Rodrigo Labarca

Characterization of Sodium‐Dependent [3H]GBR‐12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex

Abstract: High‐affinity and saturable binding sites for the diphenyl‐substituted piperazine derivative [3H]GBR‐12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR‐12935 is sodium‐dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium‐dependent [3H]GBR‐12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR‐12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r= 0.96, p < 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR‐12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR‐12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6‐hydroxydopamine results in a decrease in the number of specific [3H]GBR‐12935 binding sites in the striatum. These data indicate that [3H]GBR‐12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.

Phorbol esters inhibit agonist-induced [3H] inositol-1-phosphate accumulation in rat hippocampal slices

In rat hippocampal slices, carbachol and norepinephrine induce an accumulation of [3H]-inositol-1-phosphate which is markedly amplified in the presence of lithium. The tumor-promoting agents phorbol 12,13-dibutyrate (PDB) and 4 beta phorbol, 12 beta-myristate, 13 alpha-acetate (PMA) have no effect on [3H] inositol-1-phosphate accumulation alone, but when preincubated with hippocampal slices significantly inhibit the accumulation of [3H]-inositol-1-phosphate induced by carbachol and norepinephrine. The IC50 values for PDB and PMA are 0.2 microM and 25 microM respectively. In contrast, the weak tumor promoting agents 4-O-methylphorbol 12 myristate 13 acetate (MPMA) and phorbol 13,20-diacetate (P 13,20 DA) only slightly attenuate the agonist-induced response at concentrations less than or equal to 100 microM, whereas 4 alpha-phorbol (4 alpha-PHR), a biologically inactive phorbol, has no effect. These data suggest that phorbol ester receptor-mediated events may be negatively coupled to agonist-induced phosphatidylinositol hydrolysis.

Acute stress enhances the activity of the GABA receptor-gated chloride ion channel in brain

The effect of acute swim stress on the functional activity of the gamma-aminobutyric acid (GABA) receptor/chloride ion channel was studied using an assay to measure 36chloride (36Cl-) uptake into rat brain synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in cerebral cortex, hippocampus, but not cerebellum, was enhanced (increased potency and efficacy) in rats subjected to 10 min of swimming, compared to non-stressed, control rats. The effect of swim stress on the activity of the GABA receptor/Cl ion channel was prevented by adrenalectomy.

Characterization of neurotransmitter receptor-mediated phosphatidylinositol hydrolysis in the rat hippocampus

The stimulation of phosphatidylinositol hydrolysis by various neurotransmitter agonists was investigated in rat hippocampal slices using a rapid and sensitive radioisotopic method. Slices were preincubated with [3H]-myo-inositol and the accumulation of [3H]-myo-inositol-1-phosphate induced by various agonists was determined in the presence of 10 mM lithium. The latter resulted in a marked amplification of the response to all agonists tested. The agonist-induced accumulation of [3H]-myo-inositol-1-phosphate was dependent on tissue, lithium, [3H]-myo-inositol concentration, as well as incubation time. The hydrolysis of phosphatidylinositol in hippocampal slices is induced by carbachol, serotonin, norepinephrine and phenylephrine. The carbachol-induced response is sensitive to atropine, a muscarinic-cholinergic antagonist, but not mecamylamine a nicotinic-cholinergic antagonist, while that of norepinephrine is blocked by the alpha 1 adrenoreceptor antagonist prazosin, but not the specific alpha 2 antagonist Rx 781094. Phenylephrine, another alpha 1 adrenoreceptor agonist produced a partial or submaximal response when compared to norepinephrine. The concentration response curve for serotonin-induced phosphatidylinositol hydrolysis is bimodal and the effect is blocked by metergoline, but not mianserin, indicating that the effect of serotonin in the hippocampus may be mediated by 5HT1 receptors. Our results suggest that the measurement of agonist-induced [3H]-myo-inositol-1-phosphate accumulation, in the presence of lithium, represents a sensitive method for studying a number of receptor-mediated events in brain.

Chronic corticosterone administration in rats: Behavioral and biochemical evidence of increased central dopaminergic activity

Chronic corticosteroid treatment in humans in frequently complicated by behavioral changes. The present study suggests that chronic steroid administration in rats has distinct neurochemical consequences which are behaviorally relevant. Ten male Sprague-Dawley rats received 7 daily injections of corticosterone, following which they exhibited increased caudate homovanillic acid as well as an attenuated decline in vertical and ambulatory movement (functional measures of dopamine activity) compared to placebo-treated rats. A subgroup of steroid-treated rats which was more behaviorally responsive to corticosterone also showed increased caudate 5-hydroxyindole acetic acid and decreased prefrontal cortex dopamine and serotonin. These results are discussed in relation to the known behavioral side effects of chronic corticosteroid administration in man and the psychiatric manifestations of naturally occurring states of hypercortisolemia.

Dexamethasone increases plasma HVA but not MHPG in normal humans

Several recent studies in animals and man indicate that corticosteroids may alter catecholaminergic activity in both the peripheral and central nervous systems. We administered 1 mg of the synthetic glucocorticoid, dexamethasone, to 12 drug-free healthy volunteers and measured plasma homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). Dexamethasone was administered at 11 p.m. and blood was collected at 4 p.m. on the preceding and subsequent days. Dexamethasone administration resulted in a significant increase in plasma HVA but did not consistently affect MHPG. All subjects showed a suppression of serum cortisol to values less than 5 micrograms/dl while prolactin levels were unaltered. In an additional group of nine volunteers, we administered 2 mg of dexamethasone and observed a similar increase in plasma HVA without change in plasma MHPG, indicating a selective effect on dopamine metabolism. Implications of these findings for an understanding of the neurochemical and behavioral changes seen with steroid administration and in explaining previous results on plasma MHPG/HVA ratios in delusional depression are discussed.

Differential regulation of μ-opioid receptor mRNA in the nucleus accumbens shell and core accompanying amphetamine behavioral sensitization

Repeated amphetamine (AMPH) administration results in behavioral sensitization. To investigate the participation of the opioid system in this phenomenon, we examined the effects of acute and repeated AMPH administration on mu-opioid receptor (MOR) mRNA levels in the nucleus accumbens (NAc) and striatum (STR) of rats, by quantitative non-radioactive in situ hybridization. Five injections of d-AMPH (1.5 mg kg-1, i.p., once every other day), resulted in a sensitization response profile and a significant down-regulation of MOR mRNA levels in the NAc shell, whereas no change was observed in MOR mRNA levels in the NAc core compared to the saline controls. Conversely, MOR mRNA levels were up-regulated in the rostral STR of AMPH-sensitized rats compared to saline controls. No changes in MOR mRNA levels were observed after acute AMPH treatment in any of the brain regions studied. These results suggest that the opioid system participates in the neurobiological underpinnings of behavioral sensitization and that opioid receptor (OR) expression in the STR and NAc shell and core is differentially modulated by repeated AMPH exposure.

Autoradiographic visualization and characterization of [3H]ouabain binding to the Na+, K+-ATPase of rat brain and pineal

Ouabain binds to the catalytic subunit of Na+,K+-ATPase and specific [3H]ouabain binding can be used as a measure of the number of active enzyme molecules present in a given tissue. Specific [3H]ouabain binding can be demonstrated in frozen, cryostat sections from rat brain and pineal and these sites have the characteristics of Na+,K+-ATPase. Incubations carried out in the absence of ATP or the presence of excess unlabeled ouabain reduces specific binding by greater than or equal to 98%. The addition of K+ or omission of Mg2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [3H]ouabain binding with IC50 values of 0.73, 0.48 and 1.4 microM, respectively. Scatchard analyses of specific [3H]ouabain binding in brain sections shows a single class of non-interacting binding sites with an apparent affinity (Kd) of 339 nM and a maximal binding capacity (Bmax) of 34.9 pmol/mg protein. [3H]Ouabain binding is unevenly distributed throughout the brain with the olfactory nuclei, superior colliculus, dentate gyrus, pontine nuclei and pineal gland having a relatively high density of binding sites. The outer layers (1-3) of the cerebral cortex show more labeling than the inner layers (4-6) and most other brain areas have intermediate levels of [3H]ouabain binding sites, whereas white matter has virtually no specific binding. Computer-assisted densitometry was used to measure changes in specific [3H]ouabain binding after kainic acid injection into the caudate nucleus. An initial increase in [3H]ouabain binding was observed at 1 and 24 h after lesioning and a decrease in [3H]ouabain binding was evident by 9 days after lesioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes.

In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration-dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1-phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol.

Differential effects of haloperidol on negative symptoms in drug-naive schizophrenic patients: effects on plasma homovanillic acid

After 5 weeks of haloperidol, positive symptoms in drug-naive schizophrenic patients substantially subsided. Negative symptoms, although with a different temporal pattern, decreased after the fifth week of haloperidol treatment; specifically, a decrease was seen in anhedonia and affective flattening, whereas avolition-apathy and attentional impairment presented no changes. Alogia showed a decrease during the third week and a trend to return to placebo scores during weeks 4 and 5. Changes in affective flattening, alogia and attentional impairment correlated with changes in positive symptoms. During placebo, plasma homovanillic acid (HVA) correlated with negative symptoms and with changes presented by negative symptoms between the first and the fifth treatment week. These data show that negative symptoms respond differentially to neuroleptics and suggest that avolition-apathy may represent a different behavioral component of the schizophrenia process.