Neuronal Ensembles in the Amygdala Allow Social Information to Motivate Later Decisions.

Social experiences carry tremendous weight in our decision-making, even when social partners are not present. To determine mechanisms, we trained female mice to respond for two food reinforcers. Then, one food was paired with a novel conspecific. Mice later favored the conspecific-associated food, even in the absence of the conspecific. Chemogenetically silencing projections from the prelimbic subregion (PL) of the medial prefrontal cortex to the basolateral amygdala (BLA) obstructed this preference while leaving social discrimination intact, indicating that these projections are necessary for socially driven choice. Further, mice that performed the task had greater densities of dendritic spines on excitatory BLA neurons relative to mice that did not. We next induced chemogenetic receptors in cells active during social interactions-when mice were encoding information that impacted later behavior. BLA neurons stimulated by social experience were necessary for mice to later favor rewards associated with social conspecifics but not make other choices. This profile contrasted with that of PL neurons stimulated by social experience, which were necessary for choice behavior in social and nonsocial contexts alike. The PL may convey a generalized signal allowing mice to favor particular rewards, while units in the BLA process more specialized information, together supporting choice motivated by social information.

Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex.

Successfully navigating dynamic environments requires organisms to learn the consequences of their actions. The prelimbic prefrontal cortex (PL) formulates action-consequence memories and is modulated by addictive drugs like cocaine. We trained mice to obtain food rewards and then unexpectedly withheld reinforcement, triggering new action-consequence memory. New memory was disrupted by cocaine when delivered immediately following non-reinforcement, but not when delayed, suggesting that cocaine disrupted memory consolidation. Cocaine also rapidly inactivated cofilin, a primary regulator of the neuronal actin cytoskeleton. This observation led to the discovery that cocaine also within the time of memory consolidation elevated dendritic spine elimination and blunted spine formation rates on excitatory PL neurons, culminating in thin-type spine attrition. Training drug-naive mice to utilize inflexible response strategies also eliminated thin-type dendritic spines. Thus, cocaine may disrupt action-consequence memory, at least in part, by recapitulating neurobiological sequalae occurring in the formation of inflexible habits.

How social information impacts action in rodents and humans: the role of the prefrontal cortex and its connections.

Day-to-day choices often involve social information and can be influenced by prior social experience. When making a decision in a social context, a subject might need to: 1) recognize the other individual or individuals, 2) infer their intentions and emotions, and 3) weigh the values of all outcomes, social and non-social, prior to selecting an action. These elements of social information processing all rely, to some extent, on the medial prefrontal cortex (mPFC). Patients with neuropsychiatric disorders often have disruptions in prefrontal cortical function, likely contributing to deficits in social reasoning and decision making. To better understand these deficits, researchers have turned to rodents, which have revealed prefrontal cortical mechanisms for contending with the complex information processing demands inherent to making decisions in social contexts. Here, we first review literature regarding social decision making, and the information processing underlying it, in humans and patient populations. We then turn to research in rodents, discussing current procedures for studying social decision making, and underlying neural correlates.

Social incentivization of instrumental choice in mice requires amygdala-prelimbic cortex-nucleus accumbens connectivity.

Social experiences influence decision making, including decision making lacking explicit social content, yet mechanistic factors are unclear. We developed a new procedure, social incentivization of future choice (SIFC). Female mice are trained to nose poke for equally-preferred foods, then one food is paired with a novel conspecific, and the other with a novel object. Mice later respond more for the conspecific-associated food. Thus, prior social experience incentivizes later instrumental choice. SIFC is pervasive, occurring following multiple types of social experiences, and is not attributable to warmth or olfactory cues alone. SIFC requires the prelimbic prefrontal cortex (PL), but not the neighboring orbitofrontal cortex. Further, inputs from the basolateral amygdala to the PL and outputs to the nucleus accumbens are necessary for SIFC, but not memory for a conspecific. Basolateral amygdala→PL connections may signal the salience of social information, leading to the prioritization of coincident rewards via PL→nucleus accumbens outputs.

Cell adhesion presence during adolescence controls the architecture of projection-defined prefrontal cortical neurons and reward-related action strategies later in life.

Adolescent brain development is characterized by neuronal remodeling in the prefrontal cortex; relationships with behavior are largely undefined. Integrins are cell adhesion factors that link the extracellular matrix with intracellular actin cytoskeleton. We find that ß1-integrin presence in the prelimbic prefrontal cortex (PL) during adolescence, but not adulthood, is necessary for mice to select actions based on reward likelihood and value. As such, adult mice that lacked ß1-integrin during adolescence failed to modify response strategies when rewards lost value or failed to be delivered. This pattern suggests that ß1-integrin-mediated neuronal development is necessary for PL function in adulthood. We next visualized adolescent PL neurons, including those receiving input from the basolateral amygdala (BLA) - thought to signal salience - and projecting to the dorsomedial striatum (DMS) - the striatal output by which the PL controls goal-seeking behavior. Firstly, we found that these projection-defined neurons had a distinct morphology relative to general layer V PL neurons. Secondly, ß1-integrin loss triggered the overexpression of stubby-type dendritic spines at the expense of mature spines, including on projection-defined neurons. This phenotype was not observed when ß1-integrins were silenced before or after adolescence. Altogether, our experiments localize ß1-integrin-mediated cell adhesion within a developing di-synaptic circuit coordinating adaptive action.

ß1-Integrins in the Developing Orbitofrontal Cortex Are Necessary for Expectancy Updating in Mice.

Navigating a changing environment requires associating stimuli and actions with their likely outcomes and modifying these associations when they change. These processes involve the orbitofrontal cortex (OFC). Although some molecular mediators have been identified, developmental factors are virtually unknown. We hypothesized that the cell adhesion factor ß1-integrin is essential to OFC function, anticipating developmental windows during which ß1-integrins might be more influential than others. We discovered that OFC-selective ß1-integrin silencing before adolescence, but not later, impaired the ability of mice to extinguish conditioned fear and select actions based on their likely outcomes. Early-life knock-down also reduced the densities of dendritic spines, the primary sites of excitatory plasticity in the brain, and weakened sensitivity to cortical inputs. Notwithstanding these defects in male mice, females were resilient to OFC (but not hippocampal) ß1-integrin loss. Existing literature suggests that resilience may be explained by estradiol-mediated transactivation of ß1-integrins and tropomyosin receptor kinase B (trkB). Accordingly, we discovered that a trkB agonist administered during adolescence corrected reward-related decision making in ß1-integrin-deficient males. In sum, developmental ß1-integrins are indispensable for OFC function later in life.SIGNIFICANCE STATEMENT The orbitofrontal cortex (OFC) is a subregion of the frontal cortex that allows organisms to link behaviors and stimuli with anticipated outcomes, and to make predictions about the consequences of one's behavior. Aspects of OFC development are particularly prolonged, extending well into adolescence, likely optimizing organisms' abilities to prospectively calculate the consequences of their actions and select behaviors appropriately; these decision making strategies improve as young individuals mature into adulthood. Molecular factors are not, however, well understood. Our experiments reveal that a cell adhesion protein termed "ß1-integrin" is necessary for OFC neuronal maturation and function. Importantly, ß1-integrins operate during a critical period equivalent to early adolescence in humans to optimize the ability of organisms to update expectancies later in life.

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.

Rho-kinase inhibition has antidepressant-like efficacy and expedites dendritic spine pruning in adolescent mice.

Adolescence represents a critical period of neurodevelopment, defined by structural and synaptic pruning within the prefrontal cortex. While characteristic of typical development, this structural instability may open a window of vulnerability to developing neuropsychiatric disorders, including depression. Thus, therapeutic interventions that support or expedite neural remodeling in adolescence may be advantageous. Here, we inhibited the neuronally-expressed cytoskeletal regulatory factor Rho-kinase (ROCK), focusing primarily on the clinically-viable ROCK inhibitor fasudil. ROCK inhibition had rapid antidepressant-like effects in adolescent mice, and its efficacy was comparable to ketamine and fluoxetine. It also modified levels of the antidepressant-related signaling factors, tropomyosin/tyrosine receptor kinase B and Akt, as well as the postsynaptic marker PSD-95, in the ventromedial prefrontal cortex (vmPFC). Meanwhile, adolescent-typical dendritic spine pruning on excitatory pyramidal neurons in the vmPFC was expedited. Further, vmPFC-specific shRNA-mediated reduction of ROCK2, the dominant ROCK isoform in the brain, had antidepressant-like consequences. We cautiously suggest that ROCK inhibitors may have therapeutic potential for adolescent-onset depression.

Common basis for orofacial clefting and cortical interneuronopathy.

Orofacial clefts (OFCs) of the lip and/or palate are among the most common human birth defects. Current treatment strategies focus on functional and cosmetic repair but even when this care is available, individuals born with OFCs are at high risk for persistent neurobehavioral problems. In addition to learning disabilities and reduced academic achievement, recent evidence associates OFCs with elevated risk for a constellation of psychiatric outcomes including anxiety disorders, autism spectrum disorder, and schizophrenia. The relationship between these outcomes and OFCs is poorly understood and controversial. Recent neuroimaging studies in humans and mice demonstrate subtle morphological brain abnormalities that co-occur with OFCs but specific molecular and cellular mechanisms have not been investigated. Here, we provide the first evidence directly linking OFC pathogenesis to abnormal development of GABAergic cortical interneurons (cINs). Lineage tracing revealed that the structures that form the upper lip and palate develop in molecular synchrony and spatiotemporal proximity to cINs, suggesting these populations may have shared sensitivity to genetic and/or teratogenic insult. Examination of cIN development in a mouse model of nonsyndromic OFCs revealed significant disruptions in cIN proliferation and migration, culminating in misspecification of the somatostatin-expressing subgroup. These findings reveal a unified developmental basis for orofacial clefting and disrupted cIN development, and may explain the significant overlap in neurobehavioral and psychiatric outcomes associated with OFCs and cIN dysfunction. This emerging mechanistic understanding for increased prevalence of adverse neurobehavioral outcomes in OFC patients is the entry-point for developing evidence-based therapies to improve patient outcomes.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.

Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate.

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15th to 24th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in "non-syndromic" orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.

The teratogenic effects of prenatal ethanol exposure are exacerbated by Sonic Hedgehog or GLI2 haploinsufficiency in the mouse.

Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh(+/-) or Gli2(+/-) male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two i.p. doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh(+/-) and Gli2(+/-) groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh(+/-) and Gli2(+/-) mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.