Effects of intra-striatal GDNF on motor coordination and striatal electrophysiology in aged F344 rats.

The purpose of this study was to examine whether improvement in motor function could be demonstrated in old rats, and to see if GDNF affected post-synaptic DA function. Aged (20 month old) versus young rats were tested following GDNF treatment for postural control by using an inclined balance beam and a wire grip strength test. Rats were also examined electrophysiologically for spontaneous striatal cell firing rate alone and in the presence of DA receptor agonists, and histologically for the intensity of striatal TH staining, and number of DA containing nigral cells. Behavior was significantly improved in the aged animals who received central GDNF infusions, although the extent of improvement was less than what has been observed in 16-month-old rats. There was no effect of GDNF treatment in the aged animals on spontaneous firing rate in the striatum, or on the post synaptic response to locally applied D(1) and D(2) receptor family agonists. However, there was an effect of age alone on firing rate, and on the response to locally applied SKF 38393 and quinpirole. By using unbiased cell counting we observed no age-related decline in the number of TH positive cells in the substantia nigra. There was no effect of GDNF on the number of TH positive cells in the substantia nigra in either young or aged rats, although there were morphological improvements in DA neurons of the GDNF treated aged rats. These results replicate earlier studies showing an effect of age on striatal firing rate and dopamine receptor function, and suggest that the GDNF mediated improvement in behavior may be located other than post synaptically within the striatum.

Effects of ethanol on striatal dopamine overflow and clearance: an in vivo electrochemical study.

Previous studies have shown that the neurotransmitter dopamine (DA) is implicated in the reinforcing effects of ethanol and other abused drugs. Ethanol also alters DA overflow and uptake in vivo. Further studies of the role of DA in the behavioral and neurochemical effects of ethanol may help explain the pharmacological mechanisms by which these effects are produced. In these studies we used in vivo electrochemical recordings to investigate the effects of ethanol (EtOH) on the dynamics of evoked DA overflow and DA uptake in rat dorsal striatum. Local applications of EtOH from a multibarrel micropipette did not produce detectable changes in extracellular levels of endogenous DA in the dorsal striatum. EtOH application did attenuate potassium (K+)-evoked overflow of DA in a time-dependent fashion. In contrast, tyramine-induced DA overflow, a calcium-independent process thought to be carrier mediated, was not altered by local EtOH application in the dorsal striatum. Striatal uptake of locally applied exogenous DA was decreased by nomifensine, an effect that was attenuated by locally applied EtOH. Taken together, these data suggest that one of the effects of EtOH on DA-containing nerve endings in the rat striatum involves functional changes in the high-affinity DA transporter associated with these nerve endings.

Caloric restriction enhances evoked DA overflow in striatum and nucleus accumbens of aged Fischer 344 rats.

Previous studies have shown deficits in DA neuronal systems in senescence. Other studies indicate that prolonged dietary restriction can attenuate many of the detrimental effects of age. We have shown previously using in vivo electrochemistry that K+-evoked striatal DA overflow decreases as a function of age. This was a regional effect that appeared to be due to functional changes in DA neurons, rather than a decrease in the storage and synthesis of DA. In the present studies, we used in vivo electrochemistry to investigate the effects of caloric restriction on age related decreases in K+-evoked DA overflow along a dorsal to ventral axis in the striatum of aged female Fischer 344 rats. Aged (26-28-month-old) diet restricted animals (DRF) showed evoked DA overflow that was significantly greater in amplitude and duration compared to aged (26-28-month-old) ad lib fed animals (ALF). These results provide additional evidence that decreased DA neuronal function resulting from age is improved by caloric restriction.

Effects of ethanol and nomifensine on NE clearance in the cerebellum of young and aged Fischer 344 rats.

Rapid chronoamperometric recordings coupled with local application of drugs by pressure ejection were used to investigate the effects of nomifensine and ethanol (EtOH) on exogenous norepinephrine (NE) clearance in the cerebellum of young (5-month-old) and aged (24-26-month-old) male Fischer 344 rats. In the young rats, local nomifensine application prolonged exogenous NE clearance, indicating transporter mediated uptake inhibition. NE clearance was modestly but significantly prolonged in the aged rats as compared to the young rats, suggesting less efficient uptake. Consistent with this, there was little effect of nomifensine on NE clearance in the aged rats. In contrast to the effect of nomifensine, EtOH inhibited NE clearance in both young and aged rats. These data further support the hypothesis that one effect of EtOH in cerebellar NE systems is inhibition of NE uptake into NE-containing nerve terminals, and they also demonstrate that the effect of nomifensine on exogenous NE clearance in vivo in the cerebellum is altered by the aging process, while the effect of EtOH is not.

Medial dorsal striatum is more sensitive than lateral dorsal striatum to cocaine inhibition of exogenous dopamine clearance: relation to [3H]mazindol binding, but not striosome/matrix.

Previous studies have shown that acute systemic cocaine inhibits dopamine (DA) transport and thereby produces dose-dependent changes in exogenous DA clearance in the brain. This measure reflects the dynamic activity of the DA transporter. There are also differential effects of cocaine on DA clearance in dorsal and ventral striatum, and evidence from many studies suggests that even within the dorsal striatum, the interaction of cocaine with the DA transporter may not be homogeneous. A greater understanding of how cocaine interacts with the striatal DA transporter will help clarify the role of the striatum in mediating effects of cocaine. In these studies we used in vivo electrochemical recording to examine the effect of ip cocaine on exogenous DA clearance in the medial and lateral dorsal striatum with respect to both DA transporter binding sites and electrode localization to striosomes or matrix. Baseline exogenous DA clearance was different in medial and lateral dorsal striatum in the absence of cocaine. Systemic cocaine produced a more pronounced inhibition of DA clearance in medial dorsal striatum than in lateral dorsal striatum. [3H]Mazindol binding to DA transporters was lower in medial dorsal striatum, but was not in register with striosome or matrix compartments. There was no notable difference between cocaine's effects on DA clearance in striosome or matrix that was due to this compartmentalization. Others have demonstrated that medial dorsal striatum receives proportionally more innervation from the mesolimbic, as opposed to the nigrostriatal, DA system. Taken together, these and previous in vivo results suggest that this differential sensitivity to cocaine reflects the unique properties of the ascending mesolimbic DA projection and the lower density of DA transporters associated with it.

Occupancy of the serotonin transporter by fluoxetine, paroxetine, and sertraline: in vivo studies with [125I]RTI-55.

[125I]RTI-55 was used tracer doses to label serotonin (5-HT) transporters in vivo in the mouse brain. Fluoxetine, paroxetine, and sertraline, potent antidepressants and selective inhibitors of serotonin transporter sites, were administered in various doses and at various times. The doses and times that result in significant binding of the drugs to transporters correspond to doses and times where they are reported to have physiological effects. Estimates of occupancy rate and duration of binding to serotonin transporters were made. The rate of occupancy of the 5-HT transporter site was fastest for sertraline, intermediate for paroxetine and slowest for fluoxetine. Similarly, the duration of occupancy was significantly shorter for sertraline and paroxetine (approximately 10 h) than for fluoxetine (approximately 50 h). The results indicate that in competition studies, [125I]RTI-55 can be used to identify doses of drugs that are physiologically effective, to determine their relative rate of occupancy, and most importantly, to measure the residency time on the central serotonin transporter in vivo.

Repeated intravenous cocaine administration to rats produces behavioral sensitization without changing brain cocaine levels.

Repeated intraperitoneal (i.p.) administration of cocaine to rats results in behavioral sensitization. However, augmented brain cocaine levels are also produced by this treatment. In the present study, a 43% increase in cocaine levels was observed in striatum in response to eight once-daily i.p. injections of cocaine (10 mg/kg). It has been suggested that this dispositional change does not occur with intravenous (i.v.) cocaine administration. In agreement with this suggestion, the striatal cocaine levels observed following either a single i.v. injection or eight once-daily i.v. injections of cocaine (1 mg/kg) were similar. Nonetheless, the rats became behaviorally sensitized in response to the repeated i.v. cocaine administration. These results suggest that the increased brain levels of cocaine observed following repeated i.p. cocaine administration cannot completely account for behavioral sensitization.

In vivo binding of [125I]RTI-55 to dopamine transporters: pharmacology and regional distribution with autoradiography.

Previous studies have demonstrated that para-substituted WIN 35,065-2 analogs of cocaine show high binding affinity for dopamine uptake sites both in vitro and in vivo, and inhibit DA uptake in vitro. These analogs also produce potent cocaine-like behavioral effects in various procedures. The purpose of the present studies was to evaluate the iodinated WIN 35,065-2 analog [125I]RTI-55 as an in vivo ligand for the DA transporter. Following intravenous injection in mice, [125I]RTI-55 showed highest accumulation in areas with high densities of dopamine uptake sites. Light microscopic autoradiography was used to examine binding with higher resolution. Displacement studies demonstrated that [125I]RTI-55 binding in dopamine containing regions, striatum and olfactory tubercles, was saturable and inhibited by other cocaine analogs. GBR 12909 and WIN 35,428 significantly inhibited [125I]RTI-55 binding in striatum, while paroxetine significantly inhibited hypothalamic binding but had little effect in striatum. The latter finding suggests that [125I]RTI-55 also binds to the serotonin transporter. Haloperidol had no effect on [125I]RTI-55 binding in any brain region measured. In addition, treatment of animals with the dopamine neurotoxin MPTP caused significant reductions in striatal [125I]RTI-55 binding. The results of these studies indicate that [125I]RTI-55 binds primarily to the dopamine transporter in the mouse striatum in vivo.

[123/125I]RTI-55, an in vivo label for the serotonin transporter.

[123I]RTI-55, an iodinated derivative of the cocaine analog 3 beta-phenyltropane-2 beta-carboxylic acid methyl ester, was evaluated as an agent for in vivo labeling of the serotonin transporter. Labeling of the precursor of RTI-55 with I-123 was efficient and yielded a high specific activity product. After intravenous injection of [123I]RTI-55 into rats, the tracer accumulated in regions with high densities of serotonin and dopamine uptake sites. The distribution of [123I]RTI-55 binding in areas rich in serotonin uptake sites correlated with [3H]serotonin uptake measured in vitro in the same regions. Specific [123I]RTI-55 binding to serotonin uptake sites was inhibited by paroxetine but not by GBR 12,909. Treatment of rats with neurotoxic doses of fenfluramine caused decreases of 66% (in the hypothalamus) to 83% (in the superior colliculi) of specific [125I]RTI-55 binding in all areas except in the striatum and the olfactory tubercles (regions rich in dopamine transporters). These results indicate that [123/125I]RTI-55 binds, although not selectively, to the serotonin transporter in vivo. Furthermore, they suggest that [123I]RTI-55 holds promise as a SPECT imaging agent for the study of the serotonin transporter in humans in health and disease.

Stimulus generalization from cocaine to analogs with high in vitro affinity for dopamine uptake sites.

Previous research has shown that phenyltropane derivatives of cocaine are very potent ligands for dopamine transporters in in vitro binding and uptake, and in in vivo binding assays. In the present study, these analogs were tested for their ability to substitute for cocaine in rats trained to discriminate cocaine from saline. Results indicate that these compounds are from 6-13 times more potent than cocaine in producing cocaine-appropriate responding. This provides further evidence in support of the importance of dopamine uptake inhibition for the behavioral effects of cocaine, and suggests utility of these compounds in understanding cocaine abuse.

Behavioral effects of novel cocaine analogs: a comparison with in vivo receptor binding potency.

Several novel cocaine analogs, previously shown to be very potent in in vitro binding studies, have been examined for their stimulatory effects on locomotor activity and for their ability to displace [3H]WIN 35,428 binding in vivo in mice. These compounds, like WIN 35,428, lack an ester link between the phenyl group and the tropane ring and have para-substitutions on the phenyl ring. They were much more potent than (-)-cocaine in producing increases in locomotor activity. In addition, they were more potent than (-)-cocaine in inhibiting [3H]WIN 35,428 binding in vivo in mouse striatum. Thus, these compounds demonstrate similar high potency in behavioral tests and in receptor binding assays, both in vivo and in vitro. Results support the hypothesis of a relationship between binding at the dopamine transporter and the behavioral effects of cocaine-like drugs. Further, assuming that maximal occupancy occurs with total displacement of [3H]WIN 35,428 binding in vivo, the data suggest that maximal locomotor effects occur with near total occupancy of transporter binding sites.

Localization of brainstem sites which mediate alfentanil-induced muscle rigidity in the rat.

Previous work has demonstrated that direct injections of methylnaloxonium (MN), a relatively lipophobic quaternary opiate antagonist, in the area of the nucleus raphe pontis (RPn) significantly attenuated alfentanil-induced rigidity. It was hypothesized that other hindbrain sites, particularly the other raphe nuclei, might play a role in this rigidity. Therefore, a study was performed in which 57 rats, divided into four groups, were implanted with chronic guide cannulae directed at brain sites anterior, lateral, or posterior to the RPn. After each animal was pretreated with intracerebral injections of MN, alfentanil (0.5 mg/kg) was administered subcutaneously. Electromyographic activity was recorded from the gastrocnemius muscle as a measure of hindlimb rigidity. Each animal was subsequently injected at 4 to 5 day intervals with MN two additional times at sites 1 and 2 mm deeper, respectively, than the initial injection. Data were thus obtained on animals treated with either MN or saline at 3 successive histologically identified sites which were either anterior, lateral or posterior to the RPn. The administration of MN into two specific sites in the region just lateral to the nucleus raphe pontis significantly [F(1,38) = 18.68 and 5.02 respectively, p less than 0.05] reversed the rigidity produced by systemic alfentanil administration. There was a weak effect of MN injections anterior to the RPn but this could not be localized to any one site. These results suggest that discrete brainstem regions involved in opiate action can be sensitively and selectively identified by direct intracranial injections of a lipophobic opiate antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Ketanserin pretreatment reverses alfentanil-induced muscle rigidity.

Systemic pretreatment with ketanserin, a relatively specific type-2 serotonin receptor antagonist, significantly attenuated the muscle rigidity produced in rats by the potent short-acting opiate agonist alfentanil. Following placement of subcutaneous electrodes in each animal's left gastrocnemius muscle, rigidity was assessed by analyzing root-mean-square electromyographic activity. Intraperitoneal ketanserin administration at doses of 0.63 and 2.5 mg/kg prevented the alfentanil-induced increase in electromyographic activity compared with animals pretreated with saline. Chlordiazepoxide at doses up to 10 mg/kg failed to significantly influence the rigidity produced by alfentanil. Despite the absence of rigidity, animals that received ketanserin (greater than 0.31 mg/kg i.p.) followed by alfentanil were motionless, flaccid, and less responsive to external stimuli than were animals receiving alfentanil alone. Rats that received ketanserin and alfentanil exhibited less rearing and exploratory behavior at the end of the 60-min recording period than did animals that received ketanserin alone. These results, in combination with previous work, suggest that muscle rigidity, a clinically relevant side-effect of parenteral narcotic administration, may be partly mediated via serotonergic pathways. Pretreatment with type-2 serotonin antagonists may be clinically useful in attenuating opiate-induced rigidity, although further studies will be necessary to assess the interaction of possibly enhanced CNS, cardiovascular, and respiratory depression.

Rewarding properties of beta-endorphin as measured by conditioned place preference.

The role of beta-endorphin as a possible mediator in the reinforcing properties of opiates was investigated using a conditioned place preference paradigm. Heroin, a synthetic opiate known to have reinforcing properties, produced a strong preference for an environment previously paired with heroin injection at all doses tested (0.25, 0.5, 1.0, 2.0 mg/kg SC). No such place preference was observed following saline injections. Rats also showed dose-dependent place preference for the environment paired with beta-endorphin when injected intracerebroventricularly (significant dose was 2.5 micrograms). At higher doses (5.0 and 10.0 micrograms) rats showed no preference for the paired environment, but were catatonic. Pretreatment with naloxone (0.04, 0.2, 1.0 mg/kg SC) attenuated the rewarding effect of beta-endorphin (2.5 micrograms) at all doses tested. The lowest dose of naloxone which had no aversive effect when tested alone could also significantly block the positive effect of beta-endorphin. The reinforcing dose of beta-endorphin (2.5 micrograms) also produced an increase in locomotor activity, when tested in photocell cages. This suggests that the hyperactivity induced by beta-endorphin may contribute to the preference for an environment previously paired with the same drug. The reinforcing effect of beta-endorphin is most probably mediated by the mu and/or delta opioid subtype receptor, since beta-endorphin has a high affinity for these receptors. These results demonstrate positive reinforcing properties of beta-endorphin in the central nervous system.