Neurochemical changes in rat striatum and substantia nigra following drug administration

The effects of a number of drugs that deplete catecholamines or block their receptors were investigated on the levels of free amino acids and glutamate decarboxylase activity in rat striatum and substantia nigra, in an attempt to clarify the possible links between catecholamine and amino acid transmitter function. While none of the drugs tested were significantly active in the striatum, profound changes occurred in the substantia nigra. The data are discussed in relation to the possible mechanisms involved.     

Chronic cocaine administration reduces phospholipase A(2) activity in rat brain striatum

BACKGROUND: Phospholipase A(2) (PLA(2)) catalyses the release of free fatty acids used for eicosanoid biosynthesis. We previously reported that calcium-stimulated PLA(2) activity is reduced in the brain of cocaine users and patients with schizophrenia, and have speculated that this is due to dopaminergic hyperactivity in both conditions. METHODS: To investigate these observations under controlled conditions, PLA(2) activity was measured in brain of rats exposed to cocaine and the dopamine receptor antagonist haloperidol. RESULTS: As compared with saline-treated controls, calcium-stimulated PLA(2) activity was reduced (-30%; P<0.01) in the dopamine-rich striatum of animals sacrificed 1 h after chronic (20 mg/kg/day) injection of cocaine, but was normal in haloperidol- (2 mg/kg/day) treated animals, and in the dopamine-poor cortex and cerebellum of animals treated with either drug. CONCLUSION: This confirms and extends our observations in human brain, and further suggests a link between the brain dopaminergic and phospholipid catabolic systems.     

The accumulation and disappearance of cocaine and benzoylecgonine in rat hair following prolonged administration of cocaine.

Hair samples were obtained at various time periods from male Sprague-Dawley rats following the injection of cocaine hydrochloride in doses of 5, 10, and 20 mg/kg, ip, for 28 days. Hair samples were also taken continually after the dosing was stopped until the presence of cocaine and benzoylecgonine were no longer detected in hair. Cocaine and benzoylecgonine in hair and plasma were analyzed by gas chromatography/mass spectrometry. Both cocaine and benzoylecgonine were found in hair samples 4 days after the initiation of cocaine administration. When cocaine dosing was stopped after 28 days, approximately 25 to 30 days were required for cocaine and benzoylecgonine to disappear from rat hair in the group of animals that received the highest dose of cocaine. The disappearance of cocaine and benzoylecgonine followed first-order kinetics. The mean rate constant and mean half-life for cocaine disappearance from hair were 0.212 +/- 0.005 day-1 and 3.31 +/- 0.09 days, respectively, and the mean rate constant and mean half-life for benzoylecgonine disappearance from hair were 0.098 +/- 0.006 day-1 and 6.90 +/- 0.28 days, respectively. The mean plasma concentrations of cocaine on Day 25 for the 5, 10, and 20 mg/kg doses of cocaine were 508 +/- 42, 852 +/- 95, and 2027 +/- 75 ng/mL, respectively, and the mean plasma benzoylecgonine levels for the 5, 10, and 20 mg/kg doses of cocaine were 49.9 +/- 7.0, 103.3 +/- 9.3, and 191.0 +/- 16.0 ng/mL, respectively. There was a positive correlation between the doses of cocaine hydrochloride administered and the plasma levels of both cocaine and benzoylecgonine. This study showed that cocaine and benzoylecgonine can be measured in rat hair following the administration of cocaine and that it was possible to correlate the concentrations of cocaine and benzoylecgonine found in hair with the doses of cocaine that were administered.     

Acute cocaine treatment increases thimet oligopeptidase in the striatum of rat brain

Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, α-neoendorphin, β-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects.     

Non-linear dynamics of electrocardiographic waveforms following cocaine administration

We examined dynamics of ECG waveforms preceding and following 10 mg/kg intravenous administration of cocaine in freely moving cats. Cocaine induced substantial variation in ECG waveforms, as demonstrated by non-linear procedures of phase-plane plots and Poincaré analyses. Phase-plane plots revealed that waveform variation following cocaine administration was not randomly or uniformly distributed, but instead followed patterns of clustered organization with unvisited regions. These patterns are characteristic of chaotic distributions, and support the hypothesis that ECG waveform variation following cocaine does not degenerate into random patterns; instead, the variation follows deterministic, though chaotic patterns.     

Characterization of protein-bound gold in rat urine following aurothiomalate administration and of rat and human albumin-gold-thiomalate

The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed.     

Systemic administration of d-amphetamine induces long-lasting oxidative stress in the rat striatum

The long-term effect of d-amphetamine (AMPH) on the induction of oxidative stress was examined in vivo in the rat brain. In this study, 2,3-dihydroxybenzoic acid (2,3-DHBA) and malonaldehyde (MDA) were used as the index of the hydroxyl radical and lipid peroxidation, respectively. The levels of 2,3-DHBA, MDA and dopamine (DA) in striatal homogenates were examined 7 days following injection of a single large dose of AMPH (7.5 mg/kg, i.p.) in rats pretreated with desipramine (10 mg/kg, i.p.), an agent that inhibits the metabolism of AMPH. Our results showed that 2,3-DHBA and MDA levels were significantly increased by AMPH, whereas DA and its metabolites, DOPAC and HVA were depleted in the striatum. Pretreatment with the glutamate NMDA receptor subtype antagonist MK-801 (1 mg/kg, i.p.) attenuated the increases of 2,3-DHBA and MDA, and provided partial protection against the long-lasting loss of DA produced by AMPH. Overall, the results demonstrate that AMPH could induce sustained production of free radical and oxidative damage, and lead to DA terminal degeneration in the striatum of the rat.     

The presence of cocaine and benzoylecgonine in rat cerebrospinal fluid after the intravenous administration of cocaine.

Cocaine hydrochloride, in doses of 0.5, 1.0, 2.0 and 4.0 mg/kg, iv, was administered to male Sprague-Dawley rats. Cerebrospinal fluid (CSF) was collected from the cisterna magna over a 20 min period and blood samples were obtained at 20 min after cocaine administration. In addition, blood samples for the 1 mg/kg dose of cocaine were collected at 2, 10, 20 and 30 min following drug injection. Gas chromatography/mass spectrometry was used for the analysis of cocaine and its metabolites in plasma and CSF. The disappearance of cocaine (1 mg/kg) from the plasma exhibited first order kinetics with a half-life of 18.11 +/- 3.22 min. Cocaine and benzoylecgonine were found in CSF and the concentrations of cocaine and benzoylecgonine increased in CSF as the doses of cocaine were increased. CSF flow rates were not altered by the iv administration of cocaine or benzoylecgonine. The CSF-to-plasma ratios for cocaine were quite similar to each other over the dosage range of cocaine that was administered; however, the CSF-to-plasma ratios for benzoylecgonine decreased as the concentrations of benzoylecgonine increased in plasma and CSF. When benzoylecgonine (2 mg/kg, iv) was given, the compound was detected in CSF indicating that benzoylecgonine can enter into the central nervous system from the peripheral blood. This investigation shows that cocaine and benzoylecgonine can be assayed in CSF and that the plasma levels of these compounds correlate with their concentrations in CSF.     

Metabolic responses in discrete regions of rat brain following acute administration of glutamate

Glutamate is a major excitatory neurotransmitter in the mammalian brain. Nevertheless, high extracellular levels of this amino acid have been shown to be toxic to several neuronal populations, but no data are available to show how glutamate homeostasis is altered in response to local infusion of glutamate. In the present study, 1 M of glutamate was stereotactically injected into cerebral cortex, striatum, and hippocampus of adult rat brain, and the activities of key metabolic enzymes, lactate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were evaluated by postmortem analysis in tissue homogenates. The results show that glutamate bolus, induced significant alterations in vivo glutamate and energy metabolism, as evidenced by marked alterations in these enzyme activities, whereas dizocilpine, a glutamate receptor antagonist, negated many of the effects induced by high glutamate. However, the degree of involvement of these observations in glutamate-induced neurotoxicity remains to be ascertained.     

The membrane cytoskeletal protein adducin is phosphorylated by protein kinase C in D1 neurons of the nucleus accumbens and dorsal striatum following cocaine administration

Repeated cocaine administration results in persistent changes in synaptic function in the mesolimbic dopamine system that are thought to be critical for the transition to addiction. Cytoskeletal rearrangement and actin dynamics are essential for this drug-dependent plasticity. Cocaine administration increases levels of F-actin in the nucleus accumbens and is associated with changes in the phosphorylation state of actin-binding proteins. The adducins constitute a family of proteins that interact with actin and spectrin to maintain cellular architecture. The interaction of adducin with these cytoskeletal proteins is regulated by phosphorylation, and it is therefore expected that phosphorylation of adducin may be involved in morphological changes underlying synaptic responses to drugs of abuse including cocaine. In the current study, we characterized the regulation of adducin phosphorylation in the nucleus accumbens and dorsal striatum in response to various regimen of cocaine. Our results demonstrate that adducin is phosphorylated by protein kinase C in medium spiny neurons that express the dopamine D1 receptor. These data indicate that adducin phosphorylation is a signaling event regulated by cocaine administration and further suggest that adducin may be involved in remodeling of the neuronal cytoskeleton in response to cocaine administration.     

L-dopa administration enhances exocytotic dopamine release in vivo in the rat striatum.

Peripheral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) methylester increased extracellular levels of DOPA and dopamine (DA) in the rat striatum monitored by in vivo brain microdialysis. The increase in DA levels persisted after inhibition of DA reuptake by nomifensine. Administration of blockers of voltage-dependent Na+ (tetrodotoxin) or Ca2+ (NKY-722) channels through the dialysis membrane completely eliminated the increase in DA levels. These results demonstrate that the L-DOPA-induced DA release is exocytotic in nature and hence, derived from neurons in the striatum.     

Proliferative response and renewal of hepatic function following cocaine administration in mice

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.     

Chronic cocaine administration decreases norepinephrine-induced phosphoinositide hydrolysis in rat aorta.

The effect of chronic cocaine administration on norepinephrine stimulated hydrolysis of inositol 1,4,5-trisphosphate from the membrane phosphatidylinositol phosphate pool in isolated rat aorta was investigated. Rats received saline (controls), or 10 or 20 mg/kg cocaine once a day for 15 days. This treatment resulted in a dose-dependent reduction in norepinephrine (0.36 microM) stimulated phosphoinositide hydrolysis. The effect of acute cocaine was determined by adding 30 microM cocaine to the in vitro incubation solution. When aortas were exposed to cocaine and norepinephrine simultaneously, in vitro, inositol phosphate formation doubled. By itself, cocaine did not affect phosphoinositide hydrolysis. Contraction of aortic helical strips by norepinephrine decreased in tissues from rats chronically treated with 20 mg/kg cocaine. In vitro cocaine shifted the norepinephrine concentration/response curve to the left and increased the maximum response. Neither acute nor chronic cocaine treatment affected prazosin's apparent dissociation constant, suggesting that cocaine did not affect receptor affinity. These data suggest that chronic, but not acute cocaine administration may interfere with pharmacomechanical coupling in rat aorta.     

Subcellular distribution and postnatal development of cholinergic marker proteins in the striatum of rat

The distribution of muscarinic cholinergic receptors, choline acetyl-transferase and acetylcholinesterase activities were measured in subcellular fractions of the rat striatum on the 5th and 15th days postnatally and in adulthood. The receptor density in the striatum of 5 and 15-day-old rats was 15%, respectively, of the adult value. Similar increases of the receptors could be detected in the synaptosomal and microsomal fractions in the postnatal life of rat. The activity of choline acetyltransferase on the same days was 15% and 28%. In the subcellular fractions, the enzyme activity was the highest in the microsomal fraction on both the 5th and 15th days postnatally. The activity of acetylcholinesterase in the homogenate was 6% of the adult value in the 5-day-old rat striatum, while in the synaptosomal fraction it was 11% and 47% of the adult value on the 5th and 15th days, respectively. Our results show that the development of the muscarinic cholinergic receptors precedes that of the two cholinergic enzymes in both 5 and 15-day-old rat striatum. This may suggest an early perikaryonal synthesis and the fast translocation of receptors to the axon terminals during ontogenetic development.     

Sex and strain differences in the hepatotoxic response to acute cocaine administration in the mouse

Cocaine-induced hepatotoxicity was examined in vivo in a dose-responsive manner in C57BL/6Ibg, DBA/2Ibg, C3H/2Ibg, and Balb/cJ mice. Serum glutamic-pyruvic transaminase (SGPT) activities were determined 24 hours after intraperitoneal (IP) administration of cocaine (20 to 100 mg/kg). Significant elevations (100- to 150-fold) in SGPT were observed in male mice receiving cocaine. Significant differences in sensitivity to cocaine-induced hepatotoxicity were found among males of the inbred strains, with Balb being most sensitive and C57BL being least sensitive and C3H and DBA strains exhibiting intermediate sensitivity. Female mice of the four inbred strains were more resistant than males to cocainemediated hepatotoxicity, as indicated by only twofold to tenfold elevations in SGPT values. Among the females, sensitivity of the four inbred strains—as indicated by dose response curves—fell into two categories: the sensitive strains (C3H and C57BL) and the resistant strains (Balb and DBA). Pretreatment of males of the four inbred strains with the P-450 inducer phenobarbital resulted in enhancement of cocaine-mediated hepatotoxicity in the C57BL and Balb but not the C3H and DBA mice. Phenobarbital pretreatment of females of the four inbred strains resulted in enhancement of the hepatotoxic response to cocaine in the C3H, DBA, and Balb mice. Phenobarbital-pretreated C57BL females exhibited a 100% mortality rate after the acute cocaine dose, and thus no determination of hepatotoxicity could be established for them. These data demonstrate sex and strain differences in cocaine-induced hepatotoxicity and suggest that phenobarbital pretreatment does not uniformly enhance the hepatotoxicity of cocaine.     

Dopaminergic supersensitivity in striatum and olfactory tubercle following chronic administration of haloperidol of clozapine

Mice were maintained on diets containing haloperidol or clozapine for 8–10 days. Two days after these drug-containing diets were withdrawn the effects of apomorphine were determined on locomotor activity and on the retardation of dopamine depletion produced by synthesis inhibition with α-methyltyrosine. After either neuroleptic the effects of apomorphine were enhanced when compared with mice maintained on a control diet, suggesting the development of supersensitive dopamine receptors.     

Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra

Glial cell line-derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non-human primates, in animal models of Parkinson's disease, and may improve motoric function in patients with advanced Parkinson's disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 micro g of GDNF was injected into the striatum of normal aged (24-month-old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot-immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF-treated rats, TH phosphorylation at Ser31 increased approximately 40% in striatum and approximately 250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine- and potassium-evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF-induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.     

Analgesic activity of enkephalins following intracerebral administration in the rat

Methionine- and leucine-enkephalin were found to be potent, short-acting analgesics in the tail flick test in rats following intracerebral administration, via chronically indwelling cannulae, into the midbrain periaqueductal gray. Morphine sulfate was approximately 4 times as potent as the enkephalins when infused into this same brain site. The analgesia produced by the enkephalins and by morphine was inhibited by pretreatment with naloxone.