Neurotransmission-related gene expression in the frontal pole is altered in subjects with bipolar disorder and schizophrenia.

The frontal pole (Brodmann area 10, BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning prior to the age of onset for bipolar disorder (BP) and schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ postmortem samples (n = 72). We chose this method for its high sensitivity to detect low-level expression. We then strengthened our findings by performing a meta-analysis of publicly released BA10 microarray data (n = 101) and identified sources of convergence with our qPCR results. To improve interpretation, we leveraged the unusually large database of clinical metadata accompanying our samples to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly available datasets. Using these convergent sources of evidence, we identified 20 neurotransmission-related genes that were differentially expressed in BP and SCHIZ in BA10. These results included a large diagnosis-related decrease in two important therapeutic targets with low levels of expression, HTR2B and DRD4, as well as other findings related to dopaminergic, GABAergic and astrocytic function. We also observed that therapeutics may produce a differential expression that opposes diagnosis effects. In contrast, substances of abuse showed similar effects on BA10 gene expression as BP and SCHIZ, potentially amplifying diagnosis-related dysregulation.

Genetic background and epigenetic modifications in the core of the nucleus accumbens predict addiction-like behavior in a rat model.

This study provides a demonstration in the rat of a clear genetic difference in the propensity for addiction-related behaviors following prolonged cocaine self-administration. It relies on the use of selectively bred high-responder (bHR) and low-responder (bLR) rat lines that differ in several characteristics associated with "temperament," including novelty-induced locomotion and impulsivity. We show that bHR rats exhibit behaviors reminiscent of human addiction, including persistent cocaine-seeking and increased reinstatement of cocaine seeking. To uncover potential underlying mechanisms of this differential vulnerability, we focused on the core of the nucleus accumbens and examined expression and epigenetic regulation of two transcripts previously implicated in bHR/bLR differences: fibroblast growth factor (FGF2) and the dopamine D2 receptor (D2). Relative to bHRs, bLRs had lower FGF2 mRNA levels and increased association of a repressive mark on histones (H3K9me3) at the FGF2 promoter. These differences were apparent under basal conditions and persisted even following prolonged cocaine self-administration. In contrast, bHRs had lower D2 mRNA under basal conditions, with greater association of H3K9me3 at the D2 promoter and these differences were no longer apparent following prolonged cocaine self-administration. Correlational analyses indicate that the association of H3K9me3 at D2 may be a critical substrate underlying the propensity to relapse. These findings suggest that low D2 mRNA levels in the nucleus accumbens core, likely mediated via epigenetic modifications, may render individuals more susceptible to cocaine addiction. In contrast, low FGF2 levels, which appear immutable even following prolonged cocaine exposure, may serve as a protective factor.

Antecedents and consequences of drug abuse in rats selectively bred for high and low response to novelty.

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. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Collateralized dorsal raphe nucleus projections: a mechanism for the integration of diverse functions during stress.

The midbrain dorsal raphe nucleus (DR) is the origin of the central serotonin (5-HT) system, a key neurotransmitter system that has been implicated in the expression of normal behaviors and in diverse psychiatric disorders, particularly affective disorders such as depression and anxiety. One link between the DR-5-HT system and affective disorders is exposure to stressors. Stress is a major risk factor for affective disorders, and stressors alter activity of DR neurons in an anatomically specific manner. Stress-induced changes in DR neuronal activity are transmitted to targets of the DR via ascending serotonergic projections, many of which collateralize to innervate multiple brain regions. Indeed, the collateralization of DR efferents allows for the coordination of diverse components of the stress response. This review will summarize our current understanding of the organization of the ascending DR system and its collateral projections. Using the neuropeptide corticotropin-releasing factor (CRF) system as an example of a stress-related initiator of DR activity, we will discuss how topographic specificity of afferent regulation of ascending DR circuits serves to coordinate activity in functionally diverse target regions under appropriate conditions.

Stress-induced redistribution of corticotropin-releasing factor receptor subtypes in the dorsal raphe nucleus.

BACKGROUND: The stress-related neuropeptide corticotropin-releasing factor (CRF) is involved in determining behavioral strategies for responding to stressors, in part through its regulation of the dorsal raphe (DR)-serotonin (5-HT) system. CRF(1) and CRF(2) receptor subtypes have opposing effects on this system that are associated with active versus passive coping strategies, respectively. METHODS: Immunoelectron microscopy and in vivo single-unit recordings were used to assess CRF receptor distribution and neuronal responses, respectively, in the DR of stressed and unstressed rats. RESULTS: Here we show that in unstressed rats CRF(1) and CRF(2) are differentially distributed within DR cells, with CRF(1) being prominent on the plasma membrane and CRF(2) being cytoplasmic. Stress experience reverses this distribution, such that CRF(2) is recruited to the plasma membrane and CRF(1) tends to internalize. As a consequence of this stress-induced cellular redistribution of CRF receptors, neuronal responses to CRF change from inhibition to a CRF(2)-mediated excitation. CONCLUSIONS: Given evidence that CRF(1) and CRF(2) activation are associated with distinct behavioral responses to stress, the stress-triggered reversal in receptor localization provides a cellular mechanism for switching behavioral strategies for coping with stressors.

Co-localization of corticotropin-releasing factor and vesicular glutamate transporters within axon terminals of the rat dorsal raphe nucleus.

Electrophysiological, microdialysis and behavioral studies support a modulatory role for corticotropin-releasing factor (CRF) in regulating the dorsal raphe nucleus (DRN)-serotonin (5-HT) system. CRF and 5-HT are implicated in the pathophysiology of depression, thus neuroanatomical substrates of CRF-DRN-5-HT interactions are of interest. Identification of co-transmitters within DRN CRF axon terminals is important for elucidating the complex effects underlying CRF afferent regulation of DRN neurons. This study investigated whether CRF-labeled axon terminals within the DRN contain immunoreactivity for vesicular glutamate transporters (isoforms vGlut1 and vGlut2) indicative of the excitatory neurotransmitter glutamate. Dual immunohistochemistry for CRF and either vGlut1 or vGlut2 was conducted within the same tissue section and immunofluorescence results indicated patterns of immunoreactivity consistent with previous reports. Abundant vGlut1- and vGlut2-immunoreactivity was found in puncta exhibiting a largely uniform distribution, whereas CRF-immunoreactivity was localized to topographically distributed varicose processes within the DRN. Profiles containing both CRF- and either vGlut1- or vGlut2-immunoreactivity were apparent in the DRN. Electron microscopy confirmed that immunoreactivity for CRF and vGlut1 was localized primarily to separate axon terminals in the DRN, with a subset co-localizing CRF and vGlut1. Examination of CRF and vGlut2 immunoreactivities in the DRN indicated that CRF and vGlut2 were found within the same axon terminal more frequently than CRF and vGlut1. Overall, these anatomical findings suggest that CRF may function, in part, with the excitatory neurotransmitter glutamate in the modulation of neuronal activity in the DRN.

Differential projections of dorsal raphe nucleus neurons to the lateral septum and striatum.

The dorsal raphe nucleus (DRN)-serotonin (5-HT) system has been implicated in acute responses to stress and stress-related psychiatric disorders such as anxiety and depression. Stress alters serotonin (5-HT) release in a regionally specific manner. For example, swim stress increases extracellular levels of 5-HT in the striatum and decreases levels in the lateral septum. This finding suggests that the 5-HT efferents to the striatum and lateral septum arise from distinct populations of DRN neurons that are differentially affected by swim stress. To further examine this, retrograde axonal transport of fluorescent RetroBeads was used to identify the distribution of DRN neurons projecting to the lateral septum and striatum in the rat brain. Retrograde labeling from the lateral septum was observed primarily within the more caudal portions of the DRN, while labeling from the striatum was observed in neurons located in the more rostral regions of the DRN. Few cell bodies were observed that were labeled from both the striatum and lateral septum suggesting that DRN neurons do not send collateralized projections to the septal region and striatum. Many septal- and striatal-projecting neurons in the DRN exhibited 5-HT, and collateralized projections, when observed, were immunoreactive for 5-HT. Taken together with previous microdialysis studies, these results support the existence of distinct DRN-5-HT-forebrain projections that are differentially regulated by stress.

Ultrastructural evidence for a role of gamma-aminobutyric acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system.

The dorsal raphe nucleus (DRN) serotonin (5-HT) system has been implicated in acute responses to stress and in stress-related psychiatric disorders such as anxiety and depression. Substantial findings suggest that the neuropeptide corticotropin-releasing factor (CRF) is instrumental in modulating the activity of this system during stress. Because the DRN is neurochemically heterogeneous, dual immunoelectron microscopy was used to examine cellular substrates for interactions between CRF and either 5-HT or gamma-aminobutyric acid (GABA) in the dorsolateral and ventromedial DRN. CRF immunoreactivity was identified primarily within axon terminals, where immunolabeling was particularly enriched in dense-core vesicles. Although CRF terminals targeted 5-HT-containing dendrites in the dorsolateral DRN (16%; n = 251 terminals), synaptic contacts with dendrites that lacked detectable 5-HT immunolabeling were more numerous (48%). In contrast, dual labeling for CRF and GABA (n = 240 terminals) in the dorsolateral DRN revealed that substantially more CRF terminals contacted GABA dendrites (42%) as opposed to unlabeled dendrites (29%). In the ventromedial DRN, contacts between CRF axon terminals and either 5-HT-labeled dendrites or GABA-containing dendrites were fewer than in the dorsolateral DRN. As in the dorsolateral DRN, CRF terminals more frequently contacted GABA dendrites than 5-HT dendrites (30% vs. 8%, respectively). The findings support physiological studies suggesting that CRF has both direct and indirect effects on DRN-5-HT neurons and further implicate GABA as a primary mediator by which CRF and stressors alter the activity of the DRN-5-HT system.

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system.

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.