Neuroanatomical, electrophysiological, and morphological characterization of melanin-concentrating hormone cells coexpressing cocaine- and amphetamine-regulated transcript.

Melanin-concentrating hormone (MCH) cells in the hypothalamus regulate fundamental physiological functions like energy balance, sleep, and reproduction. This diversity may be ascribed to the neurochemical heterogeneity among MCH cells. One prominent subpopulation of MCH cells coexpresses cocaine- and amphetamine-regulated transcript (CART), and as MCH and CART can have opposing actions, MCH/CART+ and MCH/CART- cells may differentially modulate behavioral outcomes. However, it is not known if there are differences in the cellular properties underlying their functional differences; thus, we compared the neuroanatomical, electrophysiological, and morphological properties of MCH cells in male and female Mch-cre;L10-Egfp reporter mice. Half of MCH cells expressed CART and were most prominent in the medial hypothalamus. Whole-cell patch-clamp recordings revealed differences in their passive and active membrane properties in a sex-dependent manner. Female MCH/CART+ cells had lower input resistances, but male cells largely differed in their firing properties. All MCH cells increased firing when stimulated, but their firing frequency decreases with sustained stimulation. MCH/CART+ cells showed stronger spike rate adaptation than MCH/CART- cells. The kinetics of excitatory events at MCH cells also differed by cell type, as the rising rate of excitatory events was slower at MCH/CART+ cells. By reconstructing the dendritic arborization of our recorded cells, we found no sex differences, but male MCH/CART+ cells had less dendritic length and fewer branch points. Overall, distinctions in topographical division and cellular properties between MCH cells add to their heterogeneity and help elucidate their response to stimuli or effect on modulating their respective neural networks.

High Intensity Interval Training can Ameliorate Hypothalamic Appetite Regulation in Male Rats with Type 2 Diabetes: The Role of Leptin.

Disruption of leptin (LEP) signaling in the hypothalamus caused by type 2 diabetes (T2D) can impair appetite regulation. The aim of this study was to investigate whether the improvement in appetite regulation induced by high-intensity interval training (HIIT) in rats with T2D can be mediated by LEP signaling. In this study, 20 male Wister rats were randomly assigned to one of four groups: CO (non-type 2 diabetes control), T2D (type 2 diabetes), EX (non-type 2 diabetes exercise), and T2D + EX (type 2 diabetes + exercise).To induce T2D, a combination of a high-fat diet for 2 months and a single dose of streptozotocin (35 mg/kg) was administered. Rats in the EX and T2D + EX groups performed 4-10 intervals of treadmill running at 80-100% of their maximum velocity (Vmax). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), serum levels of insulin (INS) and LEP (LEPS) as well as hypothalamic expression of LEP receptors (LEP-R), Janus kinase 2 (JAK-2), signal transducer and activator of transcription 3 (STAT-3), neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin cocaine (POMC), amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1) were assessed. ANOVA and Tukey post hoc tests were used to compare the results between the groups. The levels of LEPS and INS, as well as the levels of LEP-R, JAK-2, STAT-3, POMC, and CART in the hypothalamus were found to be higher in the T2D + EX group compared to the T2D group. On the other hand, the levels of HOMA-IR, NPY, AGRP, SOCS3, and FOXO1 were lower in the T2D + EX group compared to the T2D group (P < 0.0001). The findings of this study suggest that HIIT may improve appetite regulation in rats with T2D, and LEP signaling may play a crucial role in this improvement. Graphical abstract (leptin signaling in the hypothalamus), Leptin (LEP), Leptin receptor (LEP-R), Janus kinase 2 (JAK2), Signal transducer and activator of transcription 3 (STAT3), expressing Neuropeptide Y (NPY), Agouti-related protein (AGRP), anorexigenic neurons (expressing pro-opiomelanocortin cocaine (POMC), Amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1).

Chemistry, pharmacology, and metabolism of emerging drugs of abuse.

In recent years, besides the classic designer drugs of the amphetamine type, a series of new drug classes appeared on the illicit drugs market. The chemistry, pharmacology, toxicology, metabolism, and toxicokinetics is discussed of 2,5-dimethoxy amphetamines, 2,5-dimethoxy phenethylamines, beta-keto-amphetamines, phencyclidine derivatives as well as of herbal drugs, ie, Kratom. They have gained popularity and notoriety as rave drugs. The metabolic pathways, the involvement of cytochrome P450 isoenzymes in the main pathways, and their roles in hepatic clearance are also summarized.

Involvement of hypothalamic neuropeptide Y in regulating the amphetamine-induced appetite suppression in streptozotocin diabetic rats.

BACKGROUND AND AIM: Amphetamine (AMPH) is a well-known anorectic agent. In normal rats, AMPH-induced anorexia has been attributed to its inhibitory action on hypothalamic neuropeptide Y (NPY), an appetite stimulant in the brain. In diabetic rats, however, if this anorectic response of AMPH might still be observed was uncertain. METHODS: Rats (including normal, diabetic and insulin-treated diabetic rats) were given daily with saline or AMPH for 6 days. Changes in food intake, plasma glucose level (PGL) and NPY content of these rats were measured and compared. RESULTS: The AMPH-induced anorectic response was altered in diabetic rats. Although the anorectic effects of AMPH on the first day of dosing were similar between diabetic and control rats, diabetic rats developed tolerance to this anorexia more rapidly than control rats. This alteration was independent of PGL since PGL levels were not changed following AMPH treatment and PGL normalization induced by phlorizin could not restore the level of AMPH anorexia. On the other hand, this alteration was dependent on the action of NPY because NPY contents were decreased following AMPH treatment and the replacement of insulin in diabetic rats could restore both NPY content and AMPH anorexia. CONCLUSION: These results suggested that the elevated hypothalamic NPY content in diabetic rats was involved in modifying the anorectic response of AMPH.

Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake.

Neuropeptide Y (NPY) conjugated to saporin (NPY-SAP), a ribosomal inactivating toxin, is a newly developed compound designed to selectively target and lesion NPY receptor-expressing cells. We injected NPY-SAP into the basomedial hypothalamus (BMH), just dorsal to the arcuate nucleus (ARC), to investigate its neurotoxicity and to determine whether ARC NPY neurons are required for glucoprivic feeding. We found that NPY-SAP profoundly reduced NPY Y1 receptor and alpha MSH immunoreactivity, as well as NPY, Agouti gene-related protein (AGRP), and cocaine and amphetamine-related transcript mRNA expression in the BMH. NPY-SAP lesions were localized to the injection site with no evidence of retrograde transport by hindbrain NPY neurons with BMH terminals. These lesions impaired responses to intracerebroventricular (icv) leptin (5 microg/5 microl x d) and ghrelin (2 microg/5 microl), which are thought to alter feeding primarily by actions on ARC NPY/AGRP and proopiomelanocortin/cocaine and amphetamine-related transcript neurons. However, the hypothesis that NPY/AGRP neurons are required downstream mediators of glucoprivic feeding was not supported. Although NPY/AGRP neurons were destroyed by NPY-SAP, the lesion did not impair either the feeding or the hyperglycemic response to 2-deoxy-D-glucose-induced blockade of glycolysis use. Similarly, responses to glucagon-like peptide-1 (GLP-1, 5 microg/3 microl icv), NPY (5 microg/3 microl icv), cholecystokinin octapeptide (4 microg/kg ip), and beta-mercaptoacetate (68 mg/kg ip) were not altered by the NPY-SAP lesion. Thus, NPY-SAP destroyed NPY receptor-expressing neurons in the ARC and selectively disrupted controls of feeding dependent on those neurons but did not disrupt peptidergic or metabolic controls dependent upon circuitry outside the BMH.

Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant originating from industrial waste. At sublethal concentrations it induces anorexia and weight loss as part of the so-called wasting syndrome. To gain insight into its possible underlying mechanisms, mRNA expression of some key hypothalamic neuropeptides involved in the regulation of body weight was studied using in situ hybridization histochemistry in adult male Sprague-Dawley rats 6 days after single oral administration of TCDD (15 microg/kg) and in age-paired control rats. In TCDD-treated rats which displayed a decrease in body weight gain vs. controls, arcuate nucleus expression of neuropeptide Y (NPY), proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) mRNA was increased. In the lateral hypothalamic area, melanin-concentrating hormone (MCH) mRNA expression was also increased, while levels of CART and orexin/hypocretin mRNA were not significantly changed. Since TCDD is known to bind to the aryl hydrocarbon receptor (AhR), the distribution of the AhR repressor (AhRR), which is co-expressed with AhR in the same cells, was studied by immunohistochemistry in the mouse hypothalamus using mouse AhRR specific antiserum. AhRR immunoreactivity was present in the nuclei of neurons found in all main hypothalamic groups including NPY, CART, MCH and orexin/hypocretin neurons. Xenobiotic response elements were found in these neuropeptide genes with the exception of MCH. Thus changes in expression of orexigenic and anorexigenic neuropeptides after TCDD treatment may help to explain the occurrence of the TCDD-induced weight loss, which may be either directly or indirectly related to the effects of TCDD on neuropeptide expression.

The role of zinc ions in reverse transport mediated by monoamine transporters.

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.

Trastornos adictivos y otros trastornos mentales / Addictive and other mental disorders

Diferentes estudios epidemiológicos y clínicos han demostrado una elevada comorbilidad entre los trastornos relacionados con el abuso de drogas y otros trastornos psiquiátricos, como los trastornos de ansiedad, los trastornos del estado de ánimo, los trastornos de personalidad y los trastornos psicóticos. Los pacientes que demandan tratamiento en los centros específicos de drogodependencias se presentan desestructurados cognitivamente, muestran respuestas elevadas de ansiedad y diversos problemas de interacción social y familiar. En muchos casos esta situación se prolonga una vez iniciado el tratamiento y conseguida la abstinencia a drogas. Estos pacientes presentan diversos indicadores de psicopatología que ensombrecen el propósito de abstinencia a largo plazo, son más inestables, presentan un mayor índice de recaídas y realizan numerosos intentos de deshabituación. A continuación se realiza una revisión de distintos trabajos que han encontrado una asociación entre psicopatología y abuso de drogas. Nos centraremos en estudios sobre las sustancias de abuso que generan una mayor demanda de tratamiento: alcohol, heroína, cocaína y anfetaminas. El conocimiento por parte de los clínicos de la incidencia de psicopatología en este tipo de pacientes resulta crucial para planificar sus intervenciones (AU)

Changes in high K+-evoked serotonin release and serotonin 2A/2C receptor binding in the frontal cortex of rats with thioacetamide-induced hepatic encephalopathy.

Serotonergic systems were investigated in the frontal cortex of rats with thioacetamide (TAA)-induced acute hepatic encephalopathy (HE). Extracellular basal levels of 5-HT showed no difference between control and HE animals, whereas the levels of 5-HIAA were significantly increased in HE rats. Unlike basal levels, high K+-evoked 5-HT release was significantly higher in HE rats than controls. Bmax of (+/-)-1-(2,5-dimethoxy-4-[125I] iodophenyl)-2-aminopropane ([125I] DOI) binding, mainly labeling postsynaptic 5-HT2A receptors, was significantly decreased without any change in Kd in HE rats. These results suggest that there is no change in basal 5-HT release in the cortex of rats with TAA-induced HE despite the increase in intraneuronal 5-HT metabolism and in the size of releasable 5-HT pool, and that serotonergic neurotransmission via 5-HT2A receptor is altered in the brain area of rats with HE.

Time courses of changes in cerebral blood flow and blood-brain barrier integrity by focal proton radiation in the rat.

In order to know the pathophysiological mechanisms underlying radiation brain injury, cerebral blood flow and blood-brain barrier integrity were studied using N-isopropyl-p-[123l]iodoamphetamine (IMP) and [14C]-alpha-aminoisobutyric acid (AIB), respectively, in the rat focal proton radiation model (a single dose of 30 or 60 Gy radiation with 70 MeV proton beams). One, 2, 4, and 5.5 months after irradiation, [123l]IMP and [14C]AIB were intravenously injected and uptake of IMP and AIB in the cerebral cortex, striatum, hippocampus, and thalamus was measured. Significant decreases in IMP uptake were observed in the cerebral cortex and thalamus of the irradiated side at 4 and 5.5 months after 60 Gy irradiation; the effects at 5.5 months were more prominent than those at 4 months. AIB uptake markedly increased in all the brain regions of the irradiated side at 5.5 months after 60 Gy irradiation, and at 4 months, only in the hippocampus. The results suggest that there are dose- and time-dependent responses in radiation effects and regional differences in tissue vulnerability to radiation. Proton focal radiation model appears to be a useful model for studies of radiation brain injury in small animals such as rats.

Metabolism in vitro of radioiodinated N-isopropyl-p-iodoamphetamine by isolated hepatocytes.

An in vitro technique for the determination of radiopharmaceutical metabolism has been developed using isolated hepatocytes. Radioiodinated N-isopropyl-p-iodoamphetamine (IMP; iofetamine, USP) was employed a model tracer in these studies because its labeled metabolites are well-characterized. Hepatocytes isolated from the rat and human produced labeled metabolites in vitro for up to 4 h in a manner similar to that reported for humans in vivo. Identical metabolites were generated by all cell types investigated, but the rate of metabolism differed (rat >> human; female > male; fresh > frozen). The utility of this methodology for the preclinical evaluation of potential radiopharmaceuticals is described.

Failure to detect any transport of the irreversible monoamine oxidase inhibitor N-cyclopropyl-4-chloroamphetamine by the carrier of 5-hydroxytryptamine in the brain of the rat in vivo.

N-Cyclopropyl-4-chloroamphetamine (LY 93716) was examined for its potential preference for inhibiting the activity of monoamine oxidase (MAO) within serotonergic nerve terminals in the hypothalamus of the rat. Such an effect should support the hypothesis of Fuller and Perry (1977) (Neuropharmacology 16: 495-497) that this compound is transported by the uptake mechanism for 5-hydroxytryptamine (5-HT). By using a small (0.1 microM) substrate concentration of [14C]5-HT and a synaptosomal preparation incubated in the absence and presence of a selective inhibitor of the uptake of 5-HT (citalopram) it is possible to measure the activity of MAO in serotonergic nerve terminals. It was found that LY 93716 caused greater inhibition outside than inside the serotonergic nerve terminals when the activity of MAO was analysed 24 hr after the injection, but an inverted relationship was observed when analysed 72 hr after administration. Inhibition of uptake did not cause any change in the inhibition of MAO within the serotonergic nerve terminals at the former time but antagonized the inhibition observed 72 hr after the injection. It is concluded that the latter effect was due to antagonism of the neurotoxic action of LY 93716 and that no evidence was found that LY 93716 is transported by the uptake carrier for 5-HT.

Synthesis and evaluation of p-iodo-phentermine (IP) as a brain perfusion imaging agent.

p-(123I and 131I)iodo alpha,alpha-dimethylphenethylamine (p-iodophentermine, IP) as the alpha-methylated analogue of iodoamphetamine has been prepared. It is hoped that this methyl substitution will increase the lipophilicity of the agent, enhance resistance to metabolism by monoamine oxidase, and will result in increased initial uptake and slower washout from the brain as compared to N-isopropyl-p-(123I)iodoamphetamine. IP was prepared by diazotization of p-aminophentermine followed by decomposition of the diazonium salt with KI. Radioiodinated IP was prepared either by the solid-phase isotopic exchange reaction or by decomposition of the piperidinotriazene derivative with a radiochemical yield of 40-60%. Biodistribution of 131I-IP in rats showed brain uptake in the range of 1.7% dose g-1 at 5, 30 and 60 min. Imaging studies with 123I-IP in dogs showed high brain extraction and slow washout of activity.

Kinetics and metabolism of amphetamine in the brain of rats of different ages.

The kinetics and tissue distribution of amphetamine and its metabolites p-hydroxyamphetamine (p-PH-A) and p-hydroxynorephedrine (p-OH-NE) were investigated in young adult (3-4 months) and old (20-25 months) male rats, after i.p. injection of 5 mg/kg tritium labelled D-amphetamine. The concentrations of these drugs were determined in plasma, cerebral cortex, brainstem and hypothalamus, by thin layer chromatography. 1. From 60 min up to 4 hrs after injection of amphetamine the concentration of amphetamine in plasma and brain tissue of old rats was significantly (P less than 0.05 higher than in young adult animals. In both age groups the levels of amphetamine in cerebral cortex greater than brain stem greater than hypothalamus. 2. The blood-brain barrier is permutle to p-OH-A; 10 to 20 min afer i.v. injection of 10 muCi/kg of p-OH-A (10 mCi/m mole) the ratio of brain/blood plasma was found to be 1:3. The half life of p-OH-A in blood plasma was almost identical after injection of amphetamine and p-OH-A (90 min and 98 min respectively). 3. The levels of p-OH-NE in different brain areas were significantly lower (P less than 0.05) in old animals than in young adult rats 4 hrs after application of amphetamine. This metabolite of amphetamine shows a higher concentration in the hypothalamus earlier than in other brain regions.