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Biomedical subjects

M M Garcia

Publications and source records attributed to M M Garcia.

At least 19 recordsLinked to original sources
PubMed2003

Morphine induction of c-fos expression in the rat forebrain through glutamatergic mechanisms: role of non-n-methyl-D-aspartate receptors.

Acute injection of morphine induces expression of the immediate-early genes c-Fos and JunB in several forebrain regions of the rat, in part through an N-methyl-D-aspartate (NMDA) receptor-dependent mechanism. Because membrane depolarization through (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors is believed to be necessary for full activation of NMDA receptors, we determined the role of AMPA receptors in morphine-induced c-Fos expression. Rats were given the AMPA receptor antagonist GYKI-52466 (12.9 mg/kg, i.p.) 15 min before morphine (10 mg/kg, s.c.), or the AMPA receptor enhancer CX516 (30 mg/kg, i.p.) 5 min after morphine. The c-Fos response was attenuated by the antagonist and augmented by the enhancer. Using double immunocytochemistry, we found that morphine induced c-Fos in neurons containing the GluR2/3, but not the GluR1 and rarely the GluR4, subunits of the AMPA receptor. Double immunocytochemistry for mu opioid receptor and c-Fos showed that c-Fos expression was mainly absent in the patch compartment of the striatum, which is enriched in mu opioid receptors. The glutamatergic synapse often contains metabotropic receptors as well as ionotropic receptors. Type I metabotropic glutamate receptors are coupled to activation of protein kinase C, which has also been shown to mediate the immediate-early gene response to morphine. To determine if activation of metabotropic glutamate receptors is involved in rapid effects of morphine on the brain, rats were given the type I metabotropic glutamate receptor antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.2 mg/kg, i.p.) or vehicle 30 min before morphine treatment. Pretreatment with AIDA completely blocked morphine-induced c-Fos expression in the caudate-putamen.Taken together, these results demonstrate involvement of both AMPA and type I metabotropic glutamate receptors in the acute effects of morphine on the forebrain, supporting an important role for glutamatergic neurotransmission mediated by non-NMDA glutamate receptors in morphine's actions.

Animals↗
PubMed2002

Subspecies characterization of porcine Campylobacter coli and Campylobacter jejuni by multilocus enzyme electrophoresis typing.

Enteropathogenic Campylobacterjejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated foodstuffs. C jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and L-phenylalanyl-L-leucine peptidase giving 5. 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C jejuni isolates and 19 unique ETs were detected for the 23 C coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C jejuni and C coli. respectively. Alleles were shared by C jejuni and C coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C coli in porcine livers. In certain cases, up to four phenotypically different strains of C coli were isolated from one liver, indicating multiple infections.

Animals↗
PubMed2001

Isolation and polymerase chain reaction-based detection of Campylobacter jejuni and Campylobacter coli from poultry in the Philippines.

The polymerase chain reaction (PCR) and the conventional culture method of detecting thermophilic Campylobacter species in duck and chicken samples from two locations in the province of Laguna, Philippines, were compared. Three Campylobacter jejuni and five C. coli strains were isolated from a total of 135 duck and chicken samples from both methods. The PCR technique, however, was found to be more sensitive, accurate and rapid than the conventional culture method. The specificity of two sets of published primers, C442-C490 (specific for C. jejuni, C. coli and C. lari) and CL2-CR3 (specific for C. jejuni) were confirmed with reference and field strains. To improve detection, a lysate was prepared by boiling cells in Triton X-100, and then used as template for PCR to detect Campylobacter from spiked and naturally contaminated chicken rinse. For spiked chicken samples, a 17-h Meuller-Hinton Broth enrichment for the chicken rinse resulted in an improved sensitivity at 31.7 CFU/g using C442-C490. This enrichment-PCR tandem also detected thermophilic Campylobacter from 1 out of 21 native chicken samples from a wet market. To our knowledge, this is the first report of thermophilic Campylobacter isolation from poultry in the Philippines. The approaches described here could serve as a basis for future surveillance and/or epidemiological studies on this emerging foodborne pathogen.

Animals↗
PubMed2001

Involvement of nitric oxide in morphine-induced c-Fos expression in the rat striatum.

Induction of expression of immediate-early gene c-Fos in the striatum is a common effect of many drugs of abuse, including morphine. Previous studies have shown that the morphine-mediated c-Fos response is attenuated by antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. Other evidence suggests that the NDMA receptor may be coupled to the enzyme neuronal nitric oxide synthase (nNOS). NMDA receptor-mediated increases in intracellular calcium can activate nNOS, which catalyzes the formation of the signaling molecule nitric oxide. Because activation of NMDA receptors mediates morphine-induced c-Fos expression, we tested the hypothesis that activation of nNOS is involved in this cascade. Male rats were injected with the nNOS-selective inhibitor 7-nitroindazole (7-NI) or vehicle 30 min prior to injection of morphine sulfate or vehicle. Two hours later they were perfused with fixative and the brains removed for immunocytochemical analysis for c-Fos. Morphine induced c-Fos expression in the striatum, cerebral cortex, and midline/intralaminar nuclei of thalamus. Expression in the striatum, but not thalamus or cortex, was significantly blocked by 7-NI. Double-label immunocytochemistry revealed no co-localization of c-Fos and nNOS in any brain region. These results support a role for nNOS in the neural circuits activated by morphine.

Animals↗
PubMed2001

The effect of proteolytic enzymes on the alpha9-nicotinic receptor-mediated response in isolated frog vestibular hair cells.

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells ( approximately 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the alpha9-nicotinic response, suggesting an enzymatic interaction with the alpha9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha9nAChR, SK, or both.

Acetylcholine↗
PubMed2001

Intrafamily conflict in relation to boys' adjustment at school.

Several theoretical models of child development have posited the spread of conflict within families; however, few researchers have studied this process in association with relationships children have with teachers and peers at school. The present study examined the direct, additive, and interactive contributions of interparental, parent-child, and sibling conflict in relation to teacher-child and child-peer conflict in a sample of 117 low-income boys from ages 3.5 to 6 years. Overall, the results suggest that while conflict in any one dyadic family relationship is only modestly associated with later conflictual relationships at school, risk for conflict in relationships with teachers and peers increases when multiple forms of early family conflict are experienced. Results are discussed in terms of the development of conflict across early relationships.

Child↗
PubMed2001

5-hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach.

To examine the role of the 5-hydroxytryptamine(1B) (5-HT1B) and 5-HT3 receptor subtypes in the analgesia produced by 5-HT (serotonin) agonists, we assessed the effect of antisense oligodeoxynucleotides (AODNs) designed to "knock down" the number of these receptor subtypes on analgesia produced by intrathecal (i.t.) 5-HT, the 5-HT1B receptor agonist, 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline maleate (CGS-12066A), and the 5-HT3 receptor agonist, 2-methyl-5-HT. Groups of mice (n = 17-20) were injected i.t. on days 1, 3, and 5 with one of the AODNs, a mismatch oligo, or saline. On day 6, all mice were injected i.t. with 70.5 nmol of 5-HT, 44.4 nmol of CGS-12066A, or 49 nmol of 2-methyl-5-HT by lumbar puncture. Following testing, spinal cords were rapidly removed and prepared for receptor binding assays. Treatment with AODN for 5-HT1B receptors produced a 70% reduction in ligand binding to this receptor subtype. After treatment with AODN for 5-HT3 receptors, ligand binding to this receptor subtype was undetectable. In mice tested with i.t. 5-HT, tail-flick analgesia was attenuated only in mice treated with the 5-HT3 receptor AODN. Mice treated with the AODN designed to knock down 5-HT(1B) receptors or with its mismatch oligo were not significantly different from controls. In mice tested with i.t. administration of CGS-12066A, none of the oligo treatments produced a significant attenuation of analgesia. In mice tested with i.t. administration of 2-methyl-5-HT, only 5-HT3 receptor AODN attenuated analgesia. Thus, 5-HT and 2-methyl-5-HT analgesia are mediated by the 5-HT3 receptor subtype. However, spinal CGS-12066A analgesia appears not to be mediated by either the 5-HT1B or the 5-HT3 receptor subtypes.

Animals↗
PubMed2000

Androgenic-anabolic steroids blunt morphine-induced c-fos expression in the rat striatum: possible role of beta-endorphin.

Self-administration of large doses of androgenic-anabolic steroids (AAS) in a significant portion of the population suggests that these agents are drugs of abuse. However, acute administration of AAS did not induce striatal immediate-early genes (IEG) expression in male rats, indicating that AAS do not share a common mechanism of action with other drugs of abuse. Surveys have indicated that people who abuse AAS are more likely to self-administer other drugs of abuse than do people who do not take AAS. In the present study, chronic administration of AAS blunted the striatal c-fos response to morphine, indicating that AAS can alter the molecular responses to at least one drug of abuse. Chronic administration of AAS also increased the content of beta-endorphin in the midline thalamus, suggesting a possible mechanism by which AAS may modulate the response to morphine through regulation of thalamo-striatal neurons.

Anabolic Agents↗
PubMed2000

Deafferentation-induced changes in protein kinase C expression in the rat cochlear nucleus.

Isoforms of the signal transducing molecule, protein kinase C (PKC), may play a role in neural plasticity following sensory deafferentation. To explore the role of PKC in central auditory plasticity, we studied the effect of auditory deafferentation on the expression of PKC betaI, betaII, gamma, and delta in the rat dorsal (DCN) and ventral cochlear nucleus (VCN), using immunocytochemistry. Male rats were treated with kanamycin and furosemide to induce hair cell loss. At various intervals post-treatment, brains were perfusion-fixed and processed for immunocytochemistry. Following deafferentation, we observed a gradual increase in PKC betaI immunoreactivity (ir) in the deepest layers of the DCN, possibly representing synapses of primary afferents or parallel fibers on unlabeled neurons. Correlated with this, we observed an increase in the number of neurons in the deep DCN that showed PKC delta ir. In controls, we observed PKC gamma ir in small ovoid cells concentrated in the middle layer of the DCN. From days 4 through 14 after deafferentation, we found an increase in the intensity of staining of these cells, with a return toward control levels by day 28. Finally, Purkinje-like cells (PLC) in the VCN, which express only PKC delta in control rats, began to express PKC gamma after deafferentation, correlated with increased expression of calbindin D28k in PLC. Thus PKC isoforms are differentially regulated in the CN following deafferentation, supporting a role for PKC in auditory plasticity.

Animals↗
PubMed2000

Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy.

We developed a method of double immunocytochemistry (ICC) that can be used with conventional light microscopy for localizing two different nuclear proteins. The procedure involves two sequential rounds of ICC that both employ the avidin and biotin conjugated enzyme (ABC) amplification method, separated by an Avidin D and biotin blocking step to reduce non-specific avidin-biotin reactions. Round one of ICC employs the use of avidin and biotin conjugated alkaline phosphatase (ABC-AP) and the Vector Red (VR) substrate, which produces a red colorimetric reaction product. The second round of ICC makes use of avidin and biotin conjugated peroxidase (ABC-HRP) and the Vector(R) SG substrate, which produces a gray colorimetric reaction product. Neuronal nuclei that are double-labeled for both proteins appear red with a gray core. This protocol allows the simultaneous detection of two proteins within the same subcellular compartment of a single neuron, without the need for epifluorescence or scanning confocal laser microscopy.

Alkaline Phosphatase↗
PubMed2000

Destructive sibling conflict and the development of conduct problems in young boys.

Little empirical work has explored the relation between destructive sibling conflict and conduct problems in children. This study used a measure of observed sibling conflict to examine its relations with maternal and teacher report of conduct problems in a low-income sample of 180 five-year-old boys and their close-age siblings. Early report of behavior problems and rejecting parenting were added to the analyses to control for these predictors and to examine interactive effects. The interaction between destructive sibling conflict and rejecting parenting predicted aggressive behavior problems across time and informants such that a rise in aggression scores was evident for children who had high levels of both sibling conflict and rejecting parenting. Sibling conflict was also directly related longitudinally to the Child Behavior Checklist Delinquency factor. Results are discussed in terms of additive risk models and G. R. Patterson's (1984, 1986) theory of coercion.

Aggression↗
PubMed1999

Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay.

To design efficient culture strategies for use with immunoassays to detect Salmonella in food, the growth of these organisms was investigated according to the Bacteriological Analytical Manual (BAM) and enrichment-immunoassay (EI) culture procedures. The cultures were further evaluated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The BAM procedure includes pre-enrichment in nutrient broth (NB) for 16 h followed by selective enrichment in either Rappaport-Vassiliadis (RV) or tetrathionate brilliant green (TBG) broth for 16 h. The EI procedure includes pre-enrichment in NB for 4 h, selective enrichment in RV for 16 h and post-enrichment in NB for 4 h. The effects of different incubation times for pre- and post-enrichment, and different culture media for selective enrichment (TBG and RV) and post-enrichment in NB and Brain Heart Infusion broth (BHI) on the growth of the bacteria and ELISA titers in the EI procedure were also investigated. Salmonella enteritidis and S. typhimurium inoculated at different initial concentrations between 0.1 and 35 CFU/ml grew to similar concentrations of 10(7) to 10(8) colony forming unit (CFU)/ml in pure culture and generally 2 to 4 fold lower concentrations (P<0.05) in mixed culture using spiked chicken rinse. In the BAM procedure, the concentration of Salmonella cultured in RV was higher (P<0.01) than that in TBG. The cultures in TBG showed positive results for ELISA, but those in RV were generally negative. In the EI procedure, the ELISA titers from cultures post-enriched in NB or BHI were higher (P<0.01) when TBG, as compared to RV, was used for selective enrichment. Post-enrichment in BHI yielded higher numbers of Salmonella and higher ELISA titers than those in NB (P<0.05) for post-enrichment. This study demonstrated that in both culture procedures small numbers of Salmonella could be increased to at least 10(7) CFU/ml which is detectable by most ELISAs, and that the type of the culture media used may have a significant impact on ELISA results.

Animals↗
PubMed1999

Role of the dorsal raphe nucleus in morphine-induced immediate early gene expression in the rat striatum.

Serotonin (5-HT) is thought to be involved in morphine action in the brain. To determine if the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN) are involved in morphine-induced c-Fos and JunB expression in the caudate-putamen (CPu), the mu receptor antagonist, beta-funaltrexamine (beta-FNA), was unilaterally infused into the PAG adjacent to DRN prior to morphine. Behaviorally, beta-FNA prevented morphine-induced loss of righting and Straub tail. In the CPu of beta-FNA treated rats, morphine-induced c-Fos and JunB were attenuated compared to vehicle-infused rats. These results suggest that morphine acts within the PAG-DRN to exert rapid behavioral effects and to induce c-Fos and JunB in the striatum.

Analgesics, Opioid↗
PubMed1999

Infusion of beta-FNA into the thalamus attenuates morphine-induced c-Fos induction in the rat caudate putamen.

The medial thalamus contains mu opioid receptors and sends a glutamatergic projection to the caudate putamen (CPu) in rat. Morphine-induced c-Fos expression in the CPu has been shown to be blocked by pretreatment with antagonists to N-methyl-D-aspartate receptors, indicating the involvement of glutamate in this morphine-induced response. The importance of the glutamatergic projections from the thalamus was assessed by infusing the mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), prior to systemic morphine injection. Infusion of beta-FNA near specific medial thalamic nuclei attenuated morphine-induced c-Fos expression in the CPu.

Analgesics, Opioid↗
PubMed1999

Chronic administration of morphine alters immediate-early gene expression in the forebrain of post-dependent rats.

Previous studies from this laboratory have demonstrated that acute, systemic administration of morphine results in an induction of the immediate-early gene (IEG) proteins, c-Fos and Jun-B, in the dorsomedial portion of the rat caudate-putamen (CPu). These studies have also shown that morphine can induce c-Fos in the central medial nucleus of the thalamus (CM). To determine whether this response is altered in post-dependent rats, twice-daily injections of an ascending dose of morphine were administered for 5 days, followed by a withdrawal period of 7 or 14 days. A challenge injection of morphine (10 mg/kg) was administered on the last day of withdrawal. As compared to an acute dose of morphine in a naive animal, the induction of c-Fos was increased in the dorsolateral CPu following challenge injection at 7 days, but not at 14 days. Induction of c-Fos in the CM following the challenge injection was blunted following 7 day, but not at 14 days, of withdrawal. An increase in the IEG protein, Jun-B, was also seen following 7 but not 14 days of withdrawal in both the dorsomedial and dorsolateral CPu. These findings demonstrate that a chronic treatment of morphine can result in altered patterns of IEG expression upon challenge with acute morphine, in a time-dependent manner, within the rat CPu and CM.

Analgesics, Opioid↗
PubMed1999

Sexual dimorphism in the response to N-methyl-D-aspartate receptor antagonists and morphine on behavior and c-Fos induction in the rat brain.

It has been suggested that there are sex differences in the neural response to drugs of abuse. Previous studies have shown that, upon administration of morphine, the immediate early gene c-Fos is induced in the striatum, nucleus accumbens and cortex of the rat brain. This induction of c-Fos is reduced by administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate. However, in studies using immunocytochemistry, we found that the pattern of this expression differed markedly between the sexes. In male rats treated with morphine (10 mg/kg, s.c.) and killed 2 h later, there was an induction of c-Fos in the dorsomedial caudate-putamen, the nucleus accumbens and in the intralaminar nuclei of the thalamus. Administration of dizocilpine maleate (0.2 mg/kg, i.p.; 30 min before morphine) partially blocked the response in the caudate-putamen, but not in the thalamus. In females, morphine induced c-Fos in the caudate-putamen, but with more inter-animal variability than in males. In the midline intralaminar thalamic nuclei, female rats showed less induction than males. In male rats, dizocilpine maleate alone caused negligible induction of c-Fos, whereas in female rats, it caused a large induction in the rhomboid, reuniens and central medial nuclei of the thalamus, and in the cortex. Whereas dizocilpine maleate partially blocked the morphine-induced c-Fos expression in the caudate-putamen of males, it completely blocked this response in females. With dizocilpine maleate alone, there was little or no effect on behavior in male rats, whereas in female rats, it caused head bobbing, thrashing, hyperactivity and uncoordinated movements. These behavioral sex differences were not seen on treatment of rats with the competitive N-methyl-D-aspartate receptor antagonist 2R,4R,5S-2-amino-4,5-(1,2-cyclohexyl)-7-phosphoheptanoic acid (NPC-17742; 10 mg/kg, i.p.) and this drug did not induce c-Fos expression in either sex. In the caudate-putamen, morphine-induced c-Fos expression was significantly reduced by NPC-17742 (30 min before morphine) in males and completely blocked in females. These results suggest that the responses to both morphine and N-methyl-D-aspartate receptor antagonists differ between the sexes and emphasize that glutamate is involved in morphine-induced immediate early gene expression in the brain. These studies thus have important implications for gender differences in drug addiction.

Amino Acids↗
PubMed1998

The 5-HT3 receptor antagonist, MDL 72222, dose-dependently potentiates morphine-induced immediate-early gene expression in the rat caudate putamen.

Previous studies from this laboratory have demonstrated that acute administration of morphine induces the immediate-early genes (IEGs) c-Fos and JunB in the rat caudate putamen (CPu). In the present study, we tested the hypothesis that the serotonin-3 receptor (5-HT3R) is involved in morphine-induced IEG expression, using the selective antagonist to the 5-HT3R, MDL 72222. Rats were divided into three pretreatment groups: MDL 72222, 1 mg/kg or 10 mg/kg; or vehicle (DMSO). Thirty minutes following the pretreatment, the rats were administered either morphine (10 mg/kg) or vehicle. Morphine significantly induced c-Fos expression in the dorsomedial CPu, as we have reported previously. Whereas MDL 72222 alone did not induce c-Fos, it potentiated the morphine-induced c-Fos expression. Morphine also induced JunB expression in the same region of the dorsomedial CPu. At 1 mg/kg, MDL 72222 both induced JunB expression and potentiated the response induced by morphine. At 10 mg/kg, MDL 72222 had no effect on basal JunB levels, but augmented the response to morphine. These findings demonstrate that the 5-HT3R antagonist, MDL 72222, can positively modulate morphine-induced IEG expression in the rat CPu in a dose dependent manner, in contrast to the reported suppressive effect observed when this antagonist is administered prior to amphetamine.

Animals↗