Cell adhesion factors in the orbitofrontal cortex control cue-induced reinstatement of cocaine seeking and amygdala-dependent goal seeking.

Repeated cocaine exposure causes dendritic spine loss in the orbitofrontal cortex, which might contribute to poor orbitofrontal cortical function following drug exposure. One challenge, however, has been verifying links between neuronal structural plasticity and behavior, if any. Here we report that cocaine self-administration triggers the loss of dendritic spines on excitatory neurons in the orbitofrontal cortex of male and female mice (as has been reported in rats). To understand functional consequences, we locally ablated neuronal ß1-integrins, cell adhesion receptors that adhere cells to the extracellular matrix and thus support dendritic spine stability. Degradation of ß1-integrin tone: 1) caused dendritic spine loss; 2) exaggerated cocaine-seeking responses in a cue-induced reinstatement test; and 3) impaired the ability of mice to integrate new learning into familiar routines - a key function of the orbitofrontal cortex. Stimulating Abl-related gene (Arg) kinase, over-expressing Proline-rich tyrosine kinase (Pyk2), and inhibiting Rho-associated coiled-coil containing kinase (ROCK) corrected response strategies, uncovering a ß1-integrin-mediated signaling axis that controls orbitofrontal cortical function. Finally, use of a combinatorial gene silencing/chemogenetic strategy revealed that ß1-integrins support the ability of mice to integrate new information into established behaviors by sustaining orbitofrontal cortical connections with the basolateral amygdala.SIGNIFICANCE STATEMENTCocaine degenerates dendritic spines in the orbitofrontal cortex, a region of the brain involved in interlacing new information into established behaviors. One challenge has been verifying links between cellular structural stability and behavior, if any. In this second of two related investigations, we study integrin family receptors, which adhere cells to the extracellular matrix and thereby stabilize dendritic spines (see also DePoy et al., 2019, Journal of Neuroscience). We reveal that ß1-integrins in the orbitofrontal cortex control food- and cocaine-seeking behaviors. For instance, ß1-integrin loss amplifies cocaine-seeking behavior and impairs the ability of mice to integrate new learning into familiar routines. We identify likely intracellular signaling partners by which ß1-integrins support orbitofrontal cortical function and connectivity with the basolateral amygdala.

Reward-Related Expectations Trigger Dendritic Spine Plasticity in the Mouse Ventrolateral Orbitofrontal Cortex.

An essential aspect of goal-directed decision-making is selecting actions based on anticipated consequences, a process that involves the orbitofrontal cortex (OFC) and potentially, the plasticity of dendritic spines in this region. To investigate this possibility, we trained male and female mice to nose poke for food reinforcers, or we delivered the same number of food reinforcers non-contingently to separate mice. We then decreased the likelihood of reinforcement for trained mice, requiring them to modify action-outcome expectations. In a separate experiment, we blocked action-outcome updating via chemogenetic inactivation of the OFC. In both cases, successfully selecting actions based on their likely consequences was associated with fewer immature, thin-shaped dendritic spines and a greater proportion of mature, mushroom-shaped spines in the ventrolateral OFC. This pattern was distinct from spine loss associated with aging, and we identified no effects on hippocampal CA1 neurons. Given that the OFC is involved in prospective calculations of likely outcomes, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for solidifying durable expectations. To investigate causal relationships, we inhibited the RNA-binding protein fragile X mental retardation protein (encoded by Fmr1), which constrains dendritic spine turnover. Ventrolateral OFC-selective Fmr1 knockdown recapitulated the behavioral effects of inducible OFC inactivation (and lesions; also shown here), impairing action-outcome conditioning, and caused dendritic spine excess. Our findings suggest that a proper balance of dendritic spine plasticity within the OFC is necessary for one's ability to select actions based on anticipated consequences.SIGNIFICANCE STATEMENT Navigating a changing environment requires associating actions with their likely outcomes and updating these associations when they change. Dendritic spine plasticity is likely involved, yet relationships are unconfirmed. Using behavioral, chemogenetic, and viral-mediated gene silencing strategies and high-resolution microscopy, we find that modifying action-outcome expectations is associated with fewer immature spines and a greater proportion of mature spines in the ventrolateral orbitofrontal cortex (OFC). Given that the OFC is involved in prospectively calculating the likely outcomes of one's behavior, even when they are not observable, constraining spinogenesis while preserving mature spines may be important for maintaining durable expectations.

Selective role of the catalytic PI3K subunit p110ß in impaired higher order cognition in fragile X syndrome.

Distinct isoforms of the PI3K catalytic subunit have specialized functions in the brain, but their role in cognition is unknown. Here, we show that the catalytic subunit p110ß plays an important role in prefrontal cortex (PFC)-dependent cognitive defects in mouse models of Fragile X syndrome (FXS), an inherited intellectual disability. FXS is caused by loss of function of the fragile X mental retardation protein (FMRP), which binds and translationally represses mRNAs. PFC-selective knockdown of p110ß, an FMRP target that is translationally upregulated in FXS, reverses deficits in higher cognition in Fmr1 knockout mice. Genetic full-body reduction of p110ß in Fmr1 knockout mice normalizes excessive PI3K activity, restores stimulus-induced protein synthesis, and corrects increased dendritic spine density and behavior. Notably, adult-onset PFC-selective Fmr1 knockdown mice show impaired cognition, which is rescued by simultaneous p110ß knockdown. Our results suggest that FMRP-mediated control of p110ß is crucial for neuronal protein synthesis and cognition.

Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.

Dysfunctions in serotonin (5-hydroxytryptamine, 5-HT) systems have been associated with several psychiatric illnesses, including anxiety, depression, obsessive-compulsive disorders and autism spectrum disorders. Convergent evidence from genetic analyses of human subjects has implicated the integrin ß3 subunit gene (ITGB3) as a modulator of serotonergic systems via genetic interactions with the 5-HT transporter gene (SLC6A4, SERT). While genetic interactions may result from contributions of each gene at several levels, we hypothesize that ITGB3 modulates the 5-HT system at the level of the synapse, through the actions of integrin αvß3. Here we utilized a genetic approach in mouse models to examine Itgb3 contributions to SERT function both in the context of normal and reduced SERT expression. As integrin αvß3 is expressed in postsynaptic membranes, we isolated synaptoneurosomes, which maintain intact pre- and post-synaptic associations. Citalopram binding revealed significant Slc6a4-driven reductions in SERT expression in midbrain synapses, whereas no significant changes were observed in hippocampal or cortical projections. Expecting corresponding changes to SERT function, we also measured 5-HT uptake activity in synaptoneurosomal preparations. Itgb3 single heterozygous mice displayed significant reductions in 5-HT Vmax, with no changes in Km, in midbrain preparations. However, in the presence of both Itgb3 and Slc6a4 heterozygozity, 5-HT uptake was similar to wild-type levels, revealing a significant Slc6a4 by Itgb3 genetic interaction in the midbrain. Similar findings were observed in cortical preparations, whereas in the hippocampus, most Vmax changes were driven solely by Slc6a4. Our findings provide evidence that integrin αvß3 is involved in the regulation of serotonergic systems in some, but not all 5-HT synapses, revealing novel contributions to synaptic specificity within the central nervous system.

Glucocorticoid receptor regulation of action selection and prefrontal cortical dendritic spines.

We recently reported that prolonged exposure to the glucocorticoid receptor (GR) ligand corticosterone impairs decision-making that is dependent on the predictive relationship between an action and its outcome (Gourley et al.; Proceedings of the National Academy of Sciences, 2012). Additionally, acute GR blockade, when paired with action-outcome conditioning, also blocks new learning. We then showed that dendritic spines in the prelimbic prefrontal cortex remodeled under both conditions. Nonetheless, the relationship between deep-layer dendritic spines and outcome-based decision-making remains opaque. We report here that a history of prolonged corticosterone exposure increases dendritic spine density in deep-layer prelimbic cortex. When spines are imaged simultaneously with corticosteroid exposure (i.e., without a washout period), dendritic spine densities are, however, reduced. Thus, the morphological response of deep-layer prelimbic cortical neurons to prolonged corticosteroid exposure may be quite dynamic, with spine elimination during a period of chronic exposure and spine proliferation during a subsequent washout period. We provide evidence, using a Rho-kinase inhibitor, that GR-mediated dendritic spine remodeling is causally related to complex decision-making. Finally, we conclude this report with evidence that a history of early-life (adolescent) GR blockade, unlike acute blockade in adulthood, enhances subsequent outcome-based decision-making. Together, our findings suggest that physiological levels of GR binding enable an organism to learn about the predictive relationship between an action and its outcome, but a history of GR blockade may, under some circumstances, also have beneficial consequences.