Joanna Peris

Cocaine in utero enhances the behavioral response to cocaine in adult rats

The effects of cocaine exposure in utero on cocaine-induced behaviors and dopamine (DA) transmission in the nigrostriatal and mesolimbic pathways were measured in adult rats. Pregnant rats received either saline or cocaine (1 or 3 mg/kg, IV) daily throughout gestation. When offspring were 3 months of age, locomotor and stereotypic behaviors were rated after an injection of either saline or cocaine (10 mg/kg, IP). Cocaine in utero increased the response to cocaine in adult offspring and increased basal locomotion in female offspring. Cocaine in utero increased amphetamine-stimulated release in female offspring but decreased release in males. On the other hand, male rats that had received cocaine in utero exhibited greater basal tritium release. One injection of cocaine increased amphetamine-stimulated [3H]DA release from striatal slices of male rats but not female rats. Neither cocaine in utero nor in vivo affected D2 DA receptor binding in striatum nor nucleus accumbens. Thus, cocaine in utero behaviorally sensitized animals to subsequent cocaine exposure and increased [3H]DA release from nigrostriatal endings, but the relationship of these two variables depended upon gender.

In vivo monitoring of amino acids by microdialysis sampling with on-line derivatization by naphthalene-2,3-dicarboxyaldehyde and rapid micellar electrokinetic capillary chromatography

An analytical method was developed to monitor amino acids collected by in vivo microdialysis. Microdialysate was continuously derivatized on-line by mixing 6 mM naphthalene-2,3-dicarboxyaldehyde (NDA) and 10 mM potassium cyanide with the dialysate stream in a fused silica capillary to form fluorescent products. Reaction time, determined by the flow rate and volume of reaction capillary, was 3 min. Derivatized amino acids were continuously delivered into a flow-gated interface and periodically injected onto a capillary electrophoresis unit equipped with a laser-induced fluorescence detection based on a commercial microscope. Separation was performed in the micellar electrokinetic chromatography mode using 30 mM sodium dodecyl sulfate in 15 mM phosphate buffer at pH 8.0 as the separation media. An electric field of 1.3 kV/cm was applied across a 10 cm long, 10 microm internal diameter separation capillary. These conditions allowed 17 amino acid derivatives to be resolved in less than 30 s. On-line injections could be performed at 30 s intervals for in vivo samples. Detection limits were from 10 to 30 nM for the amino acids. The method was applied to monitor the acute ethanol-induced amino acid level changes in freely moving rats. The results demonstrate the utility of the method to reveal dynamics of amino acid concentration in vivo.

A Murine Model for Human Sepiapterin-Reductase Deficiency

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

Behavioral and neurochemical changes caused by repeated ethanol and cocaine administration

Combined cocaine and ethanol abuse has become increasingly popular, yet research on the behavioral and neurochemical interactions of these two substances is limited. Four groups of male rats received either daily cocaine (10 mg/kg, IP) or saline injections with either water (groups C and S) or only 15% ethanol to drink (groups CE and E). Initially, locomotor activity was increased equally by ethanol or cocaine and to the greatest extent by both. After 2 weeks of drug treatment, group C exhibited behavioral sensitization to cocaine, group E exhibited ethanol tolerance and group CE exhibited greater cocaine sensitization with no indication of ethanol tolerance. In support of enhanced sensitization to cocaine, amphetamine-stimulated3H-dopamine (DA) release in striatum and D2 DA receptor binding in the nucleus accumbens (NAC) were increased in group CE compared to group C. In support of a loss of ethanol tolerance, increases in striatal D2 DA and35S-TBPS binding seen in group E (which exhibited ethanol tolerance) were absent in group CE (which did not exhibit tolerance). Thus, the synergistic effect of ethanol and cocaine on behavior may be due to complex interactions of these two drugs both on DA and GABA transmission in mesolimbic and nigrostriatal areas.

Microdialysis as a tool in local pharmacodynamics.

In many cases the clinical outcome of therapy needs to be determined by the drug concentration in the tissue compartment in which the pharmacological effect occurs rather than in the plasma. Microdialysis is an in vivo technique that allows direct measurement of unbound tissue concentrations and permits monitoring of the biochemical and physiological effects of drugs throughout the body. Microdialysis was first used in pharmacodynamic research to study neurotransmission, and this remains its most common application in the field. In this review, we give an overview of the principles, techniques, and applications of microdialysis in pharmacodynamic studies of local physiological events, including measurement of endogenous substances such as acetylcholine, catecholamines, serotonin, amino acids, peptides, glucose, lactate, glycerol, and hormones. Microdialysis coupled with systemic drug administration also permits the more intensive examination of the pharmacotherapeutic effect of drugs on extracellular levels of endogenous substances in peripheral compartments and blood. Selected examples of the physiological effects and mechanisms of action of drugs are also discussed, as are the advantages and limitations of this method. It is concluded that microdialysis is a reliable technique for the measurement of local events, which makes it an attractive tool for local pharmacodynamic research.

Differential Increase in Taurine Levels by Low-Dose Ethanol in the Dorsal and Ventral Striatum Revealed by Microdialysis With On-Line Capillary Electrophoresis

Ethanol increases taurine efflux in the nucleus accumbens or ventral striatum (VS), a dopaminergic terminal region involved in positive reinforcement. However, this has been found only at ethanol doses above 1 g/kg intraperitoneally, which is higher than what most rats will self-administer. We used a sensitive on-line assay of microdialysate content to test whether lower doses of ethanol selectively increase taurine efflux in VS as opposed to other dopaminergic regions not involved in reinforcement (e.g., dorsal striatum; DS). Adult male rats with microdialysis probes in VS or DS were injected with ethanol (0, 0.5, 1, and 2 g/kg intraperitoneally), and the amino acid content of the dialysate was measured every 11 sec using capillary electrophoresis and laser-induced fluorescence detection. In VS, 0.5 g/kg ethanol significantly increased taurine levels by 20% for 10 min. A similar increase was seen after 1 g/kg ethanol, which lasted for about 20 min after injection. A two-phased taurine efflux was observed with the 2.0 g/kg dose, where taurine was increased by 2-fold after 5 min but it remained elevated by 30% for at least 60 min. In contrast, DS exhibited much smaller dose-related increases in taurine. Glycine, glutamate, serine, and gamma-aminobutyric acid were not systematically affected by lower doses of ethanol; however, 2 g/kg slowly decreased these amino acids in both brain regions during the hour after injection. These data implicate a possible role of taurine in the mechanism of action of ethanol in the VS. The high sensitivity and time resolution afforded by capillary electrophoresis and laser-induced fluorescence detection will be useful for detecting subtle changes of neuronally active amino acids levels due to low doses of ethanol.

Oxytocin receptors are expressed on dopamine and glutamate neurons in the mouse ventral tegmental area that project to nucleus accumbens and other mesolimbic targets

The mesolimbic dopamine (DA) circuitry determines which behaviors are positively reinforcing and therefore should be encoded in the memory to become a part of the behavioral repertoire. Natural reinforcers, like food and sex, activate this pathway, thereby increasing the likelihood of further consummatory, social, and sexual behaviors. Oxytocin (OT) has been implicated in mediating natural reward and OT‐synthesizing neurons project to the ventral tegmental area (VTA) and nucleus accumbens (NAc); however, direct neuroanatomical evidence of OT regulation of DA neurons within the VTA is sparse. To phenotype OT‐receptor (OTR) expressing neurons originating within the VTA, we delivered Cre‐inducible adeno‐associated virus that drives the expression of fluorescent marker into the VTA of male mice that had Cre‐recombinase driven by OTR gene expression. OTR‐expressing VTA neurons project to NAc, prefrontal cortex, the extended amygdala, and other forebrain regions but less than 10% of these OTR‐expressing neurons were identified as DA neurons (defined by tyrosine hydroxylase colocalization). Instead, almost 50% of OTR‐expressing cells in the VTA were glutamate (GLU) neurons, as indicated by expression of mRNA for the vesicular GLU transporter (vGluT). About one‐third of OTR‐expressing VTA neurons did not colocalize with either DA or GLU phenotypic markers. Thus, OTR expression by VTA neurons implicates that OT regulation of reward circuitry is more complex than a direct action on DA neurotransmission. J. Comp. Neurol. 525:1094–1108, 2017.

Endogenous GABA release is reduced in the striatum of cocaine‐sensitized rats

The magnitude of behavioral sensitization to cocaine is correlated with decreased striatal GABAA receptor function. We examined whether GABA release from striatal slices is also altered in cocaine‐treated rats. Behavioral sensitization was measured in rats receiving either saline or cocaine (15 mg kg‐1) daily for 14 days. Cocaine‐treated rats showed a significant increase in locomotion and stereotypy over days. Potassium‐stimulated endogenous GABA release was measured from superfused striatal slices of these rats. GABA release was significantly decreased in cocaine‐treated rats. However, striatal slices preloaded with [3H]GABA exhibited a slight but significant increase in release after cocaine sensitization. Similar treatment with a nonsensitizing dose of cocaine (7.5 mg kg‐1) did not change endogenous GABA release. Saline‐ and cocaine‐treated rats showed no differences in striatal glutamic acid decarboxylase activity at either a saturating or Km concentration of glutamate. Therefore, the decrease in endogenous GABA release is not due to a decrease in GABA synthesis, but may reflect changes in GABA storage pools. These data are consistent with an overall decrease in GABA transmission, both pre‐ and postsynaptically, in the striatum of sensitized rats, which could contribute to enhanced striatal output and behavioral sensitization. Synapse 34:103–110, 1999.

Peripheral oxytocin administration reduces ethanol consumption in rats

The neuropeptide oxytocin interacts with mesolimbic dopamine neurons to mediate reward associated with filial behaviors, but also other rewarding behaviors such as eating or taking drugs of abuse. Based on its efficacy to decrease intake of other abused substances, oxytocin administration is implicated as a possible treatment for excessive alcohol consumption. We tested this hypothesis by measuring ethanol intake in male Sprague-Dawley rats injected with oxytocin or saline using two different ethanol self-administration paradigms. First, a dose-response curve was constructed for oxytocin inhibition of fluid intake using a modified drinking-in-the-dark model with three bottles containing .05% saccharine, 10% ethanol in saccharine, and 15% ethanol in saccharine. Doses of oxytocin tested were 0.05, 0.1, 0.3, and 0.5mg/kg (I.P.). Next, rats received 0.3mg/kg oxytocin preceding operant sessions in which they were trained to lever-press for either plain gelatin or ethanol gelatin in order to compare oxytocin inhibition of ethanol intake versus caloric intake. For the three-bottle choice study, rats consumed significantly less ethanol when treated with the three higher doses of oxytocin on the injection day. In the operant study, 0.3mg/kg oxytocin significantly decreased ethanol gel consumption to a greater extent than plain gel consumption, both in terms of the amount of gel eaten and calories consumed. These data affirm oxytocins efficacy for decreasing ethanol intake in rats, and confirm clinical studies suggesting oxytocin as a potential treatment for alcoholism.

The novel neurotensin analog NT69L blocks phencyclidine (PCP)-induced increases in locomotor activity and PCP-induced increases in monoamine and amino acids levels in the medial prefrontal cortex

Schizophrenia is a life-long, severe, and disabling brain disorder that requires chronic pharmacotherapy. Because current antipsychotic drugs do not provide optimal therapy, we have been developing novel treatments that focus on receptors for the neuropeptide neurotensin (NT). NT69L, an analog of neurotensin(8-13), acts like an atypical antipsychotic drug in several dopamine-based animal models used to study schizophrenia. Another current animal model utilizes non-competitive antagonists of the NMDA/glutamate receptor, such as the psychotomimetic phencyclidine (PCP). In the present study, we investigated the effects of NT69L on PCP-induced behavioral and biochemical changes in the rat. The top of an activity chamber was modified to allow us to perform microdialysis in rat brain, while simultaneously recording the locomotor activity of a rat. PCP injection significantly increased activity as well as the extracellular concentration of norepinephrine (NE), 5-HT, dopamine (DA), and glutamate in the medial prefrontal cortex (mPFC). Pretreating with NT69L blocked the PCP-induced hyperactivity as well as the increase of DA, 5-HT, NE, and glutamate in mPFC. Interestingly and unexpectedly, NT69L markedly increased glycine levels, while PCP was without effect on glycine levels. Thus, NT69L showed antipsychotic-like effects in this glutamate-based animal model for studying schizophrenia. Previous work from our group suggests that NT69L also has antipsychotic-like effects in dopaminergic and serotonergic rodent models. Taken together, these data suggest that NT69L in particular and NT receptor agonists in general, will be useful as broad-spectrum antipsychotic drugs.