Long-term treatment of male F344 rats with deprenyl: assessment of effects on longevity, behavior, and brain function.

L-Deprenyl (selegiline) was chronically administered to male Fischer 344 rats via their drinking water beginning at 54 weeks of age (estimated daily dose: 0.5 mg/kg/day). Beginning at 84 weeks of age, the rats were behaviorally evaluated using a sensorimotor battery, a motor-learning task, and the Morris water maze. At 118 weeks of age, cerebellar noradrenergic function was evaluated in the surviving rats using in vivo electrochemistry. The rats were then sacrificed to measure brain monoamine oxidase activity and perform quantitative autoradiography to evaluate the effect of chronic deprenyl treatment on beta-adrenergic receptors in the cerebellum, alpha 2-adrenergic receptors several brain regions, and D1 and D2 dopamine receptors in the striatum. Deprenyl treatment reduced brain monoamine oxidase B activity by 85%, but had no effect on brain monoamine oxidase A. A clear effect of chronic deprenyl treatment upon longevity was not observed. Several measures of CNS function were altered in the deprenyl-treated animals: 1) spatial learning in the Morris water maze was improved; 2) electrochemical signals recorded following local application of NE were reduced, and the responsiveness to the reuptake blocker nomifensine was enhanced, in the cerebellum; 3) beta-adrenergic receptor binding affinity was increased in the cerebellum; 4) alpha 2-adrenergic receptor density was increased in the inferior colliculus; and 5) striatal D1 dopamine receptor density was reduced but binding affinity was enhanced. In contrast, chronic deprenyl treatment did not cause changes in: 1) sensorimotor function, as evaluated by balance beam, inclined screen, or wire hang tasks; 2) motor learning; 3) alpha 2-adrenergic receptor density in any region examined except for the inferior colliculus, or binding affinity in any region examined; or 4) striatal D2 dopamine receptor number or affinity. Thus, long-term oral administration of deprenyl extended the functional life span of rats with respect to cognitive, but not motor, performance.

Reduced clearance of exogenous dopamine in rat nucleus accumbens, but not in dorsal striatum, following cocaine challenge in rats withdrawn from repeated cocaine administration.

We investigated whether changes in the dopamine transporter in the nucleus accumbens or striatum are involved in cocaine-induced behavioral sensitization by using in vivo electrochemistry to monitor the clearance of locally applied dopamine in anesthetized rats. Rats were injected with cocaine-HCl (10 mg/kg i.p.) or saline daily for 7 consecutive days and then withdrawn for 7 days. Pressure ejection of a finite amount of dopamine at 5-min intervals from a micropipette adjacent to the electrochemical recording electrode produced transient and reproducible dopamine signals. After a challenge injection of cocaine (10 mg/kg i.p.), the signals in the nucleus accumbens of cocaine-treated animals became prolonged and the clearance rate of the dopamine decreased, indicating significant inhibition of the dopamine transporter. In contrast, simultaneous measurements in the dorsal striatum indicated a transient increase in both the amplitude of the signals and the clearance rate of the dopamine. The signals in both brain regions in the saline-treated animals given the cocaine challenge were similar to those in untreated animals given an acute injection of cocaine (10 mg/kg i.p.) or saline. Behaviorally, not all of the cocaine-treated animals were sensitized; however, both sensitized and nonsensitized animals displayed similar changes in dopamine clearance rate. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine and the density of binding sites were similar in cocaine- and saline-treated rats. The decrease in dopamine clearance rate observed in the nucleus accumbens of the cocaine-treated rats after a challenge injection of cocaine is consistent with increased dopaminergic transmission, but does not appear to be sufficient in itself for producing behavioral sensitization.

GABAA receptor function and regional analysis of subunit mRNAs in long-sleep and short-sleep mouse brain.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

Differences in dopamine clearance and diffusion in rat striatum and nucleus accumbens following systemic cocaine administration.

Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCl (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopamine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine.

Terbutaline-induced desensitization of polymorphonuclear leukocyte beta 2-adrenergic receptors in young and elderly subjects.

Potential age-related differences in cardiovascular responsiveness and receptor regulation induced by short-term administration of a selective beta 2-adrenergic receptor agonist were investigated. Young (age range, 23 to 31 years) and elderly (age range, 64 to 73 years) healthy subjects were treated with terbutaline (5 mg, three times daily) for 5 days. Similar plasma terbutaline concentrations were achieved in the two age groups. The elderly group had higher baseline plasma norepinephrine concentrations and mean arterial pressures, neither of which were altered by terbutaline administration. During terbutaline treatment, heart rate increased in both age groups while subjects were supine but consistently increased only in the young group while subjects were standing. In both age groups, the density of beta 2-adrenergic receptors on polymorphonuclear leukocyte membranes was reduced by 50% during terbutaline administration and returned to baseline values at similar rates after drug administration was stopped. Isoproterenol-stimulated cyclic adenosine monophosphate accumulation in polymorphonuclear leukocytes from elderly subjects was regulated similarly. These findings suggest that the ability of terbutaline to increase standing heart rate is selectively impaired in the elderly, whereas the ability of polymorphonuclear leukocyte beta 2-adrenergic receptors to down-regulate and to return to baseline values is not.

Persistence of neurochemical changes in dopamine systems after repeated cocaine administration.

The purpose of this study was to test whether persistent changes consistent with behavioral sensitization occur in dopamine (DA) uptake, release or receptors following repeated cocaine administration. Our neurochemical experiments focused primarily on the striatum; however, quantitative autoradiography was used to measure D-1 and D-2 DA receptors in both cell body and terminal regions of the nigrostriatal and mesolimbic dopaminergic pathways. After receiving eight once-daily injections of cocaine (10 mg/kg, i.p.), rats remained behaviorally sensitized for 1 week. This repeated treatment with cocaine induced two changes consistent with increased dopaminergic transmission. Postsynaptic D-2 DA receptors were selectively increased in nucleus accumbens one day after termination of the repeated cocaine administration; however, these receptors returned to control levels one week after cocaine administration had been terminated. In contrast, amphetamine-stimulated [3H] DA release from striatal slices was increased in rats receiving repeated cocaine injections, but this increase was not apparent until 1 week after the drug administration had been terminated. While neither of these two changes is sufficient to explain cocaine-induced behavioral sensitization, both are consistent with increased dopaminergic responsiveness and may contribute to sensitization.

Regulation of adrenergic receptors in intraocular hippocampal transplants: role of noradrenergic innervation.

Hippocampal tissue transplanted into the anterior chamber of the eye offers a unique system in which development can be studied in the absence of the noradrenergic innervation. This system was used to determine the extent to which noradrenergic innervation regulates the development of adrenergic receptors. In addition to examining single denervated transplants, transplants grown with innervation from the superior cervical ganglia of the host rat or from locus coeruleus cotransplants were also examined to determine whether the source of norepinephrine and extent of innervation in oculo regulate the development and density of adrenergic receptors. In vitro autoradiographic analysis of ligand binding to both alpha 1- and beta-adrenergic receptors with 125I-BE 2254 and 125I-pindolol, respectively, was used to characterize adrenergic receptors in the intraocular transplants. Quantitative analysis of the receptors showed an up-regulation of both alpha 1- and beta-adrenergic receptors in tissue grown in the absence of norepinephrine, but in general there was not a high degree of correlation between norepinephrine content and receptor density. Although high-performance liquid chromatography (HPLC) analysis of catecholamines revealed higher than normal amounts of norepinephrine in hippocampal transplants innervated by the superior cervical ganglia or a locus coeruleus cotransplant, the density of alpha 1 and beta receptors was quite comparable with values found in the literature for normal adult hippocampus. These results suggest that the relationship between receptor number and density of innervation may differ significantly from what is observed in response to pharmacological manipulation of norepinephrine systems in the adult brain.

A new densitometric procedure to measure protein levels in tissue slices used in quantitative autoradiography.

A new quantitative staining technique for the assay of protein in tissue sections using the dye Coomassie brilliant blue G 250 is discussed. This densitometric procedure uses commercially available hardware and software employed in the quantitation of receptor autoradiographs. Rather than assuming a homogeneous distribution of protein in the tissue section, regional levels of protein are measured in the same tissue slice used to produce the autoradiograph. Additionally, the process of staining tissue with Coomassie blue for protein is reversible; the tissue can be destained after the measurement of protein is complete and then restained with a standard histological stain such as Cresyl violet. This technique is a more reliable method for normalizing receptor densities by tissue protein levels and allows for a more accurate comparison between QAR and membrane binding techniques.

Sensitization to cocaine in the nigrostriatal dopamine system.

Behavioral sensitization involving the nigrostriatal dopaminergic tract is manifested after treatment with only a single dose of cocaine and is augmented following repeated treatment. One neurochemical change observed that is consistent with behavioral sensitization is the increase in amphetamine-induced 3H-DA release from striatal slices seen after one injection of cocaine. One day after repeated administration of cocaine, however, the increase is no longer evident. It is possible that transient compensatory changes, such as increased D-2 autoreceptor inhibition, may obscure this effect when it is measured at relatively short times after the repeated administration has been terminated. One day after cessation of repeated cocaine administration, D-2 autoreceptors in both striatum and substantia nigra compacta were upregulated consistent with a compensatory mechanism and the development of behavioral tolerance rather than sensitization. In contrast, DA content, neuronal DA uptake, and postsynaptic D-2 DA receptors in striatum were not regulated by this treatment. Likewise, D-1 DA receptors in striatum and substantia nigra were unaffected. In the mesolimbic system, both the pre- and postsynaptic receptor changes are consistent with sensitization. Amphetamine-stimulated release from nucleus accumbens has not yet been measured in cocaine-sensitized animals. It is possible that changes similar to those seen in striatum may occur in this area. It is interesting that, in general, presynaptic parameters associated with the DA neuron, with the notable exception of the uptake pump, appear to be more sensitive to regulation by cocaine administration than do postsynaptic parameters. The long-lasting effects of a single moderate dose of cocaine are also surprising. It will be important to determine the molecular mechanisms underlying this regulation and whether or not similar changes are induced in mesolimbic dopaminergic systems by single and repeated administration of cocaine.

Induction of mRNA for tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase synthesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents.

When rat pheochromocytoma PC18 cells are exposed to the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, there is a marked increase in the level of a single RNA species that hybridizes to the recombinant plasmid pTH.4, which contains sequences complementary to the RNA coding for tyrosine hydroxylase. This RNA species is 1800-1900 nucleotides in length and is presumably identical to an RNA species of similar size, isolated from rat pheochromocytoma PC8b cells and shown to code for tyrosine hydroxylase. Using RNA dot hybridization to quantitate the relative level of this tyrosine mRNA species, time course studies show that this mRNA increases relatively rapidly in PC18 cells treated with either 8-bromocyclic AMP or dexamethasone. A new steady state level of tyrosine hydroxylase mRNA is achieved after 6 hr or 12-24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, respectively. The changes in the level of the mRNA slightly precede the changes in the rate of synthesis of tyrosine hydroxylase in cells treated with these inducing agents. After 24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, the increases in the level of tyrosine hydroxylase mRNA are identical to the increases in the rate of synthesis of the enzyme in the cells. In cells treated simultaneously with both 8-bromocyclic AMP and dexamethasone, the increases in the enzyme level and rate of synthesis of tyrosine hydroxylase are approximately equal to the sum of the increases in these parameters observed in cells treated with either inducing agent alone. In contrast, there is not an additive increase in the level of tyrosine hydroxylase mRNA in cells treated with both inducing agents. This lack of an additive increase in mRNA for tyrosine hydroxylase is observed in total cellular RNA samples or in cytoplasmic RNA samples. Our results suggest that in cells exposed to elevated levels of either cyclic AMP or glucocorticoids, tyrosine hydroxylase is induced by a mechanism which increases the level of its mRNA, resulting in an increased rate of synthesis of the enzyme. However, in cells exposed to elevated levels of both cyclic AMP and dexamethasone, tyrosine hydroxylase enzyme levels are regulated by multiple mechanisms, one of which regulates the rate of synthesis of the enzyme without affecting the level of its mRNA.

Induction of tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: effect of the inducing agents alone or in combination on the enzyme levels and rate of synthesis of tyrosine hydroxylase.

The enzymatic activity of tyrosine hydroxylase (EC 1.14.16.2) increases in rat pheochromocytoma PC18 cells exposed to either elevated levels of cyclic AMP or glucocorticoids. The cyclic AMP-mediated increase in activity is elicited by cyclic AMP analogs or by compounds which activate adenylate cyclase or inhibit phosphodiesterase. The glucocorticoid-mediated increase is elicited only by glucocorticoid steroid hormones; nonglucocorticoid steroid hormones have no effect on tyrosine hydroxylase. In PC18 cells exposed simultaneously to both cyclic AMP-elevating agents and glucocorticoids, the increase in tyrosine hydroxylase activity is greater than that observed in cells treated with optimal concentrations of either inducing agent alone. Immunochemical titration experiments demonstrate that the increases in tyrosine hydroxylase activity observed in cells treated with the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, are due to increases in enzyme protein. Time course studies show that in cells treated with either 8-bromocyclic AMP or dexamethasone, the enzyme level increases slowly to a level 5-7-fold greater than that observed in untreated cells after 4 days of treatment. In cells treated with both of these inducing agents simultaneously, the enzyme level increases to a level 10-12-fold greater than that observed in control cells after 4 days of treatment. This additive increase in activity in cells treated with both inducing agents is observed at all time points. The rates of synthesis and degradation of tyrosine hydroxylase have also been measured in PC18 cells, using an antiserum to tyrosine hydroxylase to rapidly isolate radiolabeled enzyme from cells that have been incubated in the presence of [3H]leucine. The apparent half-life of tyrosine hydroxylase in the PC18 cells is approximately 30 hr. In PC18 cells incubated in the presence of radiolabeled leucine for 60 min, 0.2-0.3% of the total soluble protein synthesized is identified as tyrosine hydroxylase. In cells treated with either 8-bromocyclic AMP or dexamethasone for 24 hr, there is a 6-8-fold increase in the rate of synthesis of the enzyme. In cells treated with both inducing agents simultaneously, there is a 10-12-fold increase in the rate of synthesis; thus, the additive increase in enzyme level observed in cells treated with both inducing agents is paralleled by an additive increase in the rate of synthesis of the enzyme in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)