Dose-effect functions for cocaine self-administration: effects of schedule and dosing procedure.

Research related to determining how procedural variables can alter dose-effect functions for cocaine self-administration is limited. Toward clarifying the role of procedural variables, responding was maintained in rats under either variable-interval (VI) or fixed-ratio (FR) schedules of cocaine infusion. In addition to free-operant FR schedules, discrete-trial FR schedules were evaluated. The dose-effect functions were obtained by either substituting a dose for the usual daily dose, instituting a particular dose for several sessions, or making all doses available within a session. Dose-effect functions for response rate (or number of trials with infusions for the discrete-trial FR) were bitonic for the VI and discrete-trial FR schedules but tended to be strictly decreasing for the free-operant FR schedules. Responding was maintained under FR schedules by a low dose (0.083 mg/infusion) if the dose was substituted for a higher daily dose but not when made available daily. Rate of response was higher under ratio schedules at 0.17 mg/infusion when this dose occurred within the context of other higher doses within a session than when the dose was simply substituted for a higher daily dose. These data indicate that procedural variables can alter dose-response curves for cocaine self-administration.

Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum.

A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) procedure has been developed which selectively amplifies and quantifies the two isoforms of the dopamine D2 receptor. Variability is corrected by the inclusion of a D2 dopamine receptor mRNA standard within each reaction. The internal standard was generated by introducing a point mutation within a D2 cDNA clone that created a unique restriction site within the amplified region. An in vitro transcribed RNA for the internal mutant control is added to the RNA isolated from brain tissue and the mixture is subjected to RT-PCR, digestion with the restriction enzyme, separation of the products by PAGE, and quantification by direct analysis of radioactivity incorporated during the PCR step. The standard is amplified, in the same reaction as the experimental RNA, using the same primers and RT-PCR conditions. In this manner, the effects of contaminants of the RNA preparation which could affect the amplification procedure are assessed. To insure that the amplification is linear, the number of PCR cycles is minimized. This adaptation avoids 'competitive PCR' and provides for a linear response. Moreover, to obviate non-specific co-amplification, primer annealing steps are performed at or above the melting temperature for the primers, thus increasing signal-to-noise ratios. Finally, primer pairs have been designed which permit amplification of specific fragments for each of the five rat dopamine receptor subtypes. These fragments have unique sizes and so can be differentiated when simultaneously amplified in the same RNA preparations.

Chronic cocaine administration increases CNS tyrosine hydroxylase enzyme activity and mRNA levels and tryptophan hydroxylase enzyme activity levels.

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striatum and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.

Radioenzymatic assay for tryptophan hydroxylase: [3H]H2O release assessed by charcoal adsorption.

The rate-limiting reaction in the biosynthesis of the neurotransmitter, serotonin, is catalyzed by the enzyme, tryptophan hydroxylase. Studies on the characteristics of this enzyme have been hampered by its relative instability and paucity in the brain. We have modified a charcoal adsorption radioenzymatic assay used for the measurement of tyrosine hydroxylase to assess rat brain tryptophan hydroxylase activity. This protocol is based on the principle that aromatic amino acid hydroxylases are mixed-function oxygenases and will utilize O2 and reduced pterin to convert tritiated amino acid substrate to product and tritiated H2O. All product and unreacted substrate are adsorbed by acidified charcoal. The [3H]H2O is analyzed by liquid scintillation spectrometry and is indicative (stoichiometrically) of the amount of product formed and, thus, the activity of the enzyme. This assay has a high signal-to-noise ratio and is sensitive enough to determine enzymatic activity in homogenates of individual raphe nuclei. In addition, its simplicity in design allows for the simultaneous testing of large numbers of samples. The enzyme activity and kinetic determinations derived from this protocol agree with those of other investigators using more lengthy, involved procedures.

Chronic selegiline administration transiently decreases tyrosine hydroxylase activity and mRNA in the rat nigrostriatal pathway.

Selegiline, a selective monoamine oxidase type B inhibitor, is beneficial in the treatment of Parkinson's disease. However, this beneficial effect is only transient, and patients must ultimately resort to treatment with standard levodopa therapy. We studied the effects of chronic selegiline treatment on the rat nigrostriatal pathway, to elucidate a neurochemical correlate for this adaptive clinical response. Selegiline treatment for 3, 7, 14, or 21 days decreased tyrosine hydroxylase (the enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis) activity in the cell body regions (substantia nigra) of the nigrostriatal pathway. However, tyrosine hydroxylase activity measurements in the major terminal field region (corpus striatum) of the pathway did not correspond to those in the substantia nigra; in the corpus striatum, tyrosine hydroxylase activity was decreased at 3 and 7 days of treatment and recovered by 14 days. We tested whether the decrease in tyrosine hydroxylase activity was mediated by a decrease in tyrosine hydroxylase mRNA. Northern blot and RNA dot blot analyses (using a tyrosine hydroxylase-specific cDNA probe) of substantia nigra homogenates revealed a significant decrease in tyrosine hydroxylase mRNA at 3, 7, and 14 days of selegiline treatment, compared with controls. Conversely, after 21 days of selegiline, tyrosine hydroxylase mRNA levels were significantly higher (3-fold) than controls; this finding was not reflected in substantia nigra tyrosine hydroxylase activity. The 21-day increase in mRNA may be associated with the rebound in tyrosine hydroxylase activity observed in the corpus striatum. Thus, it is possible that the recovery in tyrosine hydroxylase activity in the corpus striatum is mediated through an increase in tyrosine hydroxylase protein transport from the substantia nigra to the corpus striatum and/or that the tyrosine hydroxylase enzyme exists in a more stabilized state during this period of time. These results demonstrate that monoamine oxidase type B-selective inhibitory doses of selegiline are capable of inducing transient decreases in tyrosine hydroxylase activity and tyrosine hydroxylase mRNA levels. Furthermore, these reversible effects may represent adaptive responses associated with pharmacological tolerance and the transient beneficial actions of this drug in Parkinson's disease.