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Evidence suggests a predictive link between elevated basal activity within reward-related networks (e.g., cortico-basal ganglia-thalamic networks) and vulnerability for alcoholism. Both calcium channel function and cyclic adenosine monophosphate (cAMP)/protein kinase A-mediated signaling are critical modulators of reward neurocircuitry and reward-related behaviors. Calcium/calmodulin-stimulated adenylyl cyclases (AC) 1 and 8 are sensitive to activity-dependent increases in intracellular calcium and catalyze cAMP production. Therefore, we hypothesized AC1 and 8 regulate brain activity in reward regions of the cortico-basal ganglia-thalamic circuit and that this regulatory influence predicts voluntary ethanol drinking responses. This hypothesis was evaluated by manganese-enhanced magnetic resonance imaging and chronic, intermittent ethanol access procedures. Ethanol-naive mice with genetic deletion of both AC1 and 8 (DKO mice) exhibited bilateral reductions in baseline activity within cortico-basal ganglia-thalamic regions associated with reward processing compared to wild-type controls (WT, C57BL/6 mice). Significant activity changes were not evident in regions either outside of the cortico-basal ganglia-thalamic network or within the network that are not associated with reward processing. Parallel studies demonstrated that reward network hypoactivity in DKO mice predicted a significant attenuation in consumption and preference levels to escalating ethanol concentrations (12, 20 and 30%) compared to WT mice, an effect that was maintained over extended access (14 sessions) to 20% ethanol. Summarizing, these data support a contribution of AC1 and 8 in cortico-basal ganglia-thalamic activity and the predictive value of this regulatory influence on ethanol drinking behavior, which merits the future evaluation of calcium-stimulated ACs in the neural processes that engender vulnerability to maladaptive alcohol drinking.

The cannabinoid system is independently affected by stress and chronic ethanol exposure. However, the extent to which co-occurrence of traumatic stress and chronic ethanol exposure modulates the cannabinoid system remains unclear. We examined levels of cannabinoid system components, anandamide, 2-arachidonoylglycerol, fatty acid amide hydrolase, and monoacylglycerol lipase after mouse single-prolonged stress (mSPS) or non-mSPS (Control) exposure, with chronic intermittent ethanol (CIE) vapor or without CIE vapor (Air) across several brain regions using ultra-high-performance liquid chromatography tandem mass spectrometry or immunoblotting. Compared to mSPS-Air mice, anandamide and 2-arachidonoylglycerol levels in the anterior striatum were increased in mSPS-CIE mice. In the dorsal hippocampus, anandamide content was increased in Control-CIE mice compared to Control-Air, mSPS-Air, or mSPS-CIE mice. Finally, amygdalar anandamide content was increased in Control-CIE mice compared to Control-Air, or mSPS-CIE mice, but the anandamide content was decreased in mSPS-CIE compared to mSPS-Air mice. Based on these data we conclude that the effects of combined traumatic stress and chronic ethanol exposure on the cannabinoid system in reward pathway regions are driven by CIE exposure and that traumatic stress affects the cannabinoid components in limbic regions, warranting future investigation of neurotherapeutic treatment to attenuate these effects.

Current functional imaging techniques, such as functional magnetic resonance imaging, rely upon activity-induced blood flood changes to neurons. This indirect measurement of neuronal activity inherently limits image resolution and specificity, however, advances in transgenic technology and Photoacoustic (PA) methodology have offered new solutions. We previously demonstrated using PA imaging in conjunction with a Fos-LacZ rat model to map activated neurons through selective formation of PA-active X-gal product within Fos-expressing neurons. While we have previously used intracranial injections for successful delivery of X-gal to detect neurons that are highly-active after classical fear conditioning using PA imaging, now, we have further advanced this technique by using an intrathecal route of administration of X-gal combined with in vivo imaging to improve the translatability of this approach and whole-brain imaging capabilities. We subjected Fos/LacZ rats to fear conditioning, or control conditions. Ninety minutes post stimulus presentation, X-gal was delivered via intrathecal or intravenous injection. After 24 hours, a cranial window was made in animals and they were PA imaged in vivo. PA intensity within the medial prefrontal cortex (mPFC) of acquired images was quantified using ImageJ software. We presently report quantification of PA images of rat brains stained with X-gal product generated in vivo within activated neurons. Detailed PA signal differences are reported between mPFC subregions. We discuss the feasibility and translatability of this reporter method for neuronal activity based on our acquired images. With this technique, we propose a method of longitudinally monitoring activated neurons in vivo with high resolution and specificity.

Neuronal origins of behavioral disorders have been examined for decades to construct frameworks for understanding psychiatric diseases and developing useful therapeutic strategies with clinical application. Despite abundant anecdotal evidence for white matter etiologies, including altered tractography in neuroimaging and diminished oligodendrocyte-specific gene expression in autopsy studies, mechanistic data demonstrating that dysfunctional myelin sheaths can cause behavioral deficits and perturb neurotransmitter biochemistry have not been forthcoming. At least in part, this impasse stems from difficulties in identifying model systems free of degenerative pathology to enable unambiguous assessment of neuron biology and behavior in a background of myelin dysfunction. Herein we examine myelin mutant mice lacking expression of the Claudin11 gene in oligodendrocytes and characterize two behavioral endophenotypes: perturbed auditory processing and reduced anxiety/avoidance. Importantly, these behaviors are associated with increased transmission time along myelinated fibers as well as glutamate and GABA neurotransmitter imbalances in auditory brainstem and amygdala, in the absence of neurodegeneration. Thus, our findings broaden the etiology of neuropsychiatric disease to include dysfunctional myelin, and identify a preclinical model for the development of novel disease-modifying therapies.

Highlights • We developed a photoacoustic modality for direct imaging of Fos-expressing neuronal ensembles in Fos-LacZ transgenic rats. • X-gal product provided exogenous contrast for imaging of neuronal ensembles. • We detected Fos-expressing activated neuronal ensembles after either cocaine administration or a shock-tone pairing paradigm., Measuring neuroactivity underlying complex behaviors facilitates understanding the microcircuitry that supports these behaviors. We have developed a functional and molecular photoacoustic tomography (F/M-PAT) system which allows direct imaging of Fos-expressing neuronal ensembles in Fos-LacZ transgenic rats with a large field-of-view and high spatial resolution. F/M-PAT measures the beta-galactosidase catalyzed enzymatic product of exogenous chromophore X-gal within ensemble neurons. We used an ex vivo imaging method in the Wistar Fos-LacZ transgenic rat, to detect neuronal ensembles in medial prefrontal cortex (mPFC) following cocaine administration or a shock-tone paired stimulus. Robust and selective F/M-PAT signal was detected in mPFC neurons after both conditions (compare to naive controls) demonstrating successful and direct detection of Fos-expressing neuronal ensembles using this approach. The results of this study indicate that F/M-PAT can be used in conjunction with Fos-LacZ rats to monitor neuronal ensembles that underlie a range of behavioral processes, such as fear learning or addiction.

Individual variation in the attribution of motivational salience to reward-related cues is believed to underlie addiction vulnerability. Pavlovian conditioned approach measures individual variation in motivational salience by identifying rats that are attracted to and motivated by reward cues (sign-trackers) or motivationally fixed on the reward itself (goal-trackers). Previously, it has been demonstrated that sign-trackers are more vulnerable to addiction-like behavior. Moreover, sign-trackers release more dopamine in the nucleus accumbens than goal-trackers in response to reward-related cues, and sign- but not goal-tracking behavior is dopamine-dependent. In the present study, we investigated whether the ventral hippocampus, a potent driver of dopaminergic activity in the nucleus accumbens, modulates the acquisition and expression of Pavlovian conditioned approach behavior. In Experiment 1, lesions of the ventral, but not dorsal or total hippocampus, decreased sign-tracking behavior. In Experiment 2, lesions of the ventral hippocampus did not affect the expression of sign- or goal-tracking behaviors nor conditioned reinforcement. In addition, temporary inactivation of the ventral subiculum, the main output pathway of the ventral hippocampus, did not affect the expression of sign- or goal-tracking behaviors. High-pressure liquid chromatography of nucleus accumbens tissue punches revealed that ventral hippocampal lesions decreased levels of homovanillic acid and the homovanillic acid/dopamine ratio (a marker of dopamine release and metabolism) in only sign-trackers, and decreased accumbal norepinephrine levels in both sign- and goal-trackers. These results suggest that the ventral hippocampus is important for the acquisition but not expression of sign-tracking behavior, possibly as a result of altered dopamine and norepinephrine in the nucleus accumbens. © 2016 Wiley Periodicals, Inc.

The cAMP/protein kinase A pathway regulates methamphetamine (METH)‐induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild‐type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH‐induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)‐evoked accumbal dialysate dopamine levels were similar between genotypes, saline‐treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH‐sensitized DKO mice compared to saline‐treated controls, an effect not observed in WT mice. Notably, saline‐treated DKO mice had significantly increased phosphorylated Dopamine‐ and cAMP‐regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug‐induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin‐stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine‐induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP‐32 activation. These findings indicate AC 1/8 signaling regulates the sensitivity of the nigrostriatal pathway subserving stimulant and neuroadaptive sensitizing effects of methamphetamine. 3‐MT, 3‐methoxytyramine; Ca2+, calcium; CaM, calmodulin; cdk5; cyclin‐dependent kinase 5; DA, dopamine; DARPP‐32, dopamine‐ and cAMP‐regulated phosphoprotein; D1R, dopamine D1 receptor; HVA, homovanillic acid; PKA, protein kinase A.

Posttraumatic stress disorder (PTSD) is often comorbid with substance use disorders (SUD). Single prolonged stress (SPS) is a well-validated rat model of PTSD that provides a framework to investigate drug-induced behaviors as a preclinical model of the comorbidity. We hypothesized that cocaine sensitization and self-administration would be increased following exposure to SPS. Male Sprague-Dawley rats were exposed to SPS or control treatment. After SPS, cocaine (0, 10 or 20 mg/kg, i.p.) was administered for 5 consecutive days and locomotor activity was measured. Another cohort was assessed for cocaine self-administration (0.1 or 0.32 mg/kg/i.v.) after SPS. Rats were tested for acquisition, extinction and cue-induced reinstatement behaviors. Control animals showed a dose-dependent increase in cocaine-induced locomotor activity after acute cocaine whereas SPS rats did not. Using a sub-threshold sensitization paradigm, control rats did not exhibit enhanced locomotor activity at Day 5 and therefore did not develop behavioral sensitization, as expected. However, compared to control rats on Day 5 the locomotor response to 20mg/kg repeated cocaine was greatly enhanced in SPS-treated rats, which exhibited enhanced cocaine locomotor sensitization. The effect of SPS on locomotor activity was unique in that SPS did not modify cocaine self-administration behaviors under a simple schedule of reinforcement. These data show that SPS differentially affects cocaine-mediated behaviors causing no effect to cocaine self-administration, under a simple schedule of reinforcement, but significantly augmenting cocaine locomotor sensitization. These results suggest that SPS shares common neurocircuitry with stimulant-induced plasticity, but dissociable from that underlying psychostimulant-induced reinforcement.

Appropriate animal models of posttraumatic stress disorder (PTSD) are needed because human studies remain limited in their ability to probe the underlying neurobiology of PTSD. Although the single prolonged stress (SPS) model is an established rat model of PTSD, the development of a similarly-validated mouse model emphasizes the benefits and cross-species utility of rodent PTSD models and offers unique methodological advantages to that of the rat. Therefore, the aims of this study were to develop and describe a SPS model for mice and to provide data that support current mechanisms relevant to PTSD. The mouse single prolonged stress (mSPS) paradigm, involves exposing C57Bl/6 mice to a series of severe, multimodal stressors, including 2h restraint, 10 min group forced swim, exposure to soiled rat bedding scent, and exposure to ether until unconsciousness. Following a 7-day undisturbed period, mice were tested for cue-induced fear behavior, effects of paroxetine on cue-induced fear behavior, extinction retention of a previously extinguished fear memory, dexamethasone suppression of corticosterone (CORT) response, dorsal hippocampal glucocorticoid receptor protein and mRNA expression, and prefrontal cortex glutamate levels. Exposure to mSPS enhanced cue-induced fear, which was attenuated by oral paroxetine treatment. mSPS also disrupted extinction retention, enhanced suppression of stress-induced CORT response, increased mRNA expression of dorsal hippocampal glucocorticoid receptors and decreased prefrontal cortex glutamate levels. These data suggest that the mSPS model is a translationally-relevant model for future PTSD research with strong face, construct, and predictive validity. In summary, mSPS models characteristics relevant to PTSD and this severe, multimodal stress modifies fear learning in mice that coincides with changes in the hypothalamo-pituitary-adrenal (HPA) axis, brain glucocorticoid systems, and glutamatergic signaling in the prefrontal cortex.

Pavlovian conditioned approach (PCA) is a behavioral procedure that can be used to assess individual differences in the addiction vulnerability of drug-naive rats and identify addiction vulnerability factors. Using proton magnetic resonance spectroscopy (1 H-MRS) ex vivo, we simultaneously analyzed concentrations of multiple neurochemicals throughout the mesocorticolimbic system 2 weeks after PCA training in order to identify potential vulnerability factors to addiction in drug-naive rats for future investigations. Levels of myo-inositol (Ins), a 1 H-MRS-detectable marker of glial activity/proliferation, were increased in the nucleus accumbens (NAc) and ventral hippocampus, but not dorsal hippocampus or medial prefrontal cortex, of sign-trackers compared to goal-trackers or intermediate responders. In addition, Ins levels positively correlated with PCA behavior in the NAc and ventral hippocampus. Because the sign-tracker phenotype is associated with increased drug-seeking behavior, these results observed in drug-naive rats suggest that alterations in glial activity/proliferation within these regions may represent an addiction vulnerability factor. Sign-tracking rats preferentially approach reward cues during Pavlovian conditioning, while goal-trackers instead approach the location of impending reward. Sign-trackers are also more prone to cue-induced drug-seeking behavior. We used magnetic resonance spectroscopy to show that myo-inositol levels are higher in the ventral hippocampus and nucleus accumbens of sign-trackers relative to goal-trackers. Thus, elevated myo-inositol may be a vulnerability factor for addiction.

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.

Maternal care is an indispensable component of offspring survival and development in all mammals and necessary for reproductive success. Although brain areas regulating maternal behaviors are innervated by serotonergic afferents, very little is known about the role of this neurotransmitter in these behaviors. To evaluate the contribution of serotonin to maternal care, we used mice with a null mutation in the gene for tryptophan hydroxylase-2 (TPH2), which results in a genetic depletion of brain serotonin, and tested them in a wide range of maternal behavior paradigms. We found that litters born to and reared by TPH2(-/-) mothers showed decreased survival, lower weaning weights and increased cannibalization. In addition, TPH2(-/-) mothers performed poorly in pup retrieval, huddling, nest construction and high-arched back nursing. Aggression in TPH2(-/-) dams was not triggered by lactation and was steadily high. Survival and weaning weight deficits of TPH2(-/-) pups were rescued by cross-fostering and in litters of mixed genotype (TPH2(-/-) and TPH2(-/+) ). However, the maternal behaviors of TPH2(-/-) dams did not improve when rearing either TPH2(+/+) pups or mixed-genotype litters. In addition, TPH2(-/-) pups significantly worsened the behavior of TPH2(+/+) dams with respect to cannibalism, weaning weight and latency to attack. Olfactory and auditory functions of TPH2(-/-) females or anxiety-like behaviors did not account for these maternal alterations as they were equal to their TPH2(+/+) counterparts. These findings illustrate a profound influence of brain serotonin on virtually all elements of maternal behavior and establish that TPH2(-/-) pups can engender maladaptive mothering in dams of both genotypes.

Gamma-aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the brain and is primarily responsible for modulating excitatory tone. Clinical neuroimaging studies show decreased GABA levels in the anterior cingulate of patients with mood disorders, including major depressive disorder. Chronic unpredictable stress (CUS) is an animal model thought to mimic the stressful events that may precipitate clinical depression in humans. In this study male Sprague-Dawley rats were subjected to a modified CUS paradigm that used a random pattern of unpredictable stressors twice daily for 10 days to explore the early developmental stages of depression-like endophenotypes. Control rats were handled daily for 10 days. Some rats from each treatment group received an injection of ketamine (40 mg/kg) after the final stressor. One day following the final stressor rats were tested for behavioral effects in the forced swim test and then euthanized to collect trunk blood and anterior cingulate brain samples. GABA levels were measured in anterior cingulate samples ex vivo using proton magnetic resonance spectroscopy ((1)H-MRS) at 11.7 T. Animals subjected to CUS had lower body weights, higher levels of blood corticosterone, and increased immobility in the forced swim test; all of which suggest that the stress paradigm induced a depression-like phenotype. GABA levels in the anterior cingulate were significantly increased in the stressed animals compared to controls. Administration of ketamine on the last day of treatment blunted the depression-like behavior and increased GABA levels in the anterior cingulate following CUS. These data indicate that stress disrupts GABAergic signaling, which may over time lead to symptoms of depression and ultimately lower basal levels of cortical (1)H-MRS GABA that are seen in humans with depression. Furthermore, the data suggests that ketamine modulates cortical GABA levels as a mechanism of its antidepressant activity.

Working memory, which is dependent on higher-order executive function in the prefrontal cortex, is often disrupted in patients exposed to blast overpressure. In this study, we evaluated working memory and medial prefrontal neurochemical status in a rat model of blast neurotrauma. Adult male Sprague–Dawley rats were anesthetized with 3% isoflurane and exposed to calibrated blast overpressure (17 psi, 117 kPa) while sham animals received only anesthesia. Early neurochemical effects in the prefrontal cortex included a significant decrease in betaine (trimethylglycine) and an increase in GABA at 24 h, and significant increases in glycerophosphorylcholine, phosphorylethanolamine, as well as glutamate/creatine and lactate/creatine ratios at 48 h. Seven days after blast, only myo-inositol levels were altered showing a 15% increase. Compared to controls, short-term memory in the novel object recognition task was significantly impaired in animals exposed to blast overpressure. Working memory in control animals was negatively correlated with myo-inositol levels (r = − .759, p

Single prolonged stress (SPS) is a rodent model of posttraumatic stress disorder (PTSD)-like characteristics. Given that PTSD is frequently comorbid with substance abuse and dependence, including methamphetamine (METH), the current study sought to investigate the effects of SPS on METH-induced behavioral sensitization.In experiment 1, Sprague-Dawley rats were subject to SPS or control treatment and subsequently tested across four sessions of an escalating METH dosing paradigm. METH was injected (i.p.) in escalating doses (0, 0.032, 0.1, 0.32, 1.0, and 3.2mg/kg; dissolved in saline) every 15min and ambulatory activity was recorded. In experiment 2, SPS and control treated rats were injected (i.p.) with either saline or METH (5mg/kg) for five consecutive daily sessions and tested for stereotypy as well as ambulatory activity. Two days later, all animals were injected with a challenge dose of METH (2.5mg/kg) and again tested for activity.No differences in the acute response to METH were observed between SPS and controls. SPS enhanced METH induced ambulatory activity across sessions, compared to controls. METH-induced stereotypy increased across sessions, indicative of behavioral sensitization; however, SPS attenuated, not enhanced, this effect suggesting that SPS may prevent the development of stereotypy sensitization.Collectively, results show that SPS increases repeated METH-induced ambulatory activity while preventing the transition across sessions from ambulatory activity to stereotypy. These findings suggest that SPS alters drug-induced neuroplasticity associated with behavioral sensitization to METH, which may reflect an effect on the shared neurocircuitry underlying PTSD and substance dependence.

The high comorbidity of anxiety and depression suggests a potential degree of commonality in their etiologies. The chronic unpredictable stress (CUS) model effectively replicates depressive-like phenotypes; however, the ability of CUS to produce anxiety-like behaviors has not been adequately addressed. Using the CUS paradigm (2 stressors per day for 10 days) in adult Sprague-Dawley rats we identified behavioral, hormonal, and neurochemical changes one day after the cessation of treatment. Stress attenuated weight gain throughout the study and increased locomotor activity one day after treatment, but had no effect on anxiety-behavior as measured by the elevated plus maze. In addition, plasma corticosterone levels were positively correlated with hypothalamic serotonin (5-HT) activity one day after stress treatment as determined by the ratio of the metabolite 5-hydroxyindoleacetic acid (5-HIAA) to the parent compound (5-HIAA/5-HT ratio). These data suggest behavioral phenotypes associated with depression, but not comorbid anxiety, emerge in the immediate period after cessation of stress and that stress related physiology is related to 5-HT activity in the hypothalamus.

Application of single prolonged stress (SPS) in rats induces changes in neuroendocrine function and arousal that are characteristic of post traumatic stress disorder (PTSD). PTSD, in humans, is associated with decreased neural activity in the prefrontal cortex, increased neural activity in the amygdala complex, and reduced neuronal integrity in the hippocampus. However, the extent to which SPS models these aspects of PTSD has not been established. In order to address this, we used high-resolution magic angle spinning proton magnetic resonance spectroscopy (HR-MAS (1)H MRS) ex vivo to assay levels of neurochemicals critical for energy metabolism (creatine and lactate), excitatory (glutamate and glutamine) and inhibitory (gamma amino butyric acid (GABA)) neurotransmission, and neuronal integrity (N-acetylaspartate (NAA)) in the medial prefrontal cortex (mPFC), amygdala complex, and hippocampus of SPS and control rats. Glutamate, glutamine, and creatine levels were decreased in the mPFC of SPS rats when compared to controls, which suggests decreased excitatory tone in this region. SPS did not alter the neurochemical profiles of either the hippocampus or amygdala. These data suggest that SPS selectively attenuates excitatory tone, without a disruption of neuronal integrity, in the mPFC.

Despite the high incidence of toluene abuse in adolescents, little is known regarding the effect of binge exposure on neurochemical profiles during this developmental stage. In the current study, the effects of binge toluene exposure during adolescence on neurotransmitter levels were determined using high-resolution proton magnetic resonance spectroscopy ex vivo at 11.7 T. Adolescent male Sprague-Dawley rats were exposed to toluene (0, 8,000 , or 12,000 ppm) for 15 min twice daily from postnatal day 28 (P28) through P34 and then euthanized either one or seven days later (on P35 or P42) to assess glutamate, glutamine, and GABA levels in intact tissue punches from the medial prefrontal cortex (mPFC), anterior striatum and hippocampus. In the mPFC, toluene reduced glutamate one day after exposure, with no effect on GABA, while after seven days, glutamate was no longer affected but there was an increase in GABA levels. In the hippocampus, neither GABA nor glutamate was altered one day after exposure, whereas seven days after exposure, increases were observed in GABA and glutamate. Striatal glutamate and GABA levels measured after either one or seven days were not altered after toluene exposure. These findings show that one week of binge toluene inhalation selectively alters these neurotransmitters in the mPFC and hippocampus in adolescent rats, and that some of these effects endure at least one week after the exposure. The results suggest that age-dependent, differential neurochemical responses to toluene may contribute to the unique behavioral patterns associated with drug abuse among older children and young teens.

Despite the potential for deleterious (even fatal) effects on cardiac physiology, 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse abounds driven mainly by its euphoric effects. Acute exposure to MDMA has profound cardiovascular effects on blood pressure and heart rate in humans and animals. To determine the effects of MDMA on cardiac metabolites in rats, MDMA (0, 5, or 10 mg/kg) was injected every 2 h for a total of four injections; animals were sacrificed 2 h after the last injection (8 h drug exposure), and their hearts removed and tissue samples from left ventricular wall dissected. High resolution magic angle spinning proton magnetic resonance spectroscopy ((1)H-MRS) at 11.7 T, a specialized version of MRS aptly suited for analysis of semi-solid materials such as intact tissue samples, was used to measure the cardiac metabolomic profile, including alanine, lactate, succinate, creatine, and carnitine, in heart tissue from rats treated with MDMA. MDMA effects on MR-visible choline, glutamate, glutamine, and taurine were also determined. Body temperature was measured following each MDMA administration and serotonin and norepinephrine (NE) levels were measured by high pressure liquid chromatography (HPLC) in heart tissue from treated animals. MDMA significantly and dose-dependently increased body temperature, a hallmark of amphetamines. Serotonin, but not NE, levels were significantly and dose-dependently decreased by MDMA in the heart wall. MDMA significantly altered the MR-visible profile with an increase in carnitine and no change in other key compounds involved in cardiomyocyte energy metabolomics. Finally, choline levels were significantly decreased by MDMA in heart. The results are consistent with the notion that MDMA has significant effects on cardiovascular serotonergic tone and disrupts the metabolic homeostasis of energy regulation in cardiac tissue, potentially increasing utilization of fatty acid metabolism. The contributions of serotonergic signaling on MDMA-induced changes in cardiac metabolism remain to be determined.

Previous studies have shown that tachykinin peptide ligands of the tachykinin NK1 receptor exhibit functional selectivity with respect to signal activation and desensitization. The differences are most dramatic between the naturally occurring peptides substance P (RPKPQQFFGLM-NH2) and ranatachykinin C (HNPASFIGLM-NH2). To understand the structural features of the peptides that underlie these differences, four peptide analogs have been designed and tested. The analogs were designed to assess the major structural differences between substance P and ranatachykinin C, including the role of the N-terminal Arg and the substitution of the mid-region Glns with Ala and Ser (Q5 replaced with A and/or Q6 replaced with S). Receptor binding, receptor activation of intracellular calcium fluxes, and receptor desensitization of the rat tachykinin NK1 receptor were quantified for each ligand. All of the peptides bound to the rat tachykinin NK1 receptor with high affinity, produced robust calcium signal activation, and led to agonist-induced receptor desensitization. It was found that deletion of the N-terminal Arg of substance P or replacement of either or both Q5 and Q6 altered the functional selectivity of substance P based on the relationship of receptor binding to receptor activation and activation to desensitization. When considered in light of our previously published nuclear magnetic resonance structure data, the data presented herein suggest that the one, five and six positions of the substance P backbone are key structural residues that govern the relative degree of tachykinin peptide-mediated receptor signaling and desensitization.

Previous studies have shown differences in the biological activity and the structure of two naturally occurring tachykinin peptides, substance P (SP, RPKPQQFFGLM-NH2) and ranatachykinin C (RTKC, HNPASFIGLM-NH2). To further understand the basis for these differences, four analogs that selectively incorporate the amino acid differences between SP and RTKC have been synthesized for study. The four peptide analogs studied have the following amino acid sequences: SP2-11, also known as des-Arg SP (PKPQQFFGLM-NH2); Q5A-SP (RPKPAQFFGLM-NH2); Q6S-SP (RPKPQSFFGLM-NH2); and Q5AQ6S-SP (RPKPASFFGLM-NH2). Nuclear magnetic resonance spectroscopy and molecular modeling calculations were performed on SP, RTKC, SP2-11, Q5A-SP, Q6S-SP, and Q5AQ6S-SP to compare their conformational differences and similarities in the presence of the membrane mimetic system sodium dodecyl sulfate. The molecular modeling data of the analogs Q5A-SP and Q6S-SP show residues 1-3 have a random conformation and residues 4-8 have a helical structure, while the C-terminus contains a poly C7 conformation that is similar to SP but different from RTKC. The molecular modeling data of the analogs SP2-11 and Q5AQ6S-SP show a continuous helix conformation for residues 4-11 at the C-terminus, which is different from SP but similar to RTKC. These structural differences are related to the functional differences of binding of the peptides at the SP receptor (NK1).

The chloride channel agents are activators and inhibitors of various subtypes of chloride channels. Most are used solely for research purposes. Included in this group are 5 amino 2 (2 furyl) 7 (2 phenylethyl)pyrazolo[4,3 e][1,2,4]triazolo[1,5 c]pyrimidine; 5 nitro 2 (3 phenylpropylamino)benzoic acid; 9-AC; DIDS; DPC; Gossypol; GTPgammaS; levamisole; NS004; niflumic acid; SIT S. There have been a number of review articles describing the chloride channels affected by these substances …

5 nitro 2 (3 phenylpropylamino)benzoic acid (NPPB) is a chloride channel blocker derived from anthracene-9-carboxylate …

Following agonist binding, neurokinin-1 receptors undergo rapid desensitization followed by internalization and recycling. Desensitization requires receptor phosphorylation but does not require internalization, whereas resensitization is thought to require internalization and recycling. Our previous data, however, have suggested that, following activation and desensitization, the return of responsiveness to the neurokinin-1 agonist substance P (termed "resensitization") occurs hours before internalized receptors are recycled back to the plasma membrane. To further investigate this novel mechanism of neurokinin-1 receptor resensitization, we have studied the time courses of neurokinin-1 receptor responsiveness, recycling, and dephosphorylation by measuring cellular Ca(2+) responses, ligand-receptor binding, and receptor phosphorylation, respectively. Concentration-response curves and competition binding curves were obtained at various times following desensitization. The effects of the nonhydrolyzable GTP analog Gpp(NH)p on substance P binding were also studied to assess receptor-G protein coupling. After receptor activation and desensitization, Ca(2+) signaling in response to substance P occurred within 90 min, whereas the return of receptor binding required 240 min. Receptor dephosphorylation was greater than 90% complete 20 min after agonist washout. In addition, the return of substance P responsiveness coincided with a return in sensitivity of substance P binding to Gpp(NH)p, indicating a return in receptor-G protein coupling. These data show that the resensitization of responsiveness to substance P precedes receptor recycling. This may result from a conversion of nonfunctional neurokinin-1 receptors to functional receptors at the plasma membrane.

Prolonged or repeated activation of many G protein-coupled receptors induces rapid desensitization followed by a period during which receptors are resensitized. In this study, concanavalin A (Con A) and monensin were used to investigate the mechanisms of desensitization and resensitization of the neurokinin-1 receptor. Con A inhibits internalization, whereas monensin prevents receptor recycling. The effects of Con A and monensin on desensitization, resensitization, receptor phosphorylation, endocytosis, and recycling of the neurokinin-1 receptor were assessed. Desensitization was defined as the decrease in the ability of substance P (SP) to elicit an intracellular Ca2+ response after a prolonged SP exposure. Resensitization was characterized as the return of SP responsiveness. Under control conditions, desensitization occurred after a 5-min exposure to agonist. Resensitization was evident 30 min after agonist washout. Neither monensin nor Con A prevented desensitization. Monensin completely inhibited resensitization, whereas Con A decreased but did not completely block resensitization. Receptor phosphorylation was increased after agonist activation and returned to basal levels after a recovery period. Neither Con A nor monensin altered the amount of agonist-specific receptor phosphorylation. Receptor binding analysis showed that plasma membrane receptors were internalized after a 5-min agonist exposure. Receptor recycling was not observed after a 1-h recovery period; however, resensitization was apparent. Taken together, these results suggest that rapid neurokinin-1 receptor desensitization can occur without receptor internalization and that resensitization occurs before receptor recycling.