortical activity that can account for sex differences in cognitive consequences. Decreased LC-MOR function in females could contribute to greater stress-induced activation of the LC and increased vulnerability of females to hyperarousal symptoms of stress-related neuropsychiatric pathologies.

Polysubstance use in the U.S. opioid crisis.

Interventions to address the U.S. opioid crisis primarily target opioid use, misuse, and addiction, but because the opioid crisis includes multiple substances, the opioid specificity of interventions may limit their ability to address the broader problem of polysubstance use. Overlap of opioids with other substances ranges from shifts among the substances used across the lifespan to simultaneous co-use of substances that span similar and disparate pharmacological categories. Evidence suggests that nonmedical opioid users quite commonly use other drugs, and this polysubstance use contributes to increasing morbidity and mortality. Reasons for adding other substances to opioids include enhancement of the high (additive or synergistic reward), compensation for undesired effects of one drug by taking another, compensation for negative internal states, or a common predisposition that is related to all substance consumption. But consumption of multiple substances may itself have unique effects. To achieve the maximum benefit, addressing the overlap of opioids with multiple other substances is needed across the spectrum of prevention and treatment interventions, overdose reversal, public health surveillance, and research. By addressing the multiple patterns of consumption and the reasons that people mix opioids with other substances, interventions and research may be enhanced.

The Emerging Science of Interoception: Sensing, Integrating, Interpreting, and Regulating Signals within the Self.

Interoception refers to the representation of the internal states of an organism, and includes the processes by which it senses, interprets, integrates, and regulates signals from within itself. This review presents a unified research framework and attempts to offer definitions for key terms to describe the processes involved in interoception. We elaborate on these definitions through illustrative research findings, and provide brief overviews of central aspects of interoception, including the anatomy and function of neural and non-neural pathways, diseases and disorders, manipulations and interventions, and predictive modeling. We conclude with discussions about major research gaps and challenges.

Locus coeruleus: a new look at the blue spot.

The locus coeruleus (LC), or 'blue spot', is a small nucleus located deep in the brainstem that provides the far-reaching noradrenergic neurotransmitter system of the brain. This phylogenetically conserved nucleus has proved relatively intractable to full characterization, despite more than 60 years of concerted efforts by investigators. Recently, an array of powerful new neuroscience tools have provided unprecedented access to this elusive nucleus, revealing new levels of organization and function. We are currently at the threshold of major discoveries regarding how this tiny brainstem structure exerts such varied and significant influences over brain function and behaviour. All LC neurons receive inputs related to autonomic arousal, but distinct subpopulations of those neurons can encode specific cognitive processes, presumably through more specific inputs from the forebrain areas. This ability, combined with specific patterns of innervation of target areas and heterogeneity in receptor distributions, suggests that activation of the LC has more specific influences on target networks than had initially been imagined.

Opioid Research: Past and Future.

The International Narcotics Research Conference (INRC) has a rich history of uniting the most creative minds across the fields of chemistry, pharmacology, physiology, and behavior in the study of opioids. The Conference has been a forum for sharing knowledge, discussing controversies, introducing innovative research, and announcing landmark discoveries. In this perspective for the Special Issue commemorating the 50th anniversary of the Conference we briefly highlight how the INRC has guided the evolution of opioid research and how new tools, models, and approaches are facilitating our ability to achieve the goals of preventing and treating opioid use disorder. SIGNIFICANCE STATEMENT: This perspective highlights the important role that the International Narcotics Research Conference has played in the evolution of opioid research and emphasizes how technological advances are facilitating research toward the goals of preventing and treating opioid use disorder.

Age- and sex-dependent impact of repeated social stress on morphology of rat prefrontal cortex pyramidal neurons.

Chronic stress can lead to psychiatric illness characterized by impairments of executive function, implicating the prefrontal cortex as a target of stress-related pathology. Previous studies have shown that various types of chronic stress paradigms reduce dendritic branching, length and spines of medial prefrontal cortex (mPFC) pyramidal neurons. However, these studies largely focused on layer II/III pyramidal neurons in adult male rats with less known about layer V, the site of projection neurons. Because the prefrontal cortex develops throughout adolescence, stress during adolescence may have a greater impact on structure and function than stress occurring during adulthood. Furthermore, females display greater risk of stress-related psychiatric disorders, indicating sex-specific responses to stress. In this study, male and female adolescent (42-48 days old, 4 rats per group) or adult (68-72 days old, 4 rats per group) Sprague-Dawley rats were exposed to 5 days of repeated social stress in the resident-intruder paradigm or control manipulation. We examined dendritic morphology of cells in the mPFC in both layer II/III and Layer V. Repeated social stress resulted in decreased dendritic branching in layer II/III apical dendrites regardless of sex or age. In apical layer V dendrites, stress increased branching in adult males but decreased it in all other groups. Stress resulted in a decrease in dendritic spines in layer V apical dendrites for male adolescents and female adults, and this was mostly due to a decrease in filopodial and mushroom spines for male adolescents, but stubby spines for female adults. In sum, these results suggest that repeated stress reduces complexity and synaptic connectivity in adolescents and female adults in both input and output layers of prelimbic mPFC, but not in male adults. These changes may represent a potential underlying mechanism as to why adolescents and females are more susceptible to the negative cognitive effects of repeated or chronic stress.

Corticotropin-Releasing Factor (CRF) circuit modulation of cognition and motivation.

The neuropeptide, corticotropin-releasing factor (CRF), is a key modulator of physiological, endocrine, and behavioral responses during stress. Dysfunction of the CRF system has been observed in stress-related affective disorders including post-traumatic stress disorder, depression, and anxiety. Beyond affective symptoms, these disorders are also characterized by impaired cognition, for which current pharmacological treatments are lacking. Thus, there is a need for pro-cognitive treatments to improve quality of life for individuals suffering from mental illness. In this review, we highlight research demonstrating that CRF elicits potent modulatory effects on higher-order cognition via actions within the prefrontal cortex and subcortical monoaminergic and cholinergic systems. Additionally, we identify questions for future preclinical research on this topic, such as the need to investigate sex differences in the cognitive and microcircuit actions of CRF, and whether CRF may represent a pharmacological target to treat cognitive dysfunction. Addressing these questions will provide new insight into pathophysiology underlying cognitive dysfunction and may lead to improved treatments for neuropsychiatric disorders.

Neurochemically distinct circuitry regulates locus coeruleus activity during female social stress depending on coping style.

Stress-related psychiatric diseases are nearly twice as prevalent in women compared to men. We recently showed in male rats that the resident-intruder model of social stress differentially engages stress-related circuitry that regulates norepinephrine-containing neurons of the locus coeruleus (LC) depending on coping strategy as determined by the latency to assume a defeat posture. Here, we determined whether this social stress had similar effects in female rats. LC afferents were retrogradely labeled with Fluorogold (FG) and rats had one or five daily exposures to an aggressive resident. Sections through the nucleus paragigantocellularis (PGi), a source of enkephalin (ENK) afferents to the LC, and central nucleus of the amygdala (CeA), a source of corticotropin-releasing factor (CRF) afferents to the LC, were processed for immunocytochemical detection of c-fos, a marker of neuronal activity, FG and ENK or CRF. Like male rats, female rats defeated with a relatively short latency (SL) in response to a single resident-intruder exposure and showed significant c-fos activation of LC neurons, PGi-ENK LC afferents, and CeA-CRF-LC afferents. With repeated exposure, some rats exhibited a long latency to defeat (LL). LC neurons and CeA-CRF-LC afferents were activated in SL rats compared to control and LL, whereas PGi-ENK LC afferents were not. Conversely, in LL rats, PGi-ENK LC and CeA-CRF-LC afferents were activated compared to controls but not LC neurons. CRF type 1 receptor (CRF1) and µ-opioid receptor (MOR) expression levels in LC were decreased in LL rats. Finally, electron microscopy showed a relative increase in MOR on the plasma membrane of LL rats and a relative increase in CRF1 on the plasma membrane of SL rats. Together, these results suggest that as is the case for males, social stress engages divergent circuitry to regulate the LC in female rats depending on coping strategy, with a bias towards CRF influence in more subordinate rats and opioid influence in less subordinate rats.

Sex differences in morphine-induced trafficking of mu-opioid and corticotropin-releasing factor receptors in locus coeruleus neurons.

The locus coeruleus (LC)-norepinephrine (NE) system is a key nucleus in which endogenous opioid and stress systems intersect to regulate the stress response. LC neurons of male rats become sensitized to stress following chronic morphine administration. Whether sex dictates this pattern of opioid-induced plasticity has not been demonstrated. Delineating the neurobiological adaptations produced by chronic opioids will enhance our understanding of stress vulnerability in opioid-dependent individuals, and may reveal how stress negatively impacts addiction recovery. In the present study, the effect of chronic morphine on the subcellular distribution of mu-opioid (MOR) and CRF receptors (CRFR) was investigated in the LC of male and female rats using immunoelectron microscopy. Results showed that placebo-treated females exhibited higher MOR and CRFR cytoplasmic distribution ratio when compared to placebo-treated males. Chronic morphine exposure induced a shift in the distribution of MOR immunogold-silver particles from the plasma membrane to the cytoplasm selectively in male LC neurons. Interestingly, chronic morphine exposure induced CRFR recruitment to the plasma membrane of both male and female LC neurons. These findings provide a potential mechanism by which chronic opioid administration increases stress vulnerability in males and females via an increase in surface availability of CRFR in LC neurons. However, our results also support the notion that cellular adaptations to chronic opioids differ across the sexes as redistribution of MOR following morphine exposure was only observed in male LC neurons.

Untangling the complexity of opioid receptor function.

Mu opioid receptor agonists are among the most powerful analgesic medications but also among the most addictive. The current opioid crisis has energized a quest to develop opioid analgesics that are devoid of untoward effects. Since their discovery in the 1970's, there have been major advances in our understanding of the endogenous opioid systems that these drugs target. Yet many questions remain and the development of non-addictive opioid analgesics has not been achieved. However, access to new molecular, genetic and computational tools have begun to elucidate the structural dynamics of opioid receptors, the scaffolding that links them to intracellular signaling cascades, their cellular trafficking and the distinct ways that various opioid drugs modify them. This mini-review highlights some of the chemical and pharmacological findings and new perspectives that have arisen from studies using these tools. They reveal multiple layers of complexity of opioid receptor function, including a spatiotemporal specificity in opioid receptor-induced cellular signaling, ligand-directed biased signaling, allosteric modulation of ligand interactions, heterodimerization of different opioid receptors, and the existence of slice variants with different ligand specificity. By untangling these layers, basic research into the chemistry and pharmacology of opioid receptors is guiding the way towards deciphering the mysteries of tolerance and physical dependence that have plagued the field and is providing a platform for the development of more effective and safer opioids.

Female psychopharmacology matters! Towards a sex-specific psychopharmacology.

There is increasing recognition that women have a higher prevalence of certain psychiatric illnesses, and a differential treatment response and course of illness compared to men. Additionally, clinicians deal with a number of disorders like premenstrual syndrome, premenstrual dysphoric disorder, and postpartum depression, which affect women specifically and for which treatment and biological pathways are still unclear. In this article we highlight recent research which suggests that different biological mechanisms may underlie sex differences in responsiveness to stress. Sex differences are evident at the receptor level; where the corticotropin-releasing factor receptor shows differential coupling to adaptor proteins in males and females. The neuropeptide oxytocin also shows sex-specific effects in a range of social behaviors. It may act as a biomarker in post-traumatic stress disorder where sex differences are evident. Studies in women using hormonal contraception show that some of these oxytocin-mediated effects are likely influenced by sex hormones. In female rats rapid changes in circulating progesterone levels are associated with exaggerated behavioral responses to mild stress and blunted responses to benzodiazepines that could be prevented by acute treatment with low-dose fluoxetine. Perceived barriers in research on women have hindered progress. The development of a sex-specific psychopharmacology as a basis for translating this type of research into clinical practice is vital to improve treatment outcomes for women.