Faculty Bibliography

OBJECTIVE:Previously we mapped QTL Eac2 to mouse Chr6 and identified the first gene (Grm7) as accounting for alcohol consumption in a mammalian model. Despite the central role of glutamate receptors in addiction, the effects of Grm7 gene variants are not well known. Here we test the hypothesis that genetic variation of the distal mouse Chr6 Eac2 region, location of Grm7, controls cocaine-induced locomotor sensitization. METHOD/METHODS:C57BL/6By background and B6.C6.327.54 congenic mice were subjected to whole-genome SNP genotyping. Isogeneic (C57BL/6ByXB6.C6.327.54)F2 mice homozygous for SNPs in the BALB/c-type Eac2 region were selected to create a subcongenic strain (B6By.C6.108-120). In a 2-strain x 2-sex 2-treatment factorial design (n = 6-10) C57BL/6By and B6By.C6.108-120 mice received repeated daily cocaine or saline intraperitoneal injections, and locomotor activity was recorded for 90 minutes immediately after injection. RESULTS:C57BL/6By females with the G/G genotype of SNP rs3723352 of Grm7 responded to cocaine with significantly higher activity and greater cocaine-induced sensitization than those with the BALB/cJ-type T/T genotype in the congenic strain. CONCLUSION/CONCLUSIONS:The results are consistent with a large body of accumulated mechanistic evidence for a role of the mGlu7 receptor in the control of neurobiological responses to cocaine, and are consistent with the hypotheses that (1) natural variants of the Grm7 gene show pleiotropy and can modulate cocaine-induced behaviors in addition to alcohol consumption, (2) interactions between mGluR7 expression, estrogen receptors, and estradiol may explain phenotypic variation in females. Heritable variation of GRM7 may affect vulnerability to substance abuse in women.

Many people call for strengthening knowledge co-production between academic science and indigenous and local knowledge systems. A major barrier to cooperation seems to be a lack of experience regarding where and how traditional knowledge can be found and obtained. Our key question was whether the expert judgment of academic zoologists or a feature-based linear model is better at predicting the observed level of local familiarity with wild animal species. Neither the zoologists nor the model proved sufficiently accurate (70 and 60%, respectively), with the inaccuracy probably resulting from inadequate knowledge of the local ecological and cultural specificities of the species. This indicates that more knowledge is likely to come from local knowledge than zoologists would expect. Accuracy of targeting the relevant species for knowledge co-production could be improved through specific understanding of the local culture, provided by experts who study traditional zoological knowledge and by local knowledge holders themselves.

Recent work using a mouse model has identified the glutamate metabotropic receptor 7 (Grm7) gene as a strong candidate gene for alcohol consumption. Although there has been some work examining the effect of human glutamate metabotropic receptor 7 (GRM7) polymorphisms on human substance use disorders, the majority of the work has focused on other psychiatric disorders such as ADHD, major depressive disorder, schizophrenia, bipolar disorder, panic disorder, and autism spectrum disorders. The current study aimed to evaluate evidence for association between GRM7 and alcohol behaviors in humans using a single nucleotide polymorphism (SNP) approach, as well as a gene-based approach. Using 1803 non-Hispanic European Americans (EAs) (source: the Colorado Center on Antisocial Drug Dependence [CADD]) and 1049 EA subjects from an independent replication sample (source: the Genetics of Antisocial Drug Dependence [GADD]), two SNPs in GRM7 were examined for possible association with alcohol consumption using two family-based association tests implemented in FBAT and QTDT. Rs3749380 was suggestively associated with alcohol consumption in the CADD sample (p = 0.010) with the minor T allele conferring risk. There was no evidence for association in the GADD sample. A gene-based test using four Genome-Wide Association Studies (GWAS) revealed no association between variation in GRM7 and alcohol consumption. This study had several limitations: the SNPs chosen likely do not tag expression quantitative trait loci; a human alcohol consumption phenotype was used, complicating the interpretation with respect to rodent studies that found evidence for a cis-regulatory link between alcohol preference and Grm7; and only common SNPs imputed in all four datasets were included in the gene-based test. These limitations highlight the fact that rare variants, some potentially important common signals in the gene, and regions farther upstream were not examined.

Fetal alcohol spectrum disorders (FASD) are associated with cognitive and behavioral deficits, and decreased volume of the whole brain and cerebral cortex. Rodent models have shown that early postnatal treatments, which mimic ethanol toxicity in the third trimester of human pregnancy, acutely induce widespread apoptotic neuronal degeneration and permanent behavioral deficits. However, the lasting cellular and anatomical effects of early ethanol treatments are still incompletely understood. This study examined changes in neocortex volume, thickness, and cellular organization that persist in adult mice after postnatal day 7 (P7) ethanol treatment. Post mortem brain volumes, measured by both MRI within the skull and by fluid displacement of isolated brains, were reduced 10-13% by ethanol treatment. The cerebral cortex showed a similar reduction (12%) caused mainly by lower surface area (9%). In spite of these large changes, several features of cortical organization showed little evidence of change, including cortical thickness, overall neuron size, and laminar organization. Estimates of total neuron number showed a trend level reduction of about 8%, due mainly to reduced cortical volume but unchanged neuron density. However, counts of calretinin (CR) and parvalbumin (PV) subtypes of GABAergic neurons showed a striking >30% reduction of neuron number. Similar ethanol effects were found in male and female mice, and in C57BL/6By and BALB/cJ mouse strains. Our findings indicate that the cortex has substantial capacity to develop normal cytoarchitectonic organization after early postnatal ethanol toxicity, but there is a selective and persistent reduction of GABA cells that may contribute to the lasting cognitive and behavioral deficits in FASD.

The most prominent feature of the Basal Forebrain (BF) is the collection of large cortically projecting neurons (basal nucleus of Meynert) that serve as the primary source of cholinergic input to the entire cortical mantle. Despite its broad involvement in cortical activation, attention, and memory, the functional details of the BF are not well understood due to the anatomical complexity of the region. This study tested the hypothesis that basalocortical connections reflect cortical connectivity patterns. Distinct retrograde tracers were deposited into various frontal and posterior cortical areas, and retrogradely labeled cholinergic and noncholinergic neurons were mapped in the BF. Concurrently, we mapped retrogradely labeled cells in posterior cortical areas that project to various frontal areas, and all cell populations were combined in the same coordinate system. Our studies suggest that the cholinergic and noncholinergic projections to the neocortex are not diffuse, but instead, are organized into segregated or overlapping pools of projection neurons. The extent of overlap between BF populations projecting to the cortex depends on the degree of connectivity between the cortical targets of these projection populations. We suggest that the organization of projections from the BF may enable parallel modulation of multiple groupings of interconnected yet nonadjacent cortical areas.

Glutamatergic neurotransmission in the brain is modulated by metabotropic glutamate receptors (mGluR). In recent studies, we identified a cis-regulated variant of a gene (Grm7) which codes for mGluR subtype 7 (mGluR7), a presynaptic inhibitory receptor. The genetic variant derived from the BALB/cJ mouse strain (Grm7 BALB/cJ) codes for higher abundance of mGluR7 mRNA in the hippocampus than the C57BL/6By strain-derived variant (Grm7 C57BL/6By ). Here, we used localized in vivo 1H NMR spectroscopy to test the hypothesis that Grm7 BALB/cJ is also associated with lower glutamate concentration in the same brain region. All data were obtained on a 7.0 T Agilent (Santa Clara, CA, USA) 40-cm bore system using experimentally naive adult male inbred C57BL/6By, BALB/cJ, and congenic mice (B6By.C.6.132.54) constructed in our laboratory carrying Grm7 BALB/cJ on C57BL/6By genetic background. The voxel of interest size was 6 muL (1 x 2 x 3 mm3) placed in the hippocampal CA1 region. The results showed that the hippocampal level of glutamate in the congenic mouse strain was significantly lower than that in the background C57BL/6By strain which carried the Grm7 C57BL/6By allele. Because the two inbred strains are genetically highly similar except at the region of the Grm7 gene, the results raise the possibility that allelic variation at the Grm7 locus contributes to the strain differences in both hippocampal mRNA abundance and glutamate level which may modulate complex behavioral traits, such as learning and memory, addiction, epilepsy, and mood disorders.

Vascular calcification is implicated in the pathogenesis of atherosclerosis, diabetes and chronic kidney disease. Human vascular smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor alpha-1 (CBF-alpha1), a bone-specific transcription factor, with the subsequent induction of osteocalcin. It has been shown that heavy alcohol consumption is associated with greater calcification in coronary arteries. The goal of our study was to examine whether ethanol alters mineralization of HSMCs provoked by high Pi. Exposure of HSMCs to ethanol increased extracellular matrix calcification in a dose responsive manner, providing a significant additional calcium deposition at concentrations of >/=60 mmol/l. HSMC calcification was accompanied by further enhancement in alkaline phosphatase activity. Ethanol also provoked a significant increase in the synthesis of osteocalcin. Moreover, in cells challenged with ethanol the expression of CBF-alpha1, a transcription factor involved in the regulation of osteoblastic transformation of HSMCs, was elevated. The observed effects of ethanol were not due to alterations of phosphate uptake by HSMCs. We conclude that ethanol enhances Pi-mediated human vascular smooth muscle calcification and transition of these cells into osteoblast-like cells.

J. Neurochem. (2012) 121, 806-817. ABSTRACT: Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.

Development of addiction to alcohol or other substances can be attributed in part to exposure-dependent modifications at synaptic efficacy leading to an organism which functions at an altered homeostatic setpoint. Genetic factors may also influence setpoints and the stability of the homeostatic system of an organism. Quantitative genetic analysis of voluntary alcohol drinking, and mapping of the involved genes in the quasi-congenic Recombinant QTL Introgression strain system, identified Eac2 as a Quantitative Trait Locus (QTL) on mouse chromosome 6 which explained 18% of the variance with an effect size of 2.09 g/kg/day alcohol consumption, and Grm7 as a quantitative trait gene underlying Eac2 [Vadasz et al. in Neurochem Res 32:1099-1112, 100, Genomics 90:690-702, 102]. In earlier studies, the product of Grm7 mGluR7, a G protein-coupled receptor, has been implicated in stress systems [Mitsukawa et al. in Proc Natl Acad Sci USA 102:18712-18717, 63], anxiety-like behaviors [Cryan et al. in Eur J Neurosci 17:2409-2417, 14], memory [Holscher et al. in Learn Mem 12:450-455, 26], and psychiatric disorders (e.g., [Mick et al. in Am J Med Genet B Neuropsychiatr Genet 147B:1412-1418, 61; Ohtsuki et al. in Schizophr Res 101:9-16, 72; Pergadia et al. in Paper presented at the 38th Annual Meeting of the Behavior Genetics Association, Louisville, Kentucky, USA, 76]. Here, in experiments with mice, we show that (1) Grm7 knockout mice express increased alcohol consumption, (2) sub-congenic, and congenic mice carrying a Grm7 variant characterized by higher Grm7 mRNA drink less alcohol, and show a tendency for higher circadian dark phase motor activity in a wheel running paradigm, respectively, and (3) there are significant genetic differences in Grm7 mRNA abundance in the mouse brain between congenic and background mice identifying brain areas whose function is implicated in addiction related processes. We hypothesize that metabotropic glutamate receptors may function as regulators of homeostasis, and Grm7 (mGluR7) is involved in multiple processes (including stress, circadian activity, reward control, memory, etc.) which interact with substance use and the development of addiction. In conclusion, we suggest that mGluR7 is a significant new therapeutic target in addiction and related neurobehavioral disorders