Association between amygdala reactivity and a dopamine transporter gene polymorphism.

Essential for detection of relevant external stimuli and for fear processing, the amygdala is under modulatory influence of dopamine (DA). The DA transporter (DAT) is of fundamental importance for the regulation of DA transmission by mediating reuptake inactivation of extracellular DA. This study examined if a common functional variable number tandem repeat polymorphism in the 3' untranslated region of the DAT gene (SLC6A3) influences amygdala function during the processing of aversive emotional stimuli. Amygdala reactivity was examined by comparing regional cerebral blood flow, measured with positron emission tomography and [(15)O]water, during exposure to angry and neutral faces, respectively, in a Swedish sample comprising 32 patients with social anxiety disorder and 17 healthy volunteers. In a separate US sample, comprising 85 healthy volunteers studied with blood oxygen level-dependent functional magnetic resonance imaging, amygdala reactivity was assessed by comparing the activity during exposure to threatening faces and neutral geometric shapes, respectively. In both the Swedish and the US sample, 9-repeat carriers displayed higher amygdala reactivity than 10-repeat homozygotes. The results suggest that this polymorphism contributes to individual variability in amygdala reactivity.

Effects of verbal working memory load on corticocortical connectivity modeled by path analysis of functional magnetic resonance imaging data.

We investigated the hypothesis that there are load-related changes in the integrated function of frontoparietal working memory networks. Functional magnetic resonance imaging time-series data from 10 healthy volunteers performing a graded n-back verbal working memory task were modeled using path analysis. Seven generically activated regions were included in the model: left/right middle frontal gyri (L/R MFG), left/right inferior frontal gyri (L/R IFG), left/right posterior parietal cortex (L/R PPC), and supplementary motor area (SMA). The model provided a good fit to the 1-back (chi(2) = 7.04, df = 8, P = 0.53) and 2-back conditions (chi(2) = 9.35, df = 8, P = 0.31) but not for the 3-back condition (chi(2) = 20.60, df = 8, P = 0.008). Model parameter estimates were compared overall among conditions: there was a significant difference overall between 1-back and 2-back conditions (chi(2)(diff) = 74.77, df = 20, P < 0.001) and also between 2-back and 3-back conditions (chi(2)(diff) = 96.28, df = 20, P < 0.001). Path coefficients between LIFG and LPPC were significantly different from zero in both 1-back and 2-back conditions; in the 2-back condition, additional paths from LIFG to LPPC via SMA and to RMFG from LMFG and LPPC were also nonzero. This study demonstrated a significant change in functional integration of a neurocognitive network for working memory as a correlate of increased load. Enhanced inferior frontoparietal and prefrontoprefrontal connectivity was observed as a correlate of increasing memory load, which may reflect greater demand for maintenance and executive processes, respectively.

Opioid peptide modulation of circulatory response to hyperventilation in humans.

After hyperventilation, systolic blood pressure (SBP) significantly decreased in 10 subjects (group 1), did not change in eight (group 2) and increased in 15 (group 3). Diastolic blood pressure and heart rate increased in all groups. The decrease in SBP was associated with a decrease in plasma catecholamines and increase in beta-endorphin, whereas the increase in SBP was accompanied by an increase in catecholamine and Met-enkephalin levels. Naloxone abolished the hyperventilation-induced SBP and catecholamine decrease only in group 1. These findings show an activation of the endogenous opioid system after hyperventilation and the role of beta-endorphin in reducing SBP in response to the test.

Neural substrate of nicotine addiction as defined by functional brain maps of gene expression.

The distributed neural networks involved in the intravenous self-administration of nicotine and cocaine, and in a model of relapse of nicotine-taking after abstinence, were compared in Wistar rats. Post-mortem brain maps of c-fos-related antigens expression showed specific activation in prefrontal cortex, anterior cingulate and nucleus accumbens for both drugs, but of the anterior cingulate cortex only during relapse, suggesting that a subset of the neural network involved in drug self-administration is activated during relapse.

Transgenic mice in drug dependence research.

Transgenic mice with null mutation of specific genes of the central nervous system obtained by homologous recombination, called also knock-out mice, have been recently used by behavioural neuroscientists to understand better the relevance of certain biological mechanisms of drug dependence or addiction. This article reviews some of the main contributions to this fastly developing field. As addictive drugs exert similar reinforcing effects both in humans and other mammals, changes in behavioural performance produced by the motivational effects of the addictive drugs in knock-out mice can give important information about the relevance of that particular gene product (eg a neurotransmitter receptor) for the pathogenicity of substance abuse disorders. In same cases the deletion of a given gene for a neurotransmitter receptor involved in the action of addictive drugs is associated with a phenotype that reproduces the effects obtained by the pharmacological administration of an antagonist for the same receptor. In other instances, surprising results are obtained, the most striking being the evidence that mice lacking the dopamine transporter gene, the most important binding site of cocaine, retain the capability to self-administer cocaine intravenously. Because the gene deletion is operative during embryogenesis, some adaptive compensatory mechanisms may produce unexpected results, suggesting caution in the interpretation of these results. The advent of tissue-specific inducible knock-out mice will soon produce a second revolution in the field of substance abuse research.

Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field.

The aim of the present experiment was to study the effects of early postnatal maternal separation on behavioural and adrenocortical responses to novelty in rats tested as adults. Sprague-Dawley rat pups were exposed to daily maternal separation (5 h/day) from postnatal day 2 to 6, during the stress hyporesponsive period. Since this procedure requires physical contact with the animals, a first control group of daily handled pups was introduced. A second control group, consisting of pups never handled or separated from the mother, was also considered. At postnatal day 45, the rats were tested in a two-compartment exploratory apparatus: the maternally separated and the non-handled rats, whose behavioural performance did not differ, showed higher emotional behaviour when compared with the handled rats (P < 0.05), suggesting that the handling procedure but not maternal separation improved the capacity to cope with novelty. Corticosterone plasma levels were found to be higher in the maternally separated rats than in the other two groups (P < 0.05), either at resting conditions or at 30 min after novelty exposure (P < 0.05). Levels of nuclear glucocorticoid receptor immunoreactivity in the CA1 hippocampal field were shown to be regulated by novelty exposure, as expected, in both the handled and the non-handled rats but not in the maternally separated rats. In conclusion, repeated maternal separation periods of 5 h/day during the first week of life produced long-lasting effects on the hippocampal regulation of the hypothalamic-pituitary-adrenocortical axis, which appear to be associated with increased responsiveness to stress stimuli in adulthood.

Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine.

Release of the neurotransmitter dopamine in the mesolimbic system of the brain mediates the reinforcing properties of several drugs of abuse, including nicotine. Here we investigate the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the beta2 subunit of this receptor. We found that nicotine stimulates dopamine release in the ventral striatum of wild-type mice but not in the ventral striatum of beta2-mutant mice. Using patch-clamp recording, we show that mesencephalic dopaminergic neurons from mice without the beta2 subunit no longer respond to nicotine, and that self-administration of nicotine is attenuated in these mutant mice. Our results strongly support the idea that the beta2-containing neuronal nicotinic acetylcholine receptor is involved in mediating the reinforcing properties of nicotine.

Endogenous opioid peptides and mental stress in congestive heart failure patients.

Two groups of patients with acute congestive heart failure (CHF), New York Heart Association class III, presenting elevated plasma values of beta-endorphin, norepinephrine, atrial natriuretic factor (ANF) and endothelin-1, underwent the Mental Arithmetic Test (MAT) during placebo (n = 10) and naloxone hydrochloride (n = 10) infusion. The MAT during placebo significantly (p < 0.01) increased blood pressure, heart rate, plasma levels of Met-enkephalin, dynorphin B, beta-endorphin, norepinephrine, ANF and endothelin-1. The increases in norepinephrine, ANF and hemodynamics after the MAT during naloxone infusion were higher (p < 0.01) than those during placebo; thus, the transient upregulation of the endogenous opioid system during stress in CHF patients attenuates the hemodynamic response by reducing norepinephrine release.

Molecular and functional diversity at synapses of individual neurons in vitro.

We have quantified activity-dependent uptake of the fluorescent dye FM1-43 in combination with immunocytochemistry for synaptic vesicle-associated proteins (SVPs) at individual synapses in primary cultures of rat cortical neurons. We show that expression of synaptic proteins is highly variable and that the levels of synaptophysin (p38), synapsin I and sv2, but not synapsin II, correlate with the extent of FM1-43 labelling at synapses. The data indicate that SVP levels affect the uptake of FM1-43 with different efficacy (p38 > synapsin I > sv2 or synapsin II). We also found that the relative levels of SVPs vary at individual boutons of single neurons grown in isolation, which indicates that differential regulation of specific SVPs may contribute to the selective modulation of activity at synapses of the same neuron.

Common neural substrates for the addictive properties of nicotine and cocaine.

Regional brain activation was assessed by mapping of Fos-related protein expression in rats trained to self-administration of intravenous nicotine and cocaine. Both drugs produced specific overlapping patterns of activation in the shell and the core of the nucleus accumbens, medial prefrontal cortex, and medial caudate areas, but not in the amygdala. Thus, the reinforcing properties of cocaine and nicotine map on selected structures of the terminal fields of the mesocorticolimbic dopamine system, supporting the idea that common substrates for these addictive drugs exist.

Opioid peptide modulation of circulatory and endocrine response to mental stress in humans.

Healthy subjects were classified according to their percent increase in systolic blood pressure (SBP) after mental arithmetic test (MAT) as low (delta SBP 9.3-15.1%, n = 15) and high (delta SBP 35.1-45.4%, n = 15) responders. During MAT, low responders showed significantly (p < 0.01) increased plasma levels of beta-endorphin, cortisol, catecholamines, and atrial natriuretic factor (ANF) and decreased levels of endothelin-1, whereas high responders showed increased (p < 0.01) levels of Metenkephalin, dynorphin B, and catecholamines. Pretreatment with naloxone hydrochloride enhanced (p < 0.01) SBP, heart rate, noradrenaline, cortisol, and endothelin-1 levels, and reduced (p < 0.01) ANF in low responders in response to MAT, whereas it decreased (p < 0.01) hemodynamic parameters, noradrenaline, and endothelin-1 in high responders. The individual differences in hemodynamic and endocrine responses to MAT may depend on a different activation of the endogenous opioid system.

The reinforcing properties of nicotine are associated with a specific patterning of c-fos expression in the rat brain.

Rats were trained for nicotine intravenous infusions in a self-administration paradigm. The effect of nicotine self-administration on regional brain activity was studied by mapping changes of c-fos expression. Specific nicotine effects were determine by comparing the patterning of Fos-like immunoreactivity (Fos-Ll) in nicotine self-administering rats with that in three different control groups. Controls included rats exposed to the same manipulation as nicotine self-administering rats who received intravenous saline instead of nicotine. In addition, two groups of untrained sham-operated rats exposed daily to the same operant boxes were included: one group had the same food restriction used in the operant training, the other was fed ad libitum. Nicotine self-administration, exposure to saline and food restriction increased Fos-Ll in 43, 33 and three brain regions, respectively, when compared with the control group fed ad libitum. Computer-assisted image analysis of Fos-Ll profiles performed on 16 relevant limbic and sensory structures showed that in saline-treated rats a significant (P < 0.01) increase of Fos-Ll profiles was observed in medial prefrontal cortex, lateral septum, core and ventral shell of nucleus accumbens, claustrum, amygdaloid nuclei, paraventricular thalamic nucleus and lateral geniculate nucleus. A significant (P < 0.01) further increase produced by nicotine was found in medial prefrontal cortex and ventral shell of nucleus accumbens. Interestingly, cingulate and piriform cortex, superior colliculus and medial terminal nucleus of the accessory optic tract were specifically activated by nicotine but not saline. These results show that nicotine self-administration activates sensory structures, as well as limbic structures involved in natural rewarding pathways. The results suggest the involvement of restricted terminal regions of the mesocorticolimbic dopaminergic system in the maintenance of nicotine self-administration.

Learning impairment in transgenic mice with central overexpression of corticotropin-releasing factor.

The present studies were designed to test the learning and memory capacities of transgenic mice with central overexpression of corticotropin-releasing factor in a forced alternation water T-maze task and in the Morris water maze. In T-maze testing, littermate control mice reached a criterion of 70% correct responses after five days of trials, while the performance of transgenic subjects was still random after the same training. In Morris maze testing, control subjects reached the submerged platform significantly faster (F(1.48) = 4.51, P < 0.05) after three days of trials, while the performance of transgenic mice was unimproved over the same period. The deficit in Morris maze performance in transgenic mice was reversed when the platform was visible above the surface of the water. Pre-test administration of the benzodiazepine anxiolytic, chlordiazepoxide (10 mg/kg), before acquisition training also produced a significant (F(4.40) = 16.61, P < 0.001) and persistent improvement in Morris maze performance in transgenic mice when compared to vehicle-treated transgenic litter mates. Finally, there was no evidence of hippocampal cell loss in transgenic brains. The results suggest that corticotropin-releasing factor-overexpressing mice exhibit a profound learning deficit without sensory or motor-related impairments, and that memory plasticity can be restored by anxiolytic pre-treatment. Thus, constitutive overabundance of brain corticotropin-releasing factor may produce hyperemotionality that interferes with learned behaviors. Stress-related disorders characterized by co-morbid deficits in learning/memory may benefit from pharmacological normalization of brain corticotropin-releasing factor systems.

Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain.

In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25-kD synaptosomal-associated protein (SNAP-25a and SNAP-25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP-25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion-related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP-25b expression exceeded SNAP-25a in distribution and intensity, being present in most brain structures . Moderate or high levels of SNAP-25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP-25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP-25a mRNA was the predominant isoform, whereas SNAP-25b expression increased postnatally. The early expression of SNAP-25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP-25a mRNA, but not SNAP-25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate-induced neurotoxic damage of the hippocampal CA3-CA4 regions. Increase of SNAP-25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform-specific role of SNAP-25 in neural plasticity.

Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain.

Nicotine affects many aspects of behaviour including learning and memory through its interaction with neuronal nicotinic acetylcholine receptors (nAChR). Functional nAChRs are pentameric proteins containing at least one type of alpha-subunit and one type of beta-subunit. The involvement of a particular neuronal nicotinic subunit in pharmacology and behaviour was examined using gene targeting to mutate beta 2, the most widely expressed nAChR subunit in the central nervous system. We report here that high-affinity binding sites for nicotine are absent from the brains of mice homozygous for the beta 2-subunit mutation. Further, electrophysiological recording from brain slices reveals that thalamic neurons from these mice do not respond to nicotine application. Finally, behavioural tests demonstrate that nicotine no longer augments the performance of beta 2-1- mice on passive avoidance, a test of associative memory. Paradoxically, mutant mice are able to perform better than their non-mutant siblings on this task.

Sensitization of cocaine-stimulated increase in extracellular levels of corticotropin-releasing factor from the rat amygdala after repeated administration as determined by intracranial microdialysis.

Using intracranial microdialysis, the effect of repeated cocaine (30 mg/kg i.p.) versus saline administration for 10 consecutive days upon basal and stimulated release of corticotropin-releasing factor (CRF) was examined in the central amygdaloid nucleus (CeA) of anesthetized rats. No significant differences in basal CRF levels between daily cocaine and saline treated groups were found. However, after cocaine challenge (10 mg/kg i.p.) the increase in CRF overflow was significantly greater in cocaine- as opposed to saline-pretreated rats (266 +/- 55.4% versus 149 +/- 8.5% of basal levels). Local administration of 4-aminopyridine produced a significant increase in CRF efflux (195 +/- 58.5%) in daily cocaine-treated rats with only a weak response in the control group (127 +/- 30.9%). These data demonstrate that repeated administration of cocaine enhances cocaine-induced release of CRF in the rat CeA. The sensitization of CRF release may play a significant role in psychostimulant-induced sensitization phenomena.

Rapid induction of tumor necrosis factor alpha in the cerebrospinal fluid after intracerebroventricular injection of lipopolysaccharide revealed by a sensitive capture immuno-PCR assay.

Tumor necrosis factor alpha (TNF-alpha) is an important mediator in many pathophysiologic processes, both in the central nervous system (CNS) and in the periphery. For this study, we have designed a very sensitive immuno-PCR detection system to investigate the time course of TNF-alpha induction in the rat cerebrospinal fluid after intracerebroventricular administration of bacterial lipopolysaccharide (LPS). Immuno-PCR combines antibody specificity with PCR signal amplification and provides a sensitivity in the picomolar range. The enhanced sensitivity of this assay allowed the detection of TNF-alpha in the cerebrospinal fluid as early as 15 min after intracerebroventricular administration of LPS. The present results suggest that the ventricular compartment of the CNS, although confined within the blood-brain barrier, is highly responsive to proinflammatory stimuli such as LPS administration. Insight into the molecular mechanisms underlying this compartmentalization could be key to the pathology and treatment of many CNS diseases, especially the meningitides.

Anti-stress action of a corticotropin-releasing factor antagonist on behavioral reactivity to stressors of varying type and intensity.

Central administration of a Corticotropin-Releasing Factor (CRF) antagonist is well documented to attenuate a variety of behavioral responses to several distinct stressors; however, it is not yet clear whether the activation of CRF neurons is dependent on the type or intensity of the experimental stressor, or rather on the particular behavioral response to stress under study. To test the generality of the stress-protective effect of the CRF antagonist, alpha-helical CRF9-41, (1, 5 or 25 micrograms intracerebroventricularly), the present experiments employed a sensitive index of anxiogenic-like behavior by measuring suppression in exploration on the elevated plus-maze following exposure to social, swim, or restraint stressors. A 1 but not 5 or 25 micrograms dose of the CRF antagonist administered just prior to social, swim, or restraint stress reversed the stress-induced inhibition of exploratory behavior. Chlordiazepoxide and the steroid anesthetic, alphaxalone, also attenuated the anxiogenic-like effect of restraint stress and elevated the baseline exploratory behavior of nonstressed control groups. Although the stressors produced a graded secretion of adrenocorticotropin (ACTH) with the ranking restraint > swim > social, the relative amplitude of behavioral reactivity to social, swim, and restraint stress was comparable. The relative efficacy of the CRF antagonist to reverse the stressor effects was also comparable. These results suggest that antagonism of activated brain CRF systems attenuates the behavioral response to stress regardless of the type or intensity of the stressor as measured by ACTH secretion.

The role of corticotropin-releasing factor in the anxiogenic effects of ethanol withdrawal.

In summary, endogenous CRF has been demonstrated to play an important role in the endocrine but also autonomic and behavioral responses to a stressor and to mediate some of the signs and symptoms observed in human affective and anxiety disorders. These findings led to the hypothesis that the anxiety that characterizes drug withdrawal, such as ethanol withdrawal in humans, may be related in part to the action of CRF-producing neurons in the CNS. Indeed, rats made dependent on an ethanol liquid diet showed significant signs of enhanced stress responsiveness that was blocked by intracerebral administration of a CRF antagonist. At this time little is known about the specific site of action for endogenous CRF. However, recent studies using local administration of CRF antagonist and in vivo CRF microdialysis suggest that the central nucleus of the amygdala may be an important site for the increases in CRF activity associated with the anxiogenic effects of ethanol withdrawal. Although preliminary, these results propound that ethanol dependence may involve a prolonged dysregulation of the CRF system in the basal forebrain that may contribute to the increased motivational effect of ethanol withdrawal.