Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Abstract: Monoamine-uptake blockers were applied focally (0.1–1,000 µ M ) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.     

Extracellular Concentration and In Vivo Recovery of Dopamine in the Nucleus Accumbens Using Microdialysis

The present study compared two different in vivo microdialysis methods which estimate the extracellular concentration of analytes at a steady state where there is no effect of probe sampling efficiency. Each method was used to estimate the basal extracellular concentration of dopamine (DA) in the nucleus accumbens of the rat. In the first method, DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 2.5, 5, and 10 nM) and DA is measured in the dialysate from the brain to generate a series of points which are interpolated to determine the concentration of no net flux. Using this method, basal DA was estimated to be 4.2 +/- 0.2 nM (mean +/- SEM, n = 5). The slope of the regression gives the in vivo recovery of DA, which was 65 +/- 5%. This method was also used to estimate a basal extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) concentration in the nucleus accumbens of 5.7 +/- 0.6 microM, with an in vivo recovery of 52 +/- 11% (n = 5). A further experiment which extended the perfusate concentration range showed that the in vivo recovery of DA is significantly higher than the in vivo recovery of DOPAC (p less than 0.001), whereas the in vitro recoveries of DA and DOPAA are not significantly different from each other. The in vivo difference is thought to be caused by active processes associated with the DA nerve terminal, principally release and uptake of DA, which may alter the concentration gradient in the tissue surrounding the probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine Microinjections in the Hypothalamic Paraventricular Nucleus Increase Extracellular Dopamine and Decrease Acetylcholine in the Nucleus Accumbens: Relevance to Feeding Reinforcement

Abstract: Norepinephrine (NE) was microinjected into the paraventricular nucleus (PVN), while microdialysis was used to monitor extracellular dopamine (DA) and acetylcholine (ACh) in the nucleus accumbens (NAc). The PVN is a site where exogenously administered NE can act through α₂ receptors to elicit eating behavior and preference for carbohydrates. It was hypothesized that NE in the PVN acts on a behavior reinforcement system by altering the DA/ACh balance in the NAc. NE microinjections (80 nmol in 0.3 µl), which effectively elicited feeding in satiated rats in a separate test, caused a significant increase in extracellular DA (109%) and decrease in ACh (−27%) when the same animals were tested in the absence of food. In contrast when the food was available and ingested, ACh increased (51%) instead of decreasing. These results support the hypothesis that a functional link exists between the PVN and the NAc in which DA helps initiate and ACh helps stop appetitive behavior involved in the reinforcement of eating.     

Effects of Feeding and Drinking on Acetylcholine Release in the Nucleus Accumbens, Striatum, and Hippocampus of Freely Behaving Rats

Extracellular levels of acetylcholine (ACh) were measured in the nucleus accumbens (NAC), striatum (STR), and hippocampus (HIPP) using microdialysis in 30-min intervals before, during, and after free-feeding in 20-h food-deprived rats. The effects on ACh in the NAC and STR were also observed in response to water intake in 20-h water-deprived animals. Neostigmine was used in the perfusate to improve ACh recovery. Basal ACh was sensitive to tetrodotoxin and low calcium, and therefore largely neuronal in origin. Feeding caused a 38% increase in extracellular ACh in the NAC and no change in the STR or HIPP. Dopamine was also increased in the NAC (48%) and to a lesser extent in the STR (21%) following feeding. Drinking caused 18-20% increases in ACh release in both the NAC and STR. In a separate experiment, ACh release in the NAC was monitored in 10-min intervals during free-feeding; ACh increased in the interval immediately following maximal food intake. These results suggest a site-specific increase in ACh release following feeding that cannot be solely attributed to the activation associated with this behavior.     

GABAA Receptor Blockade in the Anterior Ventral Tegmental Area Increases Extracellular Levels of Dopamine in the Nucleus Accumbens of Rats

Abstract: Previously, it was shown that microinfusion of the GABA_A antagonist picrotoxin into the anterior ventral tegmental area (VTA) is reinforcing. It was hypothesized that this reinforcing effect of picrotoxin in the anterior VTA is mediated, at least in part, by the activation of the mesoaccumbens dopamine (DA) system. The objective of the present study was to determine if blockade of GABA_A receptors in the anterior VTA can increase extracellular levels of DA in the nucleus accumbens (ACB), using an in vivo microdialysis technique in freely moving rats. Concentrations of picrotoxin (40, 80, and 160 µ M ) that had previously been shown to produce a reinforcing effect increased the extracellular levels of DA and its major metabolites in the ACB. The increased extracellular DA levels induced by intra-VTA injection of picrotoxin was markedly attenuated by coadministration with the GABA_A agonist muscimol, whereas intra-VTA injection of muscimol alone did not have an apparent effect on extracellular DA levels in the ACB. Microinjection of another GABA_A antagonist, bicuculline, into the anterior VTA also increased the extracellular release of DA in the ACB. These results suggest that DA neurons projecting from the anterior VTA to the ACB are tonically inhibited by GABA through its actions at the GABA_A receptors.     

Low Level Lead Exposure Decreases In Vivo Release of Dopamine in the Rat Nucleus Accumbens: A Microdialysis Study

Abstract: The basal and K⁺-induced release of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were measured in microdialysate samples obtained in vivo from the nucleus accumbens region of rats subchronically exposed to 50 ppm lead for 90 days. The basal and stimulus-induced release of dopamine and the metabolites were significantly reduced in the lead-exposed rats as compared with the controls. These reductions in dopamine and its metabolites are consistent with the reports of decreased dopamine availability associated with lead-induced changes in certain behavioral indices (fixed-interval performance) in rats. Furthermore, these changes were observed at blood lead levels similar to those considered to cause impairment in cognitive functions in children.     

Differential Effects of δ- and μ-Opioid Receptor Antagonists on the Amphetamine-Induced Increase in Extracellular Dopamine in Striatum and Nucleus Accumbens

Abstract: The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine-induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective δ-opioid receptor antagonist, but not by the irreversible μ-opioid receptor antagonist β-funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 µg) or vehicle was given 15 min before and β-funaltrexamine (10 µg) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10-µl volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneous d-amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30-min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose-dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 µg) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 µg of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, β-funaltrexamine (10 µg) had no effect in the striatum but significantly attenuated the amphetamine-induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that δ-opioid receptors play a relatively larger role than μ-opioid receptors in mediating the amphetamine-induced increase in extracellular dopamine content in the striatum, whereas μ-opioid receptors play a larger role in mediating these effects in the nucleus accumbens.     

Effect of Isolation-Rearing on Conditioned Dopamine Release In Vivo in the Nucleus Accumbens of the Rat

Abstract: Intracerebral microdialysis in conjunction with HPLC coupled to electrochemical detection was used to investigate the effect of isolation-rearing in the rat on extracellular dopamine (DA) and its metabolites in vivo, in the shell region of the nucleus accumbens, in response to footshock and in relation to a conditioned emotional response. Male Lister hooded rats were reared from weaning for 6–8 weeks in either social isolation or groups of five. In the training phase, rats were exposed to a novel environment for 10 min where they experienced mild footshock. Footshock caused an immediate increase in basal extracellular DA levels in both rearing groups relative to control rats. However, the increase in extracellular DA was prolonged in the case of the isolation-reared rats and significantly greater than in group-reared rats. Exposure to the novel environment without shock (control groups) did not significantly alter basal extracellular DA in the nucleus accumbens shell; 140 min later rats were returned to the testing box (contextual stimulus) without receiving footshock. The contextual stimulus increased basal extracellular DA in the nucleus accumbens of both groups of rats with respect to controls; however, this increase was significantly greater and more prolonged in isolates. Extracellular levels of the metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid did not differ between isolation- and group-reared rats, and they were not significantly affected by either footshock or the contextual stimulus. These results suggest that exposure to footshock and a contextual stimulus are associated with increases in basal extracellular DA levels in the nucleus accumbens shell. The results also support evidence in favour of an isolation-induced enhancement in dopaminergic activity in the nucleus accumbens, which probably underlies aspects of the behavioural syndrome associated with isolation.     

Selective Increase in Dopamine Utilization in the Shell Subdivision of the Nucleus Accumbens by the Benzodiazepine Inverse Agonist FG 7142

Abstract: We examined the effects of the benzodiazepine inverse agonist FG 7142 on dopamine metabolism in the core and shell subdivisions of the nucleus accumbens. FG 7142 (15 mg/kg i.p.) or vehicle was administered to adult male rats 30 min before they were killed. Selected brain regions, including samples from the whole nucleus accumbens as well as core and shell subdivisions, were collected and assayed for tissue concentrations of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Consistent with previous reports, FG 7142 administration increased dopamine utilization in the medial prefrontal cortex but not the whole nucleus accumbens. Examination of subdivisions revealed that FG 7142 produced increased dopamine utilization in the shell subdivision of the nucleus accumbens. No effect of FG 7142 on dopamine utilization in the core region of the nucleus accumbens was observed. These data are discussed in terms of in vivo microdialysis studies reporting increased dopamine release in the nucleus accumbens after FG 7142 administration.     

Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 microM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)-sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. Intradermal injection of formalin produced a TTX-sensitive increase in Asp concentration and a non-TTX-sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.     

Repeated Cocaine Alters Glutamate Receptor Subunit Levels in the Nucleus Accumbens and Ventral Tegmental Area of Rats that Develop Behavioral Sensitization

Increased glutamate transmission in the nucleus accumbens and ventral tegmental area has been proposed as a mechanism underlying sensitized behavioral responses to repeated cocaine administration. GluR1, GluR2/3, and NMDAR1 subunits of glutamate receptors were quantified from immunoblots in these brain nuclei in rats at 24 h and 3 weeks after discontinuing 1 week of daily cocaine injections. Motor behavior was monitored after the first and last injections of daily cocaine, and those rats that showed >20% increase in motor activity after the last compared with the first injection were considered to have developed behavioral sensitization. The subjects that developed behavioral sensitization showed a significant increase in GluR1 levels in the nucleus accumbens at 3 weeks but not at 24 h of withdrawal. Conversely, sensitized animals showed a significant increase in NMDAR1 and GluR1 levels in the ventral tegmental area at 1 day but not at 3 weeks of withdrawal. None of these increases occurred in the rats exposed to daily cocaine that did not develop behavioral sensitization (<20% increase in motor activity), and no changes were measured in the level of GluR2/3 in any treatment group. The functional importance of the increases in glutamate receptor subunit levels is suggested by the fact that the changes were present only in rats that developed behavioral sensitization to repeated cocaine administration.     

Fluoxetine Increases Extracellular Dopamine in the Prefrontal Cortex by a Mechanism Not Dependent on Serotonin

Fluoxetine at 10 and 25 mg/kg increased (167 and 205%, respectively) the extracellular dopamine concentration in the prefrontal cortex, whereas 25 (but not 10) mg/kg citalopram raised (216%) dialysate dopamine. No compound modified dialysate dopamine in the nucleus accumbens. The effect of 25 mg/kg of both compounds on cortical extracellular dopamine was not significantly affected by 300 mg/kg p-chlorophenylalanine (PCPA) (fluoxetine, saline, 235%; PCPA, 230%; citalopram, saline, 179%; PCPA, 181%). PCPA depleted tissue and dialysate serotonin by approximately 90 and 50%, respectively, and prevented the effect of fluoxetine and citalopram on dialysate serotonin (fluoxetine, saline, 246%; PCPA, 110%; citalopram, saline, 155%; PCPA, 96%). Citalopram significantly raised extracellular serotonin from 0.1 to 100 microM (251-520%), whereas only 10 and 100 microM increased dialysate dopamine (143-231%). Fluoxetine similarly increased extracellular serotonin (98-336%) and dopamine (117-318%). PCPA significantly reduced basal serotonin and the effects of 100 microM fluoxetine (saline, 272%; PCPA, 203%) and citalopram (saline, 345%; PCPA, 258%) on dialysate serotonin but did not modify their effect on dopamine (fluoxetine, saline, 220%; PCPA, 202%; citalopram, saline, 191%; PCPA, 211%). The results clearly show that the effects of fluoxetine and of high concentrations of citalopram on extracellular dopamine do not depend on their effects on serotonin.     

In Vivo Microdialysis as a Technique to Monitor Drug Transport: Correlation of Extracellular Cocaine Levels and Dopamine Overflow in the Rat Brain

A sensitive and rapid HPLC-UV method for in vivo determinations of cocaine levels in extracellular fluid of specific brain regions and plasma is described. Free drug levels resulting from intravenous administration of cocaine were sampled using in vivo microdialysis probes simultaneously located in the jugular vein, nucleus accumbens, and anteromedial caudate-putamen of halothane-anesthetized rats. In a separate group of animals, the influence of cocaine on extracellular dopamine concentrations in the anteromedial caudate-putamen was also assessed. The time dependences of changes in cocaine concentration in each of the above regions were congruent, and peak concentrations were reached 10 min after the drug was administered. The half-lives of cocaine in the blood, nucleus accumbens, and anteromedial caudate-putamen were estimated to be 31.5, 29.1, and 21.4 min, respectively. A repeated injection of cocaine, given 90 min later, produced a maximal cocaine level and pharmacokinetic profile that were indistinguishable from those of the initial infusion. Cocaine was concentrated to a greater extent in brain than in blood, a feature consistent with the action of a lipophilic drug. In addition, extracellular dopamine levels measured in the anteromedial caudate-putamen following cocaine infusions closely mirrored those of cocaine itself. The ability to measure the free concentration of drugs by microdialysis should be applicable to a wide range of in vivo pharmacological studies.     

The Effects of Opioid Peptides on Dopamine Release in the Nucleus Accumbens: An In Vivo Microdialysis Study

An involvement of the mesolimbic dopamine (DA) system in mediating the motivational effects of opioids has been suggested. Accordingly, the present study employed the technique of in vivo microdialysis to examine the effects of selective mu-, delta-, and kappa- opioids on DA release in the nucleus accumbens (NAC) of anesthetized rats. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DO-PAC) and homovanillic acid (HVA), using a reverse-phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (i.c.v.) administration of selective mu-opioid [D-Ala2, N-methyl-Phe4, Gly5-ol]-enkephalin (DAMGO) or delta-opioid [D-Pen2, D-Pen5]-enkephalin (DPDPE) agonists, at doses that function as positive reinforcers in rats, resulted in an immediate and significant increase in extracellular DA. DOPAC and HVA levels were also significantly increased. The effects of DAMGO were blocked by the selective mu-antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) whereas those of DPDPE were blocked by the delta-antagonist allyl2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864). In contrast to mu- and delta-agonists, the kappa-agonist N-CH3-Tyr-Gly-Gly-Phe-Leu-Arg-N-CH3-Arg-D-Leu-NHC2H5 (E-2078), a dynorphin analog that produces aversive states, decreased DA release in a biphasic manner. Norbinaltorphimine, a selective kappa-antagonist, could block this effect. These results demonstrate that mu-, delta-, and kappa-opioid agonists differentially affect DA release in the NAC and this action is centrally mediated.     

Dopamine D2 Receptor-Mediated Regulation of Serotonin Extracellular Concentration in the Dorsal Raphe Nucleus of Freely Moving Rats

Abstract: The neuropeptide-inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain-derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15-fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin-3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival-promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.     

Amperozide, a Novel Antipsychotic Drug, Inhibits the Ability of d-Amphetamine to Increase Dopamine Release In Vivo in Rat Striatum and Nucleus Accumbens

The in vivo effects of amperozide, a novel atypical antipsychotic drug, on the release of dopamine (DA) and the output of its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were investigated in the striatum and the nucleus accumbens of awake, freely moving rats using microdialysis. Amperozide (2-10 mg/kg, s.c.) significantly increased extracellular levels of DA in both the striatum and nucleus accumbens in a dose-dependent manner. It had a similar but lesser effect on extracellular DOPAC levels in both regions. d-Amphetamine (2 mg/kg, s.c.) alone produced a very large (43-fold) increase in DA release, together with a 70% decrease in DOPAC levels in both the striatum and the nucleus accumbens. Amperozide (1-5 mg/kg, s.c.) 30 min before d-amphetamine (2 mg/kg) dose-dependently attenuated d-amphetamine-induced DA release but had no effect on the d-amphetamine-induced decrease in extracellular DOPAC levels in both regions. The effect of amperozide on d-amphetamine-induced DA release in the nucleus accumbens may explain the inhibitory effect of amperozide on amphetamine-induced locomotor activity. However, the failure of amperozide to block amphetamine-induced stereotypy, despite marked inhibition of striatal DA release, suggests the need to reexamine the importance of striatal DA for amphetamine-induced stereotypy.     

Differential Effects of Forced Locomotion, Tail-Pinch, Immobilization, and Methyl-β-Carboline Carboxylate on Extracellular 3,4-Dihydroxyphenylacetic Acid Levels in the Rat Striatum, Nucleus Accumbens, and Prefrontal Cortex: An In Vivo Voltammetric Study

In vivo voltammetry with carbon fiber electrodes was used to assess extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) levels in striatum, nucleus accumbens, and anteromedial prefrontal cortex of freely moving rats subjected to altered motor activity or anxiogenic stimuli. Forced locomotion on a rotarod for 40 min caused an increase in extracellular DOPAC levels in the striatum and to a lesser extent in the nucleus accumbens but not in the prefrontal cortex. Subcutaneous injection of the anxiogenic agent methyl-beta-carboline carboxylate (10 mg/kg) increased extracellular DOPAC levels to a similar extent in prefrontal cortex and nucleus accumbens. Immobilization for 4 min augmented dopamine (DA) metabolism preferentially in the nucleus accumbens and to a lesser extent in the prefrontal cortex. Tail-pinch caused a selective activation of DA metabolism in the nucleus accumbens. None of these stimuli altered extracellular striatal DOPAC levels. These results confirm the involvement of dopaminergic systems projecting to the striatum and nucleus accumbens in motor function and suggest that mesolimbic and mesocortical dopaminergic systems can be specifically activated by certain kinds of anxiogenic stimuli; the relative activation of either of these latter systems could depend primarily on the nature (sensory modality, intensity) of the acute stressor.     

Potentiation of the Fluoxetine-Induced Increase in Dialysate Levels of Serotonin (5-HT) in the Frontal Cortex of Freely Moving Rats by Combined Blockade of 5-HT1A and 5-HT1B Receptors with WAY 100,635 and GR 127,935

Abstract: In this study, we examined the influence of blockade of serotonin (5-HT)_1A and/or 5-HT_1B autoreceptors on the fluoxetine-induced increase in dialysate levels of 5-HT as compared with dopamine (DA) and noradrenaline (NAD) in single samples of the frontal cortex (FCx) of freely moving rats. Fluoxetine (10.0 mg/kg, s.c.) elicited a twofold increase in dialysate levels of 5-HT relative to baseline values. The selective 5-HT_1A antagonist WAY 100,635 (0.16 mg/kg, s.c.) did not influence 5-HT release alone but doubled the influence of fluoxetine on basal levels. Similarly, the selective 5-HT_1B/1D antagonist GR 127,935 (2.5 mg/kg, s.c.) did not alter basal 5-HT levels alone and doubled the fluoxetine-induced increase in 5-HT levels. Combined administration of WAY 100,635 and GR 127,935 elicited an (at least) additive rise in the fluoxetine-induced increase in 5-HT levels to eightfold basal values, without modifying resting 5-HT levels. These changes were selective for 5-HT inasmuch as the parallel (twofold) increase in DA and NAD levels provoked by fluoxetine was not potentiated. The present data demonstrate that combined blockade of 5-HT_1A and 5-HT_1B autoreceptors markedly and selectively potentiates the fluoxetine-induced increase in dialysate levels of 5-HT versus DA and NAD in the FCx of freely moving rats. These observations suggest that 5-HT_1A/1B antagonism may represent a novel strategy for the improvement in the therapeutic profile of 5-HT reuptake inhibitor antidepressant agents and that 5-HT may be primarily involved in such interactions.     

A New Inhibitor of the Chymotrypsin-Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin-Protein Conjugates in a Neuronal Cell

Abstract: Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.