A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.
prefrontal cortex; hippocampus; neurogenesis; stress; anxiety; depression
Human genetic and epidemiological studies provide evidence that only a subset of individuals who experiment with potentially addictive drugs become addicts. What renders some individuals susceptible to addiction remains to be determined, but most would agree that there is no single trait underlying the disorder. However, there is evidence in humans that addiction liability has a genetic component, and that certain personality characteristics related to temperament (e.g. the sensation-seeking trait) are associated with individual differences in addiction liability. Consequently, we have used a selective breeding strategy based on locomotor response to a novel environment to generate two lines of rats with distinct behavioral characteristics. We have found that the resulting phenotypes differ on a number of neurobehavioral dimensions relevant to addiction. Relative to bred low-responder (bLR) rats, bred high-responder (bHR) rats exhibit increased exploratory behavior, are more impulsive, more aggressive, seek stimuli associated with rewards, and show a greater tendency to relapse. We therefore utilize this unique animal model to parse the genetic, neural and environmental factors that contribute to addiction liability. Our work shows that the glucocorticoid receptor (GR), dopaminergic molecules, and members of the fibroblast growth factor family are among the neurotransmitters and neuromodulators that play a role in both the initial susceptibility to addiction as well as the altered neural responses that follow chronic drug exposure. Moreover, our findings suggest that the hippocampus plays a major role in mediating vulnerability to addiction. It is hoped that this work will emphasize the importance of personalized treatment strategies and identify novel therapeutic targets for humans suffering from addictive disorders.
addiction; selectively bred; dopamine; fibroblast growth factor (FGF); cocaine; novelty-seeking; high-responder; low-responder
The rs1344706, an intronic SNP within the zinc-finger protein 804A gene (ZNF804A), was identified as one of the most compelling risk SNPs for schizophrenia (SZ) and bipolar disorder (BD). It is however not clear by which molecular mechanisms ZNF804A increases disease risk. We evaluated the role of ZNF804A in SZ and BD by genotyping the originally associated rs1344706 SNP and an exonic SNP (rs12476147) located in exon four of ZNF804A in a sample of 428 SZ, 385 BD, and 578 controls from the isolated population of the Costa Rica Central Valley. We also investigated the rs1344706 SNP for allelic specific expression (ASE) imbalance in the dorsolateral prefrontal cortex (DLPFC) of 46 heterozygous postmortem brains.
While no significant association between rs1344706 and SZ or BD was observed in the Costa Rica sample, we observed an increased risk of SZ for the minor allele (A) of the exonic rs12476147 SNP (p =0.026). Our ASE assay detected a significant over-expression of the rs12476147 A allele in DLPFC of rs1344706 heterozygous subjects. Interestingly, cDNA allele ratios were significantly different according to the intronic rs1344706 genotypes (p-value = 0.03), with the rs1344706 A allele associated with increased ZNF804A rs12476147 A allele expression (average 1.06, p-value = 0.02, for heterozygous subjects vs. genomic DNA).
In conclusion, we have demonstrated a significant association of rs12476147 with SZ, and using a powerful within-subjects design, an allelic expression imbalance of ZNF804A exonic SNP rs12476147 in the DLPFC. Although this data does not preclude the possibility of other functional variants in ZNF804A, it provides evidence that the rs1344706 SZ risk allele is the cis-regulatory variant directly responsible for this allelic expression imbalance in adult cortex.
schizophrenia; bipolar disorder; ZNF804A; association study; rs1344706; allelic-specific expression
Individual differences in the locomotor response to novelty have been linked to basal differences in dopaminergic neurotransmission. Mesolimbic dopaminergic outputs are regulated by cholecystokinin (CCK), a neuropeptide implicated in anxiety. In turn, CCK expression is regulated by fibroblast growth factor-2 (FGF2), which has recently been identified as an endogenous regulator of anxiety. FGF2 binds to the high-affinity fibroblast growth factor receptor-1 (FGF-R1) to regulate the development and maintenance of dopamine neurons in the ventral tegmental area (VTA). However, the relationship between the FGF and CCK systems in the VTA is not well understood. Therefore, we utilized the selectively-bred low-responder (bLR; high-anxiety) and high-responder (bHR; low-anxiety) rats to examine the effects of repeated (21-day) FGF2 treatment on CCK and FGF-R1 mRNA in the rostral VTA (VTAr). In vehicle-treated controls, both CCK and FGF-R1 mRNA levels were increased in the VTAr of bLR rats relative to bHR rats. Following FGF2 treatment, however, bHR-bLR differences in CCK and FGF-R1 mRNA expression were eliminated, due to decreased CCK mRNA levels in the VTAr of bLR rats and increased FGF-R1 expression in bHR rats. Differences after FGF2 treatment may denote distinct interactions between the CCK and FGF systems in the VTAr of bHR vs. bLR rats. Indeed, significant correlations between CCK and FGF-R1 mRNA expression were found in bHR, but not bLR rats. Colocalization studies suggest that CCK and FGF-R1 are coexpressed in some VTAr neurons. Taken together, our findings suggest that the FGF system is poised to modulate both CCK and FGF-R1 expression in the VTAr, which may be associated with individual differences in mesolimbic pathways associated with anxietylike behavior.
Fibroblast growth factor-2; fibroblast growth factor receptor-1; novelty response; in situ hybridization; colocalization; individual differences
Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Student’s t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.
Anhedonia, or the inability to experience positive feelings is a hallmark of depression. However, few animal models have relied on decreased positive affect as an index of susceptibility to depression. Rats emit frequency modulated ultrasonic vocalizations (USVs), designated as “positive” calls in the 50kHz range. USVs have been associated with pharmacological activation of motivational reward circuits. Here we utilized selectively-bred rats differing in “emotionality” to ask whether there are associated differences in USVs. Rats bred based on locomotor response to novelty and classified as bred high-responders (bHRs) or bred low-responder (bLRs) exhibit inborn differences in response to environmental cues, stress responsiveness, and depression-like behavior. These animals also exhibit differences in anxiety-like behavior, which are reversed by exposure to environmental complexity (EC). Finally, these animals exhibit unique profiles of responsiveness to rewarding stimuli accompanied with distinct patterns of dopamine regulation. We investigated whether acute and chronic environmental manipulations impacted USVs in bHRs and bLRs. We found that, relative to bLRs, bHRs emitted significantly more 50 kHz USVs. However, if a bLR is accompanied by another bLR, there is a significant increase in 50kHZ USVs emitted by this phenotype. bHRs emitted increases in 50kHZ UVSs upon first exposure to EC, whereas bLRs showed a similar increase only after repeated exposure. bLRs’ increase in positive affect after chronic EC was coupled with significant positive correlations between corticosterone levels and c-fos mRNA in the accumbens. Conversely, a decline in the rate of positive calls in bHRs after chronic EC was associated with a negative correlation between corticosterone and accumbens c-fos mRNA. These studies demonstrate that inborn differences in emotionality interact with the environment to influence positive affect and underscore the potential interaction between glucocorticoids and the mesolimbic reward circuitry in modulating 50 kHz calls.
Ultrasonic vocalization; Affect; Emotionality; Individual Differences
Repeated exposure to drugs of abuse is associated with structural plasticity in brain reward pathways. Rats selectively bred for locomotor response to novelty differ on a number of neurobehavioral dimensions relevant to addiction. This unique genetic animal model was used here to examine both pre-existing differences and long-term consequences of repeated cocaine treatment on structural plasticity. Selectively bred high-responder (bHR) and low-responder (bLR) rats received repeated saline or cocaine injections for 9 consecutive days. Escalating doses of cocaine (7.5, 15 and 30 mg/kg) were administered on the first (day 1) and last (day 9) days of treatment and a single injection of the intermediate dose (15 mg/kg) was given on days 2-8. Motor activity in response to escalating doses of cocaine was compared on the first and last days of treatment to assess the acute and sensitized response to the drug. Following prolonged cocaine abstinence (28 days), spine density was examined on terminal dendrites of medium spiny neurons in the nucleus accumbens core. Relative to bLRs, bHRs exhibited increased psychomotor activation in response to both the acute and repeated effects of cocaine. There were no differences in spine density between bHR and bLR rats under basal conditions or following repeated saline treatment. However, spine density differed markedly between these two lines following prolonged cocaine abstinence. All spine types were decreased in cocaine-treated bHRs, while only mushroom spines were decreased in bLRs that received cocaine. Changes in spine density occurred specifically near the branch point of terminal dendrites. These findings indicate that structural plasticity associated with prolonged cocaine abstinence varies markedly in two selected strains of rats that vary on numerous traits relevant to addiction. Thus, genetic factors that contribute to individual variation in the behavioral response to cocaine also influence cocaine-induced structural plasticity.
cocaine; psychomotor sensitization; dendrites; prolonged abstinence; spine density; addiction
MicroRNAs (miRNAs) are small non-coding RNAs that act as potent regulators of gene expression. A recent GWAS reported the rs1625579 SNP, located downstream of miR-137, as the strongest new association with schizophrenia (Ripke et al., 2011). Prior to this GWAS finding, a schizophrenia imaging-genetic study found miR-137 target genes significantly enriched for association with activation in the dorsolateral prefrontal cortex (DLPFC) (Potkin et al., 2010).
We investigated the expression levels of miR-137 and three candidate target genes (ZNF804A, CACNA1C, TCF4) in the DLPFC of postmortem brain tissue from 2 independent cohorts: 1) 26 subjects (10 control (CTR), 7 schizophrenia (SZ), 9 bipolar disorder (BD)) collected at the UCI brain bank; and 2) 99 subjects (33 CTR, 35 SZ, 31 BD) obtained from the Stanley Medical Research Institute (SMRI). MiR-137 expression in the DLPFC did not differ between diagnoses. We also explored the relationship between rs1625579 genotypes and miR-137 expression. Significantly lower miR-137 expression levels were observed in the homozygous TT subjects compared to TG and GG subjects in the control group (30% decrease, p-value=0.03). Moreover, reduced miR-137 levels in TT subjects corresponded to increased levels of the miR-137 target gene TCF4. The miR-137 expression pattern in 9 brain regions was significant for regional effect (ANOVA p-value=1.83E-12), with amygdala and hippocampus having the highest miR-137 expression level. In conclusion, decreased miR-137 expression is associated with the SZ risk allele of rs1625579, and potential regulation of TCF4, another SZ candidate gene. This study offers additional support for involvement of miR-137 and downstream targets as mechanisms of risk for psychiatric disorders.
schizophrenia; bipolar disorder; rs1625579; miR-137; TCF4; gene expression
Exposure to early life stress dramatically impacts adult behavior, physiology, and neuroendocrine function. Using rats bred for novelty-seeking differences and known to display divergent anxiety, depression, and stress vulnerability, we examined the interaction between early life adversity and genetic predisposition for high- versus low-emotional reactivity. Thus, bred Low Novelty Responder (bLR) rats, which naturally exhibit high anxiety- and depression-like behavior, and bred High Novelty Responder (bHR) rats, which show low anxiety/depression together with elevated aggression, impulsivity, and addictive behavior, were subjected to daily 3 h maternal separation (MS) stress postnatal days 1–14. We hypothesized that MS stress would differentially impact adult bHR/bLR behavior, physiology (stress-induced defecation), and neuroendocrine reactivity. While MS stress did not impact bHR and bLR anxiety-like behavior in the open field test and elevated plus maze, it exacerbated bLRs’ already high physiological response to stress – stress-induced defecation. In both tests, MS bLR adult offspring showed exaggerated stress-induced defecation compared to bLR controls while bHR offspring were unaffected. MS also selectively impacted bLRs’ (but not bHRs’) neuroendocrine stress reactivity, producing an exaggerated corticosterone acute stress response in MS bLR versus control bLR rats. These findings highlight how genetic predisposition shapes individuals’ response to early life stress. Future work will explore neural mechanisms underlying the distinct behavioral and neuroendocrine consequences of MS in bHR/bLR animals.
bred High Responder (bHR); bred Low Responder (bLR); stress-induced defecation; maternal separation stress; corticosterone
Hippocampal plasticity (e.g., neurogenesis) likely plays an important role in maintaining addictive behavior and/or relapse. This study assessed whether rats with differential propensity to drug-seeking behavior, bred Low-Responders (bLR) and bred High-Responders (bHR) to novelty, show differential neurogenesis regulation after cocaine exposure. Using specific immunological markers, we labeled distinct populations of adult stem cells in the dentate gyrus at different time-points of the cocaine sensitization process; Ki-67 for newly born cells, NeuroD for cells born partway, and BrdU for older cells born prior to sensitization. Results show that: (1) bHRs exhibited greater psychomotor response to cocaine than bLRs. (2) Acute cocaine did not alter cell proliferation in bLR/bHR rats. (3) Chronic cocaine decreased cell proliferation in bLRs only, which became amplified through the course of abstinence. (4) Neither chronic cocaine nor cocaine abstinence affected the survival of immature neurons in either phenotype. (5) Cocaine abstinence decreased survival of mature neurons in bHRs only, an effect that paralleled the greater psychomotor response to cocaine. (6) Cocaine treatment did not affect the ratio of neurons to glia in bLR/bHR rats as most cells differentiated into neurons in both lines. Thus, cocaine exerts distinct effects on neurogenesis in bLR versus bHR rats, with a decrease in the birth of new progenitor cells in bLRs and a suppression of the survival of new neurons in bHRs which likely leads to an earlier decrease in formation of new connections. This latter effect in bHRs could contribute to their enhanced degree of cocaine-induced psychomotor behavioral sensitization.
psychomotor sensitization; cell genesis; dentate gyrus; hippocampus; bLR/bHR rats
High versus low novelty exploration predicts a variety of behavioral differences. For example, rats selectively-bred for high novelty exploration (bred High Responders, bHR) exhibit exaggerated aggression, impulsivity, and proclivity to addictive behaviors compared to low novelty-reactive rats (bred Low Responders, bLRs), which are characterized by a high anxiety/depressive-like phenotype. Since bHR/bLR rats exhibit differences in dopaminergic circuitry and differential response to rewarding stimuli (i.e., psychostimulants, food), the present study examined whether they also differ in another key hedonic behavior – sex. Thus, adult bHR/bLR males were given five 30-min opportunities to engage in sexual activity with a receptive female. Sexual behavior and motivation were examined and compared between the groups. The bHR/bLR phenotype affected both sexual motivation and behavior, with bLR males demonstrating reduced motivation for sex compared with bHR males (i.e., fewer animals copulated, longer latency to engage in sex). The bHR males required more intromissions at a faster pace per ejaculation than did bLR males. Thus, neurobiological differences that affect motivation for drugs of abuse, aggression, and impulsivity in rats also affect sexual motivation and performance.
bred High Responder (bHR); bred Low Responder (bLR); sexual performance; reward; dopamine
The onset of major depressive disorder is likely precipitated by a combination of heredity and life stress. The present study tested the hypothesis that rats selectivity bred on a trait related to emotional reactivity would show differential susceptibility or resilience to the development of depression-like signs in response to chronic mild variable intermittent stress (CMS).
Male Sprague-Dawley rats that were bred based on the trait of either high or low locomotor activity in response to a novel environment were exposed to four weeks of CMS or control conditions. Changes in hedonic behavior were assessed using weekly sucrose preference tests and anxiety-like behavior was evaluated using the novelty-suppressed feeding test.
During four weeks of CMS, bred low responder (bLR) rats became anhedonic at a faster rate and to a larger degree than bred high responder (bHR) rats, based on weekly sucrose preference tests. Measures of anxiety-like behavior in the novelty-suppressed feeding test were also significantly increased in the CMS-exposed bLR rats, though no differences were observed between CMS-exposed bHR rats and their unstressed controls.
These findings present further evidence that increased emotional reactivity is an important factor in stress susceptibility and the etiology of mood disorders, and that bHR and bLR rats provide a model of resistance or vulnerability to stress-induced depression. Furthermore, exposing bHR and bLR rats to CMS provides an excellent way to study the interaction of genetic and environmental factors in the development of depression-like behavior.
depression; chronic mild stress; high responder; low responder; selectively bred rat; vulnerability
Activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in response to the organism’s innate need for homeostasis. The glucocorticoids (GCs) that are released into the circulation upon acute activation of the HPA axis perform stress-adaptive functions and provide negative feedback to turn off the HPA axis, but can be detrimental when in excess. Long-term activation of the HPA axis (such as with chronic stress) enhances susceptibility to neuronal dysfunction and death, and increases vulnerability to Alzheimer’s disease (AD). However, little is known how components of the HPA axis, upstream of GCs, impact vulnerability to AD. This study examined basal gene expression of stress-related molecules in brains of 3xTg-AD mice during early-stage pathology. Basal glucocorticoid levels and mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and corticotropic releasing hormone (CRH) in several stress- and emotionality-related brain regions were measured in 3–4-month-old 3xTg-AD mice. Despite normal glucocorticoid levels, young 3xTg-AD mice exhibit an activated central HPA axis, with altered mRNA levels of MR and GR in the hippocampus, GR and CRH in the paraventricular nucleus of the hypothalamus, GR and CRH in the central nucleus of the amygdala, and CRH in the bed nucleus of the stria terminalis. This HPA axis activation is present during early-stage neuropathology when 3xTg-AD mice show mild behavioral changes, suggesting an ongoing neuroendocrine regulation that precedes the onset of severe AD-like pathology and behavioral deficits.
HPA axis; stress; corticotropic releasing hormone; glucocorticoid receptor; mineralocorticoid receptor; Alzheimer’s disease
Given the emergent interest in biomarkers for mood disorders, we assessed gene expression in the choroid plexus (CP), the region that produces cerebrospinal fluid (CSF), in individuals with major depressive disorder (MDD). Genes that are expressed in the CP can be secreted into the CSF and may be potential biomarker candidates. Given that we have previously shown that fibroblast growth factor family members are differentially expressed in post-mortem brain of subjects with MDD and the CP is a known source of growth factors in the brain, we posed the question whether growth factor dysregulation would be found in the CP of subjects with MDD. We performed laser capture microscopy of the CP at the level of the hippocampus in subjects with MDD and psychiatrically normal controls. We then extracted, amplified, labeled, and hybridized the cRNA to Illumina BeadChips to assess gene expression. In controls, the most highly abundant known transcript was transthyretin. Moreover, half of the 14 most highly expressed transcripts in controls encode ribosomal proteins. Using BeadStudio software, we identified 169 transcripts differentially expressed (p < 0.05) between control and MDD samples. Using pathway analysis we noted that the top network altered in subjects with MDD included multiple members of the transforming growth factor-beta (TGFβ) pathway. Quantitative real-time PCR (qRT-PCR) confirmed downregulation of several transcripts that interact with the extracellular matrix in subjects with MDD. These results suggest that there may be an altered cytoskeleton in the CP in MDD subjects that may lead to a disrupted blood-CSF-brain barrier.
cytoskeleton; mRNA; hippocampus; depression; brain
The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, “activated” cAMP signaling activity in BPD and “blunted” cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.
G-protein coupled receptor (GPCR); transcriptome; bipolar disorder; major depressive disorder; GPR37; GPRC5B; cyclic AMP; phosphatidylinositol
Gene expression profiles of postmortem brain tissue represent important resources for understanding neuropsychiatric illnesses. The impact(s) of quality covariables on the analysis and results of gene expression studies are important questions. This paper addressed critical variables which might affect gene expression in two brain regions. Four broad groups of quality indicators in gene expression profiling studies (clinical, tissue, RNA, and microarray quality) were identified. These quality control indicators were significantly correlated, however one quality variable did not account for the total variance in microarray gene expression. The data showed that agonal factors and low pH correlated with decreased integrity of extracted RNA in two brain regions. These three parameters also modulated the significance of alterations in mitochondrial-related genes. The average F-ratio summaries across all transcripts showed that RNA degradation from the AffyRNAdeg program accounted for higher variation than all other quality factors. Taken together, these findings confirmed prior studies, which indicated that quality parameters including RNA integrity, agonal factors, and pH are related to differences in gene expression profiles in postmortem brain. Individual candidate genes can be evaluated with these quality parameters in posthoc analysis to help strengthen the relevance to psychiatric disorders. We find that clinical, tissue, RNA, and microarray quality are all useful variables for collection and consideration in study design, analysis, and interpretation of gene expression results in human post-mortem studies.
Growth factors are critical in neurodevelopment and neuroplasticity, and recent studies point to their involvement in addiction. We previously reported increased levels of basic fibroblast growth factor (FGF2) in high novelty/drug-seeking rats (bred High Responders, bHR) compared to low novelty/drug-seeking rats (bred Low Responders, bLRs). The present study asked whether an early life manipulation of the FGF system (a single FGF2 injection on postnatal day 2) can impact cocaine sensitization and associated neurobiological markers in adult bHR/bLR animals. Neonatal FGF2- and vehicle-treated bHR/bLR rats were sensitized to cocaine (7 daily injections, 15 mg/kg/day, i.p.) in adulthood. Neonatal FGF2 markedly increased bLRs’ typically low psychomotor sensitization to cocaine (day 7 locomotor response to cocaine), but had little effect on bHRs’ cocaine sensitization. Gene expression studies examined dopaminergic molecules as well as FGF2 and the FGFR1 receptor in cocaine naïve animals, to investigate possible neurobiological alterations induced by neonatal FGF2 exposure that may influence behavioral response to cocaine. bLRs showed decreased tyrosine hydroxylase in the ventral tegmental area (VTA), decreased D1 and increased D2 receptor expression in the nucleus accumbens core, as well as decreased FGF2 in the VTA, substantia nigra, accumbens core, and caudate putamen compared to bHRs. Neonatal FGF2 selectively increased D1 receptor and FGF2 mRNA in the accumbens core of bLRs, which may contribute to their heightened cocaine sensitization. Our results suggest increased FGF2 in the mesodopaminergic circuit (as in baseline bHRs and neonatal FGF2-exposed bLRs vs. baseline bLRs) enhances an individual’s susceptibility to cocaine sensitization and may increase vulnerability to drug seeking and addiction.
bred High Responder; bred Low Responder; dopamine; FGF2; nucleus accumbens; cocaine
In this review we propose a broader view of the role of the fibroblast growth factor (FGF) family in modulating brain function. We suggest that some of the FGF ligands together with the FGF receptors are altered in individuals with affective disorder and modulate emotionality in animal models. Thus, we propose that members of the FGF family may be genetic predisposing factors for anxiety, depression or substance abuse; that they play a key organizing role during early development but continue to play a central role in neuroplasticity in adulthood; and that they work not only over extended time frames, but also via rapid signaling mechanisms, allowing them to exert an “on-line” influence on behavior. Therefore, the FGF family appears to be a prototype of “switch genes” that are endowed with organizational and modulatory properties across the lifespan, and that may represent molecular candidates as biomarkers and treatment targets for affective and addictive disorders.
hippocampus; drug abuse; stress; anxiety; depression
Rationale and objectives
The 5-HT2A and 5-HT2C receptors have been shown to be differentially involved in modulating cocaine-induced behaviors. In this study we investigated the effects of the 5-HT2A antagonist MDL100907 (0.3 mg/kg, i.p.) and the 5-HT2C antagonist SB242084 (0.5 mg/kg, i.p.) on development, expression, and recall of cocaine-induced conditioned place preference (CPP) in (HR) high- and (LR) low-responder rats to novelty.
First, we examined the effects of MDL100907 and SB242084 on development of cocaine-induced CPP. Our results indicated that LR, but not HR, animals conditioned with SB242084+cocaine showed a significantly higher CPP response than controls. This effect was long-lasting, as it was still present 30 days after the last conditioning session. Second, we investigated the acute effects of MDL-100907 and SB242084 on CPP expression 24h after cocaine conditioning. Again, our data showed that SB242084 significantly enhanced the expression of cocaine CPP in LR, but not, HR animals. Finally, we studied the acute effects of MDL100907 and SB242084 on CPP recall 30 days after cocaine conditioning. Neither MDL100907 nor SB242084 significantly affected the CPP response regardless of the rats’ behavioral phenotype.
This is the first study investigating the contribution of 5-HT2A and 5-HT2C receptors on development, expression and recall of cocaine-induced CPP in the HR-LR model of individual vulnerability to drug abuse. Our results show that SB242084 differentially modulates development and expression of CPP in HR vs. LR rats, and suggest that 5-HT2C receptors play a key role in individual differences on cocaine reward-related learning/memory processes.
Genetic factors and early life adversity are critical in the etiology of mood disorders and substance abuse. Because of their role in the transduction of stress responses, glucocorticoid hormones and their receptors could serve as both genetic factors and mediators of environmental influences. We have shown that constitutive overexpression of the glucocorticoid receptor (GR) in forebrain results in increased emotional reactivity and “lability” in mice. Here we asked whether there was a critical period for the emergence of this phenotype.
We generated a mouse line with inducible GR overexpression specifically in forebrain (GRov). Anxiety-like behaviors and cocaine-induced sensitization were assessed in adult mice following GR overexpression during different periods in development. The molecular basis of the behavioral phenotype was examined using microarray analyses of dentate gyrus and nucleus accumbens.
Transient overexpression of GR during early life led to increased anxiety and cocaine sensitization, paralleling the phenotype of lifelong GR overexpression. This increased emotional reactivity was not observed when GR overexpression was induced after weaning. GR overexpression in early life is sufficient to alter gene expression patterns for the rest of the animal’s life, with dentate gyrus being more responsive than nucleus accumbens. The altered transcripts are implicated in GR and axonal guidance signaling in dentate gyrus and dopamine receptor signaling in nucleus accumbens.
Transient overexpression of GR early in life is both necessary and sufficient for inducing transcriptome-wide changes in the brain and producing a lifelong increase in vulnerability to anxiety and drugs of abuse.
Glucocorticoid receptor; early life; forebrain; anxiety; sensitization; cocaine
Cues associated with rewards acquire the ability to engage the same brain systems as rewards themselves. However, reward cues have multiple properties. For example, they not only act as predictors of reward capable of evoking conditional responses (CRs), but they may also acquire incentive motivational properties. As incentive stimuli they can evoke complex emotional and motivational states. Here we sought to determine whether the predictive value of a reward cue is sufficient to engage brain reward systems, or whether the cue must also be attributed with incentive salience. We took advantage of the fact that there are large individual differences in the extent to which reward cues are attributed with incentive salience. When a cue (conditional stimulus, CS) is paired with delivery of food (unconditional stimulus, US), the cue acquires the ability to evoke a CR in all rats; that is, it is equally predictive and supports learning the CS-US association in all. However, only in a subset of rats is the cue attributed with incentive salience, becoming an attractive and desirable incentive stimulus. We used in situ hybridization histochemistry to quantify the ability of a food cue to induce c-fos mRNA expression in rats that varied in the extent to which they attributed incentive salience to the cue. We found that a food cue induced c-fos mRNA in the orbitofrontal cortex, striatum (caudate and nucleus accumbens), thalamus (paraventricular, intermediodorsal and central medial nuclei) and lateral habenula, only in rats that attributed incentive salience to the cue. Furthermore, patterns of “connectivity” between these brain regions differed markedly between rats that did or did not attribute incentive salience to the food cue. These data suggest that the predictive value of a reward cue is not sufficient to engage brain reward systems - the cue must also be attributed with incentive salience.
sign-trackers; goal-trackers; incentive salience; c-fos; mesocorticolimbic; motive circuit
We have previously demonstrated that selectively-bred High (bHR) and Low (bLR) novelty-seeking rats exhibit agonistic differences, with bHRs acting in a highly aggressive manner when facing homecage intrusion. In order to discover the specific neuronal pathways responsible for bHRs’ high levels of aggression, the present study compared c-fos mRNA expression in several forebrain regions of bHR/bLR males following this experience. bHR/bLR males were housed with female rats for two weeks, and then the females were replaced with a male intruder for 10 min. bHR/bLR residents were subsequently sacrificed by rapid decapitation, and their brains were removed and processed for c-fos in situ hybridization. Intrusion elicited robust c-fos mRNA expression in both phenotypes throughout the forebrain, including the septum, amygdala, hippocampus, cingulate cortex, and the hypothalamus. However, bHRs and bLRs exhibited distinct activation patterns in select areas. Compared to bHR rats, bLRs expressed greater c-fos in the lateral septum and within multiple hypothalamic nuclei, while bHRs showed greater activation in the arcuate hypothalamic nucleus and in the hippocampus. No bHR/bLR differences in c-fos expression were detected in the amygdala, cortical regions, and striatum. We also found divergent 5-HT1A receptor mRNA expression within some of these same areas, with bLRs having greater 5-HT1A, but not 5-HT1B, receptor mRNA levels in the septum, hippocampus and cingulate cortex. These findings, together with our earlier work, suggest that bHRs exhibit altered serotonergic functioning within select circuits during an aggressive encounter.
c-fos; bred High Responder (bHR); bred Low Responder (bLR); septum; hypothalamus; hippocampus; aggression; resident-intruder test; serotonin; 5-HT1A receptor; 5-HT1B receptor
Aggression frequently coincides with specific dimensions of emotionality, such as impulsivity, risk-taking, and drug abuse. Serotonergic (5-HTergic) neurotransmission contributes to the regulation of numerous neurobiological functions, and is thought to play a key role in modulating aggressive responses. The current study uses selectively-bred High (bHR) and Low (bLR) Responder rats that exhibit differences in emotionality and behavioral control, with bHRs exhibiting heightened novelty-induced exploration, impulsivity, and increased sensitivity to drugs of abuse, and with bLRs characterized by exaggerated depressive- and anxiety-like behaviors. Based on this behavioral profile we hypothesized that bHR rats exhibit increased aggression along with changes in testosterone and corticosterone secretion characteristic of aggression, and that these changes are accompanied by alterations in the expression of key genes that regulate 5-HTergic neurotransmission (Tph2 and Sert) as well as in the activation of 5-HTergic cell groups following aggressive encounter. Our data demonstrate that when compared to bLR rats, bHRs express increased baseline Tph2 and Sert in select brainstem nuclei, and when tested on the resident-intruder test they exhibited: 1) increased aggressive behavior; 2) potentiated corticosterone and testosterone secretion; and 3) diminished intrusion-induced c-fos expression in select 5-HTergic brainstem cell groups. The most prominent gene expression differences occurred in the B9 cell group, pontomesencephalic reticular formation, median raphe, and the gigantocellular nucleus pars α. These data are consistent with the notion that altered 5-HT neurotransmission contributes to bHRs’ heightened aggression. Furthermore, they indicate that a specific subset of brainstem 5-HTergic cell groups contributes to the regulation of intrusion-elicited behavioral responses.
emotionality; brainstem; TPH2; SERT; c-fos; resident-intruder test
The current study uses an extended access rat model of cocaine self-administration (5-h session per day, 14 days), which elicits several features manifested during the transition to human addiction, to study the neural adaptations associated with cocaine withdrawal. Given that the hippocampus is thought to have an important role in maintaining addictive behavior and appears to be especially relevant to mechanisms associated with withdrawal, this study attempted to understand how extended access to cocaine impacts the hippocampus at the cellular and molecular levels, and how these alterations change over the course of withdrawal (1, 14, and 28 days). Therefore, at the cellular level, we examined the effects of cocaine withdrawal on cell proliferation (Ki-67+ and NeuroD+ cells) in the DG. At the molecular level, we employed a ‘discovery' approach with gene expression profiling in the DG to uncover novel molecules possibly implicated in the neural adaptations that take place during cocaine withdrawal. Our results suggest that decreased hippocampal cell proliferation might participate in the adaptations associated with drug removal and identifies 14 days as a critical time-point of cocaine withdrawal. At the 14-day time-point, gene expression profiling of the DG revealed the dysregulation of several genes associated with cell fate regulation, highlighting two new neurobiological correlates (Ascl-1 and Dnmt3b) that accompany cessation of drug exposure. Moreover, the results point to Fas-Associated protein with Death Domain (FADD), a molecular marker previously associated with the propensity to substance abuse and cocaine sensitization, as a key cell fate regulator during cocaine withdrawal. Identifying molecules that may have a role in the restructuring of the hippocampus following substance abuse provides a better understanding of the adaptations associated with cocaine withdrawal and identifies novel targets for therapeutic intervention.
extended access cocaine self-administration; withdrawal; cell fate regulation; rat brain hippocampus; microarray analysis; addiction and substance abuse; animal models; psychostimulants; molecular and cellular neurobiology; extended access cocaine self-administration; withdrawal; cell fate regulation; rat brain hippocampus; microarray analysis