Andreas B. Wulff

The claustrum is among the most highly interconnected structures in the mammalian brain, with reciprocal projections spanning the cortical mantle and a growing roster of functions that range from sleep regulation to attentional control. Here we review current understanding of claustral circuit organization and function, synthesizing findings from anatomical, physiological, computational, and behavioral studies. We argue that a unifying logic underlies the apparent diversity of claustral functions: the claustrum operates through projection-defined subnetworks whose input selectivity, output targets, and local circuit interactions collectively determine their functional contribution and whose impact on cortical targets varies with arousal and ongoing brain state. Corticoclaustral connectivity is organized into matched inputoutput modules, local inhibitory circuits confer spatial filtering properties that amplify strong cortical signals while suppressing weaker ones and claustral output populations differ in their laminar targets, postsynaptic cell-type preferences, and molecular properties, producing diverse and context-dependent effects on cortical dynamics. These circuit properties translate into claustral regulation of cortical population dynamics across multiple timescales, from the moment-to-moment coordination of brain states to the consolidation of frontal control strategies. In pain and addiction, specific claustral projection populations are selectively dysregulated, and distinct subnetworks exert opposing influences on nociceptive sensitivity and drug-related behavior according to their cortical targets. Together, these findings position the claustrum as a modular subcortical hub for the selective regulation of distributed cortical networks and identify it as a candidate therapeutic target for disorders characterized by dysregulated cortical network activity, including chronic pain, addiction, and impulsivity.

Cognitive impairment is a major component of Alcohol Use Disorder. Optimal cognitive performance requires anterior cingulate input activation of the claustrum, a subcortical nucleus that orchestrates cortical activity. Yet the impact of chronic alcohol exposure on the ability for the anterior cingulate cortex to drive activity of claustrum projection neuron subtypes is unknown. In adult male and female mice, we found that the majority of non-burst firing Type 1 claustrum projection neurons did not express the vesicular glutamate transporter 2 (VGLUT2), while the majority of burst firing Type 2 projection neurons were VGLUT2-expressing. Following chronic intermittent vaporized ethanol exposure (CIE), we found that both Type 1 and VGLUT2-non-expressing neurons exhibited increased responsivity to anterior cingulate cortex input activation that was mediated by increased postsynaptic membrane excitability. In contrast, Type 2 and VGLUT2-expressing projection neurons exhibited increased responsivity to anterior cingulate cortex input due to strengthened pre- and post-synaptic transmission mechanisms. Altogether, we uncovered a hyper-excitatory drive of the claustrum by the anterior cingulate cortex following chronic alcohol exposure. The data provide a foundational resource for the complex effects of chronic alcohol exposure on the claustrum, a critical cognitive control nucleus.

The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type–specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion, and enhanced acquisition of reward learning in comparison with all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison with their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward learning.

Trauma and chronic stress exposure are the strongest predictors of lifetime neuropsychiatric disease presentation. These disorders often have significant sex biases, with females having higher incidences of affective disorders such as major depression, anxiety, and PTSD. Understanding the mechanisms by which stress exposure heightens disease vulnerability is essential for developing novel interventions. Current rodent stress models consist of a battery of sensory, homeostatic, and psychological stressors that are ultimately integrated by corticotropin-releasing factor (CRF) neurons to trigger corticosteroid release. These stress paradigms, however, often differ between research groups in the type, timing, and duration of stressors utilized. These inconsistencies, along with the variability of individual animals’ perception and response to each stressor, present challenges for reproducibility and translational relevance. Here, we hypothesized that a more direct approach using chemogenetic activation of CRF neurons would recapitulate the effects of traditional stress paradigms and provide a high-throughput method for examining stress-relevant phenotypes. Using a transgenic approach to express the Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) receptor hM3Dq in CRF-neurons, we found that the DREADD ligand clozapine-N-oxide (CNO) produced an acute and robust activation of the hypothalamic-pituitary-adrenal (HPA) axis, as predicted. Interestingly, chronic treatment with this method of direct CRF activation uncovered a novel sex-specific dissociation of glucocorticoid levels with stress-related outcomes. Despite hM3Dq-expressing females producing greater corticosterone levels in response to CNO than males, hM3Dq-expressing males showed significant typical physiological stress sensitivity with reductions in body and thymus weights. hM3Dq-expressing females while resistant to the physiological effects of chronic CRF activation, showed significant increases in baseline and fear-conditioned freezing behaviors. These data establish a novel mouse model for interrogating stress-relevant phenotypes and highlight sex-specific stress circuitry distinct for physiological and limbic control that may underlie disease risk.

The sucrose preference test (SPT) is a widely used preclinical assay for studying stress-sensitive reward behaviors and antidepressant treatments in rodents, with some face, construct, and predictive validity. However, while stress-induced loss of sucrose preference is presumed to reflect an anhedonic-like state, little detail is known about what behavioral components may influence performance in the SPT in stress-naive or stressed rodents. We analyzed the licking microstructure of mice during the SPT to evaluate how preference is expressed and lost following chronic stress. In stress-naive mice, preference is expressed as both longer and more numerous drinking bouts at the sucrose bottle, compared with the water bottle. We also found evidence that memory of the sucrose bottle location supports preference. Through manipulations of the caloric content of the sweetener or caloric need of the mouse, we found that energy demands and satiety signals do not affect either preference or the underlying drinking behavior. Both acute and chronic stress impaired sucrose location memory and reduced the number of drinking bouts at the sucrose bottle, the latter of which explained the loss of sucrose preference in stress susceptible mice compared with stress resilient mice. Female mice generally exhibited similar drinking behavior to male mice but may be less susceptible to chronic stress and display better memory performance than male mice, both before and after chronic stress. Our data suggest that chronic stress inhibits a sucrose preference by reducing reward seeking behavior without affecting palatability.

Summary: Opioid discontinuation generates a withdrawal syndrome marked by a negative emotional state. Increased anxiety and dysphoria during opioid discontinuation are a significant barrier to achieving long-term abstinence in opioid-dependent individuals. Adaptations in brain-reward circuitry are implicated in the opioid abstinence syndrome, but current knowledge is limited to changes following natural and semi-synthetic opioids. Here we report abstinence from the synthetic opioid fentanyl engenders structural, functional, and molecular plasticity in nucleus accumbens neuron subtypes (MSNs) that mediate negative emotional behaviors. We show fentanyl abstinence causes dendritic atrophy and increased excitatory drive exclusive to D1-receptor containing MSNs. Using subtype specific RNAseq and Weighted Gene Co-Expression Network Analysis, we identified molecular signatures of fentanyl abstinence in MSN subtypes. We found a network of co-expressed genes downregulated selectively in D1-MSNs, and transcriptionally co-regulated by E2F1. We show targeting abstinence-induced molecular changes protects D1-MSNs from maladaptive plasticity and alleviates negative emotional behaviors after fentanyl abstinence.

Chronic stress can increase the risk of developing a substance use disorder in vulnerable individuals. Numerous models have been developed to probe the underlying neurobiological mechanisms, however most prior work has been restricted to male rodents, conducted only in rats, or introduces physical injury that can complicate opioid studies. Here we sought to establish how chronic psychosocial stress influences fentanyl consumption in male and female C57BL/6 mice. We used chronic social defeat stress (CSDS), or the modified vicarious chronic witness defeat stress (CWDS), and used social interaction to stratify mice as stress-susceptible or resilient. We then subjected mice to a 15 day fentanyl drinking paradigm in the home cage that consisted of alternating forced and choice periods with increasing fentanyl concentrations. Male mice susceptible to either CWDS or CSDS consumed more fentanyl relative to unstressed mice, and exhibited increased preference for fentanyl. CWDS-susceptible female mice did not differ from unstressed mice during the forced periods, but showed increased preference for fentanyl. We also found decreased expression of nucleus accumbens Rho GTPases in male, but not female mice following stress and fentanyl drinking. We also compare fentanyl drinking behavior in mice that had free access to plain water throughout. Our results indicate that stress-sensitized fentanyl consumption is dependent on both sex and behavioral outcomes to stress.

Significance: Psychedelic compounds, such as psilocybin, have beneficial actions in several psychiatric diseases. They also produce strong alterations of consciousness, which may be a barrier to their widespread use. We found that psilocybin has fast-acting antidepressant-like properties in mice. Using multiple assays of hedonic behavior and an antagonist of prohallucinatory 5-HT2A receptors, we further suggest that altered perception may not be necessary for its therapeutic actions. We also showed that psilocybin strengthens connections between brain cells in regions important for processing rewards and emotions. These experiments suggest that it may be possible to retain the beneficial actions of psilocybin while minimizing the alterations in consciousness, thereby accelerating its use in the clinic.