Ronald Kuczenski

Chronic amphetamine: Is dopamine a link in or a mediator of the development of tolerance and reverse tolerance?

Rats were administered chronic multiple injections of amphetamine (AMPH) using dosage regimens which produce tolerance to the AMPH facilitation of self-stimulation responding, or reverse tolerance (sensitization) to the locomotor stimulant and stereotypy-producing effects of the drug. Subsequently rats were challenged with AMPH at behaviorally relevant doses and times and striatal and mesolimbic dopamine (DA) dynamics were assessed using the conversion of 3H-tyrosine to 3H-DA, and endogenous levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as indices of dopaminergic function. Acute administration of AMPH produced dose and time related changes in all indices of DA function in both the striatal and mesolimbic brain regions. Co-administration of haloperidol during chronic AMPH pretreatment prevented the appearance of most of the behavioral changes induced by chronic AMPH, suggesting an important role for DA systems. However, following chronic AMPH treatment, no additional biochemical changes in striatal or mesolimbic DA metabolism could be detected which would parallel the development of tolerance to AMPH facilitation of self-stimulation behavior or reverse tolerance to AMPH as reflected in enhanced post-stereotypy locomotor activity or a suggested increased intensity of stereotypy. Challenge with AMPH after chronic AMPH pretreatment did accelerate the changes in striatal but not mesolimbic DA metabolism, correlating with the more rapid onset of stereotypy induced by chronic AMPH. Thus, while DA systems appear to be a critical link, not only in the acute effects of AMPH, but also in the development of tolerance and reverse tolerance, most of the behavioral differences between acutely and chronically treated animals are not reflected by comparable differences in DA synthesis and metabolism.

Two dissociable components of behavioral sensitization following repeated amphetamine administration

The acute administration of moderate to high doses (>2 mg/kg) of amphetamine (AMPH) to rats produces a multiphasic behavioral response consisting of an initial period of locomotor activity followed by episodes of intense stereotyped behavior, and a period of post-stereotypy locomotion. Repeated administration of the drug results in a sensitization with two components: more rapid onset of stereotypy and enhancement of the post-stereotypy locomotor activity. The studies presented below provide converging evidence that the two components of the sensitization are dissociable. 1. Rats from ten different strains or suppliers all exhibited more rapid onset of stereotypy following repeated AMPH pretreatment, whereas only five of these strains or supplier groups exhibited significantly enhanced post-stereotypy locomotion. 2. The time course differed for the development of these two components of the sensitization. 3. The recovery from sensitization differed for these two components of the behavioral response. Following withdrawal of the drug, post-stereotypy motor activity diminished within 2 months while the more rapid onset of stereotypy persisted for at least 3 months. These observations have particular relevance to future studies directed at specifying the biochemical substrates of the sensitization.

Biphasic effects of amphetamine on striatal dopamine dynamics.

S(+)-Amphetamine (AMPH) was administered to rats over the dose range 0.15-10.0 mg/kg (1.74(3)mumoles/kg) at various times before sacrifice. 15 min before sacrifice rats received an intraventricular injection of 3H-tyrosine, and the accumulation of striatal 3H-dopamine (DA) was assessed as an index of striatal DA formation and utilization. 3H-DA formation exhibited a biphasic dose response to AMPH. At 16 and 31 min after AMPH administration, 3H-DA accumulation increased linearly in response to AMPH to a maximal rate of 200% of control values at 1.0 mg/kg, then declined to less than 50% of control values at AMPH doses of 5 and 10 mg/kg. As a function of time, low doses of AMPH (to 2.0 mg/kg)) only increased 3H-DA accumulation, whereas high doses of AMPH (greater than 2.5 mg/kg) only decreased 3H-DA accumulation. All doses of AMPH increased endogenous levels of striatal DA. The data are discussed in terms of compensatory adaptive mechanisms of the nigrostriatal dopaminergic pathway.

Effects of para-chlorophenylalanine on amphetamine and haloperidol-induced changes in striatal dopamine turnover

Para-chlorophenylalanine (PCPA) was administered to rats, and the animals were sacrificed 48 h later. Animals received various doses of S(+)-amphetamine (AMPH) 31 min before sacrifice or of haloperidol (HAL) 46 min before sacrifice and an intracerebral injection of [3H]tyrosine 15 min prior to sacrifice. PCPA pretreatment potentiated the increased accumulation of striatal [3H]dopamine (DA) induced by doses of AMPH to 1.5 mg/kg, and inhibited the decrease in striatal [3H]DA accumulation observed at higher doses (greater than 2.5 mg/kg) of AMPH. Further, the AMPH-induced increase in endogenous levels of striatal DA was inhibited by PCPA pretreatment. Similarly, PCPA pretreatment potentiated the HAL-induced increase in striatal [3H]DA accumulation. The data are discussed with respect to an inhibitory serotonergic influence on nigrostriatal DA function, and the relationship of nigrostriatal biochemical events to AMPH-induced behavioral changes.

Chronic amphetamine: Tolerance and reverse tolerance reflect different behavioral actions of the drug

Chronic administration of amphetamine (AMPH) has been reported to produce tolerance to the drugs behavioral effects in some paradigms (self-stimulation, discriminative stimulus, self-administration) and an enhanced effect or reverse tolerance when other behaviors are monitored (locomotor activity, stereotypy). The present study investigated whether the two phenomena are, in fact, related to the particular behavior monitored or reflect the marked differences in the injection regimens (1X vs. 3X daily injections) used to produce the phenomena. The effects of chronic AMPH administered once or three times daily on AMPH facilitation of self-stimulation responding and on the locomotor stimulant and stereotypy-producing effects of the drug were assessed. Regardless of the injection regimen used, chronic AMPH resulted in an enhancement of the locomotor stimulant effects of the drug as well as a more rapid onset and greater intensity of the stereotypy produced. In the self-stimulation paradigm, only the 3X daily regimen significantly reduced the effectiveness of a challenge dose of AMPH (tolerance), although the 1X regimen produced effects that were qualitatively similar but quantitatively less. Perhaps behavioral tasks in which tolerance develops reflect the mood-altering properties of the drug in humans whereas a process similar to reverse tolerance may underlie the increased susceptibility to psychoses elicited by the drug with repeated use.

Amphetamine-haloperidol interactions in rat striatum: failure to correlate behavioral effects with dopaminergic and cholinergic dynamics.

Previous reports have suggested that the hyperactivity and stereotypy produced by amphetamine (AMP) and the catalepsy produced by haloperidol (HAL) are mediated by striatal dopaminergic mechanisms. In the present study, we have measured the behavioral effects of AMP and HAL, and their effects on striatal dopaminergic function, using both an index of pre-synaptic activity (synaptosomal dopamine (DA) synthesis) and a parameter which we suggest will reflect post-synaptic dopaminergic function (sodium-dependent, high affinity choline uptake). Administration of 2 mg/kg AMP produces hyperactivity and causes a decrease in DA biosynthesis, both of which are blocked by 0.75 mg/kg HAL. AMP (5 mg/kg) produces stereotypy, further decreases DA biosynthesis and causes a decrease in choline uptake, consistent with stimulation of DA receptors. However, while pretreatment with 3 mg/kg HAL completely blocked the stereotypy induced by 5 mg/kg AMP it failed to reverse the effects of this dose on either DA biosynthesis or choline uptake. These data suggest that either 5 mg/kg AMP affects straital dopaminergic and cholinergic parameters by a mechanism independent of HAL sensitive receptors, or the stereotypy produced by high doses of AMP are not related to striatal dopaminergic and cholinergic function.

Dose response for amphetamine-induced changes in dopamine levels in push-pull perfusates of rat striatum.

Abstract: Levels of dopamine were determined in push‐pull perfusates of striatum in chloral hydrate‐anesthetized rats as a function of increasing systemic doses of amphetamine over the range 0.5–5.0 mg/kg. In the absence of amphetamine administration, basal dopamine levels remained stable for at least 6 h. Perfusate levels of dopamine responded in a quantitatively predictable fashion to increasing doses of amphetamine: (1) the maximal increase in perfusate levels of dopamine after amphetamine, relative to predrug levels, was directly proportional to the dose of the drug up to 3 mg/kg (fivefold after 0.5 mg/kg to 30‐fold after 3 mg/kg); (2) the duration over which perfusate levels of dopamine were significantly elevated, with respect to preamphetamine levels, was proportional to the dose of amphetamine up to 5 mg/kg; and (3) each successively higher dose of amphetamine significantly increased the perfusate level of dopamine over that observed at the next lower dose up to 3 mg/kg amphetamine. However, maximal levels of dopamine in striatal perfusates were achieved following 3 mg/kg amphetamine and were not increased further at higher doses of the drug. The data suggest that, at higher doses of amphetamine, extraneuronal metabolism of dopamine may be of sufficient capacity to limit increases in synaptic levels of dopamine. The absence of further increases in perfusate levels of dopamine as the dose of amphetamine is increased beyond 3 mg/kg is discussed in terms of potential relevance to mechanisms of amphetamine‐induced stereotyped behaviors.

Evidence that amphetamine and Na+ gradient reversal increase striatal synaptosomal dopamine synthesis through carrier-mediated efflux of dopamine.

Amphetamine (AMPH) releases dopamine (DA) from striatal synaptosomes and concomitantly increases DA synthesis. Since AMPH may release DA through carrier-mediated diffusion via reversal of the DA uptake system, the increase in DA synthesis might depend on a functioning uptake carrier. Consistent with such a mechanism, the uptake inhibitors nomifensine (NMF) and benztropine (BZT) completely prevented the AMPH-induced increase in DA synthesis at concentrations known to inhibit DA uptake. Changes in the Na+ gradient across the synaptosomal membrane also promote DA release, since DA and Na+ are cotransported by the neuronal uptake carrier. Incubation of synaptosomes in medium containing decreasing Na+ increased DA synthesis inversely proportional to Na+ over the range 128 to 20 mM. Similarly, incubations in the presence of 10(-4) M ouabain to inhibit Na+, K+-ATPase and allow intracellular accumulation of Na+ also increased DA synthesis. These changes in DA synthesis could also be prevented by BZT and were non-additive with the AMPH-induced increase in DA synthesis. However, a concentration of ouabain (10(-6) M) which by itself did not increase DA synthesis, and does not promote DA release, potentiated the AMPH-induced increase in DA synthesis. Further, the increased DA synthesis promoted by all three manipulations was only marginally dependent on the presence of Ca2+ in the incubation medium. However, at 5 and 10 mM Na+, a second component of increased DA synthesis was observed which was insensitive to BZT, but was prevented by Ca2+ removal. These results suggest that the increase in DA synthesis, and presumably DA release promoted by AMPH, lowered Na+, and ouabain, depend on the availability of the DA carrier at the internal face of the neuronal membrane and the intracellular content of Na+. The second component of increased DA synthesis which is evident at 5 and 10 mM Na+ is discussed in terms of a possible Ca2+-mediated change in DA synthesis which is independent of the DA carrier.

Striatal dopamine metabolism in response to apomorphine: the effects of repeated amphetamine pretreatment

The dose-dependent decrease in striatal dopamine (DA) metabolites following apomorphine (APO) administration was utilized as an index of changes in DA receptor sensitivity following the repeated administration of amphetamine (AMPH). The results suggest that: (a) repeated AMPH pretreatment does not alter DA autoreceptor sensitivity; and (b) interpretations of the decline in striatal DA metabolites at high doses of APO (greater than 50 micrograms/kg), in terms of activation of postsynaptic DA receptors, may require re-evaluation.

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane‐bound form. The membrane‐bound enzyme, which exhibits lower Kms for both tyrosine (7 μM) and reduced pterin cofactor (110 μM) relative to the soluble enzyme (47 μM and 940 μM, respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane‐bound tyrosine hydroxylase with C.perfringens phospholipase C increased the Km of the enzyme for tyrosine to 27 μM and the Vmax by 60% without changing the Km for cofactor. In contrast, treatment of membrane‐bound tyrosine hydroxylase with V. russelli phospholipase A2 increased the Km for tyrosine to 48 μM increased the Vmax and increased the Km for cofactor to 560 μM. The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A2 treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase‐treated, detergent‐solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.