Systemic kappa opioid receptor antagonism accelerates reinforcement learning via augmentation of novelty processing in male mice.

Selective inhibition of kappa opioid receptors (KORs) is highly anticipated as a pharmacotherapeutic intervention for substance use disorders and depression. The accepted explanation for KOR antagonist-induced amelioration of aberrant behaviors posits that KORs globally function as a negative valence system; antagonism thereby blunts the behavioral influence of negative internal states such as anhedonia and negative affect. While effects of systemic KOR manipulations have been widely reproduced, explicit evaluation of negative valence as an explanatory construct is lacking. Here, we tested a series of falsifiable hypotheses generated a priori based on the negative valence model by pairing reinforcement learning tasks with systemic pharmacological KOR blockade in male C57BL/6J mice. The negative valence model failed to predict multiple experimental outcomes: KOR blockade accelerated contingency learning during both positive and negative reinforcement without altering innate responses to appetitive or aversive stimuli. We next proposed novelty processing, which influences learning independent of valence, as an alternative explanatory construct. Hypotheses based on novelty processing predicted subsequent observations: KOR blockade increased exploration of a novel, but not habituated, environment and augmented the reinforcing efficacy of novel visual stimuli in a sensory reinforcement task. Together, these results revise and extend long-standing theories of KOR system function.

Recurrent activity within microcircuits of macaque dorsolateral prefrontal cortex tracks cognitive flexibility.

Human and non-human primate data clearly implicate the dorsolateral prefrontal cortex (dlPFC) as critical for advanced cognitive functions 1,2 . It is thought that intracortical synaptic architectures within dlPFC are the integral neurobiological substrate that gives rise to these processes, including working memory, inferential reasoning, and decision-making 3-7 . In the prevailing model, each cortical column makes up one fundamental processing unit composed of dense intrinsic connectivity, conceptualized as the 'canonical' cortical microcircuit 3,8 . Each cortical microcircuit receives sensory and cognitive information from a variety of sources which are represented by sustained activity within the microcircuit, referred to as persistent or recurrent activity 4,9 . Via recurrent connections within the microcircuit, activity can propagate for a variable length of time, thereby allowing temporary storage and computations to occur locally before ultimately passing a transformed representation to a downstream output 4,5,10 . Competing theories regarding how microcircuit activity is coordinated have proven difficult to reconcile in vivo where intercortical and intracortical computations cannot be fully dissociated 5,9,11,12 . Here, we interrogated the intrinsic features of isolated microcircuit networks using high-density calcium imaging of macaque dlPFC ex vivo . We found that spontaneous activity is intrinsically maintained by microcircuit architecture, persisting at a high rate in the absence of extrinsic connections. Further, using perisulcal stimulation to evoke persistent activity in deep layers, we found that activity propagates through stochastically assembled intracortical networks, creating predictable population-level events from largely non-overlapping ensembles. Microcircuit excitability covaried with individual cognitive performance, thus anchoring heuristic models of abstract cortical functions within quantifiable constraints imposed by the underlying synaptic architecture.

Behavioral and brain anatomical analysis of Foxg1 heterozygous mice.

FOXG1 Syndrome (FS) is a devastating neurodevelopmental disorder that is caused by a heterozygous loss-of-function (LOF) mutation of the FOXG1 gene, which encodes a transcriptional regulator important for telencephalic brain development. People with FS have marked developmental delays, impaired ambulation, movement disorders, seizures, and behavior abnormalities including autistic features. Current therapeutic approaches are entirely symptomatic, however the ability to rescue phenotypes in mouse models of other genetic neurodevelopmental disorders such as Rett syndrome, Angelman syndrome, and Phelan-McDermid syndrome by postnatal expression of gene products has led to hope that similar approaches could help modify the disease course in other neurodevelopmental disorders such as FS. While FoxG1 protein function plays a critical role in embryonic brain development, the ongoing adult expression of FoxG1 and behavioral phenotypes that present when FoxG1 function is removed postnatally provides support for opportunity for improvement with postnatal treatment. Here we generated a new mouse allele of Foxg1 that disrupts protein expression and characterized the behavioral and structural brain phenotypes in heterozygous mutant animals. These mutant animals display changes in locomotor behavior, gait, anxiety, social interaction, aggression, and learning and memory compared to littermate controls. Additionally, they have structural brain abnormalities reminiscent of people with FS. This information provides a framework for future studies to evaluate the potential for post-natal expression of FoxG1 to modify the disease course in this severe neurodevelopmental disorder.

Safety and efficacy of genetic MECP2 supplementation in the R294X mouse model of Rett syndrome.

Rett syndrome is a neurodevelopmental disorder caused predominantly by loss-of-function mutations in MECP2, encoding transcriptional modulator methyl-CpG-binding protein 2 (MeCP2). Although no disease-modifying therapies exist at this time, some proposed therapeutic strategies aim to supplement the mutant allele with a wild-type allele producing typical levels of functional MeCP2, such as gene therapy. Because MECP2 is a dosage-sensitive gene, with both loss and gain of function causing disease, these approaches must achieve a narrow therapeutic window to be both safe and effective. While MeCP2 supplementation rescues RTT-like phenotypes in mouse models, the tolerable threshold of MeCP2 is not clear, particularly for partial loss-of-function mutations. We assessed the safety of genetically supplementing full-length human MeCP2 in the context of the R294X allele, a common partial loss-of-function mutation retaining DNA-binding capacity. We assessed the potential for adverse effects from MeCP2 supplementation of a partial loss-of-function mutant and the potential for dominant negative interactions between mutant and full-length MeCP2. In male hemizygous R294X mice, MeCP2 supplementation rescued RTT-like behavioral phenotypes and did not elicit behavioral evidence of excess MeCP2. In female heterozygous R294X mice, RTT-specific phenotypes were similarly rescued. However, MeCP2 supplementation led to evidence of excess MeCP2 activity in a motor coordination assay, suggesting that the underlying motor circuitry is particularly sensitive to MeCP2 dosage in females. These results show that genetic supplementation of full-length MeCP2 is safe in males and largely so females. However, careful consideration of risk for adverse motor effects may be warranted for girls and women with RTT.

Granulocyte colony-stimulating factor (G-CSF) enhances cocaine effects in the nucleus accumbens via a dopamine release-based mechanism.

Cocaine use disorder is associated with alterations in immune function including altered expression of multiple peripheral cytokines in humans-several of which correlate with drug use. Individuals suffering from cocaine use disorder show altered immune system responses to drug-associated cues, highlighting the interaction between the brain and immune system as a critical factor in the development and expression of cocaine use disorder. We have previously demonstrated in animal models that cocaine use upregulates the expression of granulocyte colony-stimulating factor (G-CSF)-a pleiotropic cytokine-in the serum and the nucleus accumbens (NAc). G-CSF signaling has been causally linked to behavioral responses to cocaine across multiple behavioral domains. The goal of this study was to define whether increases in G-CSF alter the pharmacodynamic effects of cocaine on the dopamine system and whether this occurs via direct mechanisms within local NAc microcircuits. We find that systemic G-CSF injection increases cocaine effects on dopamine terminals. The enhanced dopamine levels in the presence of cocaine occur through a release-based mechanism, rather than through effects on the dopamine transporter-as uptake rates were unchanged following G-CSF treatment. Critically, this effect could be recapitulated by acute bath application of G-CSF to dopamine terminals, an effect that was occluded by prior G-CSF treatment, suggesting a similar mechanistic basis for direct and systemic exposures. This work highlights the critical interaction between the immune system and psychostimulant effects that can alter drug responses and may play a role in vulnerability to cocaine use disorder.

Pharmacological read-through of R294X Mecp2 in a novel mouse model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). More than 35% of affected individuals have nonsense mutations in MECP2. For these individuals, nonsense suppression has been suggested as a possible therapeutic approach. To assess the viability of this strategy, we created and characterized a mouse model with the common p.R294X mutation introduced into the endogenous Mecp2 locus (Mecp2R294X). Mecp2R294X mice exhibit phenotypic abnormalities similar to those seen in complete null mouse models; however, these occur at a later time point consistent with the reduced phenotypic severity seen in affected individuals containing this specific mutation. The delayed onset of severe phenotypes is likely due to the presence of truncated MeCP2 in Mecp2R294X mice. Supplying the MECP2 transgene in Mecp2R294X mice rescued phenotypic abnormalities including early death and demonstrated that the presence of truncated MeCP2 in these mice does not interfere with wild-type MeCP2. In vitro treatment of a cell line derived from Mecp2R294X mice with the nonsense suppression agent G418 resulted in full-length MeCP2 protein production, demonstrating feasibility of this therapeutic approach. Intraperitoneal administration of G418 in Mecp2R294X mice was sufficient to elicit full-length MeCP2 protein expression in peripheral tissues. Finally, intracranial ventricular injection of G418 in Mecp2R294X mice induced expression of full-length MeCP2 protein in the mouse brain. These experiments demonstrate that translational read-through drugs are able to suppress the Mecp2 p.R294X mutation in vivo and provide a proof of concept for future preclinical studies of nonsense suppression agents in RTT.