Adolescent impulsivity as a sex-dependent and subtype-dependent predictor of impulsivity, alcohol drinking and dopamine D2 receptor expression in adult rats.

Impulsivity is a personality trait associated with a heightened risk for drug use and other psychiatric conditions. Because impulsivity-related disorders typically emerge during adolescence, there has been interest in exploring methods for identifying adolescents that will be at risk to develop substance use disorders in adulthood. Here, we used a rodent model to assess inhibitory control (impulsive action) and impulsive decision making (impulsive choice) during adolescence (43-50 days old) or adulthood (93-100 days old) and then examined the impact of development on these impulsivity traits by re-testing rats 50 days later. Impulsive action was not stable from adolescence to adulthood in male rats and was lowest in adult male rats, relative to adolescents and female rats. Impulsive choice was stable across development and unaffected by age or sex. Next, we examined the connection between our model of impulsivity and two measures relevant to substance abuse research: the initiation of voluntary alcohol drinking and dopamine D2 receptor (D2 R) expression in the prelimbic prefrontal cortex. Consumption of saccharin-sweetened ethanol during 30-minute sessions in adulthood was associated with adolescent, but not adult, impulsive action, particularly in male rats. Prelimbic D2 R expression was reduced in individuals with high levels of impulsive choice, and this relationship appeared to be strongest among female rats. The results of this study demonstrate that impulsive choice, along with its connection to D2 R expression, is relatively unchanged by the process of development. For impulsive action, however, individual levels of impulsivity during adolescence predict drinking in adulthood despite changes in the measure during development.

SHANK1 is differentially expressed during development in CA1 hippocampal neurons and astrocytes.

Recent studies have strongly suggested a role for the synaptic scaffolding protein SHANK1 in normal synaptic structure and signaling. Global SHANK1 knockout (SHANK1-/-) mice demonstrate reduced dendritic spine density, an immature dendritic spine phenotype and impairments in various cognitive tasks. SHANK1 overexpression is associated with increased dendritic spine size and impairments in fear conditioning. These studies suggest proper regulation of SHANK1 is crucial for appropriate synaptic structure and cognition. However, little is known regarding SHANK1's developmental expression in brain regions critical for learning. The current study quantified cell specific developmental expression of SHANK1 in the hippocampus, a brain region critically involved in various learning paradigms shown to be disrupted by SHANK1 dysregulation. Consistent with prior studies, SHANK1 was found to be strongly co-expressed with dendritic markers, with significant increased co-expression at postnatal day (P) 15, an age associated with increased synaptogenesis in the hippocampus. Interestingly, SHANK1 was also found to be expressed in astrocytes and microglia. To our knowledge, this is the first demonstration of glial SHANK1 localization; therefore, these findings were further examined via a glial purified primary cell culture fraction using magnetic cell sorting. This additional analysis further demonstrated that SHANK1 was expressed in glial cells, supporting our immunofluorescence co-expression findings. Developmentally, astroglial SHANK1 co-expression was found to be significantly elevated at P5 with a reduction into adulthood, while SHANK1 microglial co-expression did not significantly change across development. These data collectively implicate a more global role for SHANK1 in mediating normal cellular signaling in the brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 363-373, 2018.

Neocortical developmental analysis of vasculature and their growth factors offer new insight into fragile X syndrome abnormalities.

Fragile X Syndrome (FXS) is the most common single gene cause for Autism Spectrum Disorder and the most prevalent form of inherited mental retardation. Our prior studies have demonstrated that adult FXS mice have abnormal blood vessel density (BVD) and elevated Vascular Endothelial Growth Factor A expression (VEGF-A). VEGF-A is one of the most prominent regulators of BVD, and its abnormal expression is the most likely cause for FXS BVD abnormalities. We have demonstrated that attenuating elevated VEGF-A expression can ameliorate many non-vascular FXS abnormalities (Belagodu, Zendeli Slater and Galvez: Dev Neurobiol 77 (2017) 14-25), suggesting that abnormal VEGF-A expression is an underlying cause for some FXS abnormalities. However, FXS is a developmental disorder and VEGF-A's potential role in mediating FXS abnormalities during development have never been explored. Furthermore, VEGF-A is one protein in a family of proteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, & PLGF) that activate one of three primary receptors (VEGFR1, VEGFR2, & VEGFR3). Abnormal expression of any of these proteins could hinder proper development. The current study demonstrated that FXS mice do not exhibit normal BVD developmental patterns, resulting in elevated adult expression, most likely due to observed elevated VEGF-A adult expression. Interestingly, all five VEGF family of proteins exhibited altered developmental expression patterns that could cause abnormal development. However, none of the receptors exhibited abnormal adult expression, but did exhibit altered developmental expression. Expanding upon our prior analyses, the current study provides additional interesting insight towards potential developmental mechanisms mediating FXS abnormalities, while offering further sites for age specific therapeutic interventions. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1321-1333, 2017.

Blocking elevated VEGF-A attenuates non-vasculature Fragile X syndrome abnormalities.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, ). One of the most prominent regulators of vascular growth is VEGF-A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF-A expression. In addition to its role in vascular regulation, VEGF-A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF-A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF-A in brain. These studies suggest that increased VEGF-A expression in FXS is contributing to non-vascular FXS abnormalities. To explore the role of VEGF-A in mediating non-vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF-A. Bevacizumab treatment was found to decrease FXS Synapsin-1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF-A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF-A is elevated in FXS brain and suggest that its expression promotes non-vascular FXS abnormalities. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 14-25, 2017.

Characterization of ultrasonic vocalizations of Fragile X mice.

Fragile X Syndrome (FXS) is the leading form of inherited intellectual disability. It is caused by the transcriptional silencing of FMR1, the gene which codes for the Fragile X Mental Retardation Protein (FMRP). Patients who have FXS exhibit numerous behavioral and cognitive impairments, such as attention-deficit/hyperactivity disorder, obsessive compulsive disorder, and autistic-like behaviors. In addition to these behavioral abnormalities, FXS patients have also been shown to exhibit various deficits in communication such as abnormal sentence structures, increased utterances, repetition of sounds and words, and reduced articulation. These deficits can dramatically hinder communication for FXS patients, exacerbating learning and cognition impairments while decreasing their quality of life. To examine the biological underpinnings of these communication abnormalities, studies have used a mouse model of the Fragile X Syndrome; however, these vocalization studies have resulted in inconsistent findings that often do not correlate with abnormalities observed in FXS patients. Interestingly, a detailed examination of frequency modulated vocalizations that are believed to be a better assessment of rodent communication has never been conducted. The following study used courtship separation to conduct a detailed examination of frequency modulated ultrasonic vocalizations (USV) in FXS mice. Our analyses of frequency modulated USVs demonstrated that adult FXS mice exhibited longer phrases and more motifs. Phrases are vocalizations consisting of multiple frequency modulated ultrasonic vocalizations, while motifs are repeated frequency modulated USV patterns. Fragile X mice had a higher proportion of "u" syllables in all USVs and phrases while their wildtype counterparts preferred isolated "h" syllables. Although the specific importance of these syllables towards communication deficits still needs to be evaluated, these findings in production of USVs are consistent with the repetitive and perseverative speech patterns observed in FXS patients. This study demonstrates that FXS mice can be used to study the underlying biological mechanism(s) mediating FXS vocalization abnormalities.