Cathepsin K inhibition reduces CTXII levels and joint pain in the guinea pig model of spontaneous osteoarthritis.

Cathepsin K is a cysteine proteinase which is believed to contribute to osteoarthritis (OA) pathogenesis. This brief report evaluates the effect of the novel selective cathepsin K inhibitor AZ12606133 on cartilage metabolism in the Dunkin-Hartley guinea pig model of spontaneous OA. In parallel, electrophysiological studies were performed to determine whether acute and chronic treatment with the cathepsin K inhibitor could alter joint nociception. Acute treatment of OA knees with AZ12606133 had no effect on joint afferent nerve activity; however, prolonged (1 month) administration of the cathepsin K inhibitor delivered via a chronically implanted osmotic pump significantly reduced mechanosensitivity in response to both non-noxious and noxious joint movements. Urinal concentrations of the cartilage breakdown products cross-linked C-telopeptides of type II collagen (CTXII) were also reduced by chronic cathepsin K inhibition. These data suggest that prolonged AZ12606133 administration can reduce cartilage turnover and joint nociception in the Dunkin-Hartley guinea pig model of spontaneous OA.

Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.

OBJECTIVE: To investigate the effects of a matrix metalloproteinase (MMP) inhibitor on joint pathology and pain behavior in the rat meniscal transection (MNX) model of osteoarthritis (OA) and evaluate which aspects of structural disease modification contribute to symptom improvement. METHODS: OA pathology was induced in male Lewis rats, by transecting the medial collateral ligament with (MNX) or without (SHAM) a full thickness cut through the meniscus. MNX animals were orally administered an equipotent MMP 2, 8, 9, 12, 13 inhibitor (0.25, 1 and 5 mg/kg/day) or vehicle from day 1. Chondropathy, osteophytosis, osteochondral vascularity were assessed from toluidine blue stained coronal sections of the total knee joint and weight-bearing asymmetry by incapacitance. Group differences were evaluated using 1-way analysis of variance (ANOVA) and associations as Spearman's correlation coefficients. RESULTS: Treatment with the MMP inhibitor reduced weight-bearing asymmetry from day 14 onwards, and attenuated chondropathy (both P<0.05). Osteochondral vascularity was elevated in MNX compared with SHAM-operated animals (P<0.001) and reduced, dose-dependently, by MMP inhibitor treatment (r=-0.89, P<0.05). Reduced osteochondral vascularity and chondropathy were associated with the amelioration of weight-bearing asymmetry (both P<0.05). CONCLUSION: Here we show that treatment with a MMP inhibitor reduces joint damage, osteochondral angiogenesis and behavioral evidence of pain. The association between osteochondral angiogenesis and pain behavior may be explained by perivascular nerve growth or stimulation of subchondral nerves following loss of osteochondral integrity. Our data suggest that targeting angiogenesis may have utility in the treatment of pain associated with structural damage in OA.

A threshold neurotoxic amphetamine exposure inhibits parietal cortex expression of synaptic plasticity-related genes.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores: flash-induced cytochrome b reduction.

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an alpha-band maximum at 562 nm in the presence of antimycin. The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood. Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation. These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides.

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides.

1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda max at 549.5 +/- 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed. 3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b-90 was observed. 4. Difference spectra attributed to (BChl)2, (Formula: see text), c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata. 5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.

Supplementary carrying compared with advice to increase responsive parenting as interventions to prevent persistent infant crying.

OBJECTIVE: To compare two interventions (supplementary carrying, increased parental responsiveness) introduced from birth for their effectiveness in reducing the amounts of crying in general community infants at 2, 6, and 12 weeks age. DESIGN AND PARTICIPANTS: Mothers and infants in newborn wards of maternity hospitals were assigned to carrying intervention, responsiveness intervention, or control groups. Follow-up measures were used to confirm that the interventions were implemented and to determine their effects on infant crying. SETTING AND MEASUREMENTS: Diary measurements completed in the home were employed to measure the aspects of parental behavior targeted by the interventions. Audio recordings, diaries, and questionnaires assessed the amounts the infants cried and the impact of the crying on their mothers and the health services. Sample sizes at 6 weeks of age were 59 (carrying intervention), 57 (responsiveness intervention), and 94 infants (control group). RESULTS: The carrying intervention successfully increased the amounts the infants were carried, particularly while settled, to the target levels. The responsiveness intervention led to more limited increases in carrying and to a modest increase in feeding frequency, but did not affect measures of parental interactiveness and play. No differences in amounts of crying and fussing were found between the three groups of infants on any of the measures. Subsidiary analyses confirmed that the dependent variable (infant fuss/crying) and main independent variable (carrying while settled) were not significantly correlated. CONCLUSION: It is not, at present, possible to recommend either supplementary carrying or increased parental responsiveness as primary, preventative interventions to reduce infant crying.

Individual differences in dopamine release but not rotational behavior correlate with extracellular amphetamine levels in caudate putamen in unlesioned rats.

It has been postulated that differences in pharmacokinetics do not contribute to the well-known individual variability in response to amphetamine (AMPH), but this is yet to be investigated thoroughly. Therefore, rotational behavior of outbred rats (Sprague-Dawley, 4 months old) was recorded during microdialysis sessions and striatal microdialysate was analyzed concomitantly for AMPH and dopamine concentrations after a single injection of 2.5 mg/kg AMPH SC. Three hours later these rats received three doses of 5 mg/kg AMPH SC (spaced 2 h apart) and their brain temperature was recorded every 20 min. The most important findings were: 1) the increase in extracellular dopamine was highly correlated with the corresponding peak AMPH levels in the microdialysate; 2) the peak dopamine level in response to 2.5 mg/kg AMPH was predictive of the hyperthermic response observed during 3 x 5 mg/kg AMPH and 3) high versus low rotators differed neither in their AMPH nor in their dopamine extracellular striatal concentrations.

Changes in mRNA levels for heat-shock/stress proteins (Hsp) and a secretory vesicle associated cysteine-string protein (Csp1) after amphetamine (AMPH) exposure.

Damage to nerve terminals, reactive gliosis and somatic degeneration can result when pronounced hyperthermia occurs during amphetamine (AMPH) exposure. The effects of AMPH-induced hyperthermia and damage on the relative mRNA levels for several heat shock/stress proteins (Hsp27, Hsp60, Hsp70 and Hsc70), as well as secretory vesicle associated cysteinestring protein (Csp1) were determined in both the striatum and substantia nigra using reverse transcriptase polymerase chain reaction (RT-PCR). These changes were compared to changes in Hsp mRNA levels seen in primary rat cerebral astrocyte cultures after heat shock/stress. Striatal Hsp70 mRNA increased about 2-fold over control levels at 16 hr after AMPH-induced hyperthermia, and was the only Hsp species to significantly increase in response to AMPH. Hsp70 mRNA levels returned to control within 14 days after AMPH. Two-fold increases in Hsp70 mRNA were also seen in primary cultures of rat cerebrum 24 hr after heat shock. In primary cultures and brain tissue, the increased Hsp70 mRNA levels were still more than 500-fold less than constitutive Hsc70 mRNA and 50-fold less than Hsp60 levels. Hsp27 mRNA was not present in the striatum, nigra and primary cell cultures. Thus, the expression of Hsp species mRNA measured was very similar in brain tissue and primary cell cultures. Because only a modest induction of Hsp 70 mRNA occurred, the Hsp species evaluated may only play a minor role in AMPH neurotoxicity. However, further studies are necessary to determine whether large increases in Hsp70 are occurring in selected neurons or glia in the striatum. RT-PCR products for Csp1 were produced in total RNA obtained from brain but not from cultured astrocytes, suggesting that the Csp1 mRNA measured by RT-PCR is of neuronal origin. Csp1 mRNA levels were acutely downregulated in neurons in the substantia nigra, possibly in response to damage, but not the striatum after AMPH exposure. A slight long-term upregulation at 4 months of Csp1 mRNA may occur in the striatum but not in nigra.

Time course of brain temperature and caudate/putamen microdialysate levels of amphetamine and dopamine in rats after multiple doses of d-amphetamine.

Brain temperature monitoring and microdialysis were performed simultaneously in the caudate/putamen (CPu) of conscious, freely moving rats dosed with d-amphetamine (AMPH). The brain temperature was determined via a thermistor inserted through a microdialysis guide cannula located in the left CPu, while the microdialysis probe was positioned in the right CPu. The peak AMPH and dopamine (DA) levels were reached 40 to 60 min after dosing, while peak brain temperature was not achieved until 20 to 40 min thereafter in rats becoming moderately hyperthermic. Those rats becoming severely hyperthermic (temperatures above 41.0 degrees C) had microdialysate concentrations of AMPH and DA almost 2-fold higher than those with moderate hyperthermia after the second dose of 5 mg/kg AMPH. However, these peaks were not reached until 60 to 80 min after dosing. This was probably due, in part, to the longer half-life of AMPH in the severely hyperthermic group. The changes in brain temperature observed after exposure to neurotoxic doses of AMPH closely paralleled core body temperature changes previously reported during AMPH exposure. Temperature plays an important role in many types of neurotoxicity, and monitoring brain temperature during microdialysis studies can be done continuously, and with less chance of damage to the microdialysis equipment than most of the traditional methods used to measure core body temperature.

Comparison of glutamine-enhanced glutamate release from slices and primary cultures of rat brain.

Increased extracellular glutamate has been associated with a wide range of effects including production of neurotoxicity. Glutamine has previously been shown to cause increased release of glutamate from a variety of preparations. Extracellular central nervous system (CNS) glutamine levels are known to increase with neurotoxin exposures, hepatic failure, renal failure, head trauma or stroke. However, the action of glutamine to enhance the release of glutamate under nondepolarizing conditions has not been well studied. Since glutamine-mediated increases in extracellular glutamate are potentially of significance in cellular damage as a result of CNS insult, further examination of this phenomenon is important. Striatal and hippocampal slices or virtually neuron-free primary striatal glial cultures were employed in studies to further elucidate the mechanism(s) of glutamine-enhanced glutamate release. Elevated extracellular glutamine caused increased glutamate release in all three preparations. In hippocampal and striatal slices elevated glutamine caused an enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated [3H]catecholamine release equivalent to that produced by high concentrations (up to 100 microM) of exogenous glutamate. In both striatal slices and primary cultures kynurenate increased glutamate release in the presence of 500 microM glutamine, while kainate either had no effect or decreased glutamate levels in the presence of glutamine. Since several presynaptic modulators of release did not affect the glutamate release produced by glutamine in slices, vesicular release of glutamate from nerve terminals was probably not involved in the effects of the exogenous glutamine. The similarities between striatal slices and primary striatal cultures indicate that enzymatic conversion of glutamine to glutamate within glia may be an important factor in the glutamine-mediated elevation of extracellular glutamate levels.

d-Fenfluramine produces neuronal degeneration in localized regions of the cortex, thalamus, and cerebellum of the rat.

d-Fenfluramine is a potent serotonin (5-HT) reuptake inhibitor/releaser and, until its recent recall, was prescribed as an anoretic agent. This study demonstrates that 10 mg/kg d-fenfluramine i.p., when administered to rats in a warm (27 degrees C) environment, produces neuronal degeneration within select brain regions. Degeneration was detected and localized using a recently developed fluorescent marker of neuronal degeneration, Fluoro-Jade. The most extensive cortical damage was in the anterior cingulate region. In the medial thalamus, degeneration was frequently seen within the intralaminar nuclei, and somewhat less frequently observed within the paraventricular nucleus, the mediodorsal nucleus, and the gelatinosis nucleus. Cerebellar damage occurred primarily in medial Purkinje cells and occasionally in granule cells or basket cells. Degeneration was not observed in either saline-injected control animals or in rats given even higher doses of 25 mg/kg d-fenfluramine but kept in a cooler environment (23 degrees C). The degeneration was clearly most prominent in animals with body temperatures of 41 degrees to 42 degrees C, but this degeneration was not seen in animals given saline that became extremely hyperthermic in a 37 degrees C environment. Behavioral signs such as tremors, myoclonus, rigidity, and splayed legs were seen in all animals with extensive neurodegeneration. The areas damaged by d-fenfluramine, when hyperthermia occurs, could play a role in the expression of the serotonin syndrome. Elevated extracellular 5-HT levels alone are probably not sufficient for neurotoxicity, and additional factors such as hyperthermia, regional specificity of 5-HT receptor subtypes, blood flow, and/or neuronal networks may be involved.

Seizure activity and hyperthermia potentiate the increases in dopamine and serotonin extracellular levels in the amygdala during exposure to d-amphetamine.

Behavioral stereotypy, hyperthermia, and convulsive activity produced by exposure to multiple doses of d-amphetamine (AMPH) were related to changes in the extracellular levels of dopamine and serotonin (5-HT) in the amygdala, using the technique of microdialysis in awake and freely moving rats. Hyperactivity and stereotypy, as well as increases in microdialysis dopamine levels ranging from 100-300% of pre-AMPH basal microdialysate levels (BL), occurred during exposure to 3 doses of 2.5 mg/kg (3 x 2.5 mg/kg) AMPH. Three doses of 5 mg/kg produced a more intense stereotypic behavior as well as hyperthermia, and resulted in large increases in the peak dopamine levels (700% BL) while 5-HT levels were increased to a lesser extent (300% BL). The highest doses tested of 3 x 15 mg/kg produced convulsive activity, seizures, intense stereotypy and hyperthermia with peak microdialysate dopamine (1300% BL) and 5-HT levels (1800% BL) that were 2-fold and 6-fold greater, respectively, than those at the 3 x 5-mg/kg doses. Microdialysate glutamate levels were not changed by AMPH exposure. Rats that did not become hyperthermic when dosed with 15 mg/kg AMPH in a cold environment (10 degrees C) exhibited some hyperactivity and stereotypic behavior, but not overt convulsive behavior. Dopamine and 5-HT levels in these rats were significantly reduced by about 75% and 60%, respectively, compared to the room-temperature group. Inclusion of 2 microM tetrodotoxin (TTX) in the microdialysis buffer significantly reduced the 15-mg/kg AMPH-induced increases in dopamine by 30% and the increase in 5-HT levels by 70% at room temperature. These results indicate that a smaller portion of the dopamine release evoked by doses of AMPH that induce seizure activity is neuronal impulse-dependent while the majority of 5-HT released is impulse-dependent. Irrespective of impulse activity, the hyperthermia alone markedly potentiated dopamine release but had a lesser effect on 5-HT release. Thus, there are differences in the regulation of dopamine and serotonin release in the amygdala from high doses of AMPH, which are known to produce neurotoxicity. Further studies are necessary to determine the impact of these differences in release on AMPH neurotoxicity.

An evaluation of l-ephedrine neurotoxicity with respect to hyperthermia and caudate/putamen microdialysate levels of ephedrine, dopamine, serotonin, and glutamate.

l-Ephedrine is an active ingredient in several herbal formulations with a mechanism of action similar to amphetamine and methamphetamine. However, its potential to damage dopaminergic terminals in the caudate/putamen (CPu) has yet to be fully evaluated. The studies here used in vivo brain microdialysis experiments to determine the systemic doses and extracellular brain levels of l-ephedrine necessary to produce similar increases in CPu extracellular dopamine and marked hyperthermia that were previously shown necessary for amphetamine-induced neurotoxicity in male Sprague-Dawley rats. At an environmental temperature of 23 degrees C, a single 40 mg/kg intraperitoneal (ip) dose of l-ephedrine produced marked hyperthermia (>/= 40 degrees C), peak microdialysate ephedrine levels of 7.3 +/- 1.2 microM, and a 20-fold increase in microdialysate dopamine levels. Twenty-five mg/kg produced a lesser degree of hyperthermia, peak microdialysate ephedrine levels of 2.6 +/- 0.4 microM, and a 10-fold increase in dopamine levels. Three doses of 40 mg/kg given at 3-h intervals or 4 doses of 25 mg/kg l-ephedrine given at 2-h intervals were compared with 4 doses of 5 mg/kg d-amphetamine given at 2-h intervals. Multiple doses of either ephedrine or amphetamine caused severe hyperthermia (>/= 41.3 degrees C) but striatal tissue levels of dopamine 7 days after dosing were reduced only 25% or less by ephedrine compared to the 75% reductions produced by amphetamine. The increases in CPu microdialysate levels of serotonin produced by either 4 x 25 mg/kg l-ephedrine or 4 x 5 mg/kg d-amphetamine did not significantly differ, but elevation of dopamine levels by d-amphetamine were over 2-fold times the level caused by l-ephedrine. Microdialysate glutamate levels were elevated to the same extent by either 25 mg/kg l-ephedrine or 4 x 5 mg/kg d-amphetamine. l-Ephedrine may not be as neurotoxic to dopaminergic terminals as d-amphetamine, because non-lethal doses of l-ephedrine do not sufficiently increase the CPu dopamine levels within nerve terminals or the extracellular space to those necessary for a more pronounced long-term dopamine depletion.

Neuronal degeneration in the limbic system of weanling rats exposed to saline, hyperthermia or d-amphetamine.

Neuronal degeneration was detected in the tenia tecta and other regions of the anterior limbic system of male weanling rats 3 days after four doses of 5 mg/kg d-amphetamine (4 x 5 mg/kg AMPH) when seizures occurred during AMPH exposure. Neurodegeneration in the parietal cortex, loss of tyrosine hydroxylase immunoreactivity in the caudate-putamen (CPu) and decreases in CPu tissue dopamine levels in weanlings was much less than those previously observed in adults. The neurotoxicity seen in the parietal cortex and CPu of the weanlings was much less than previously seen in adults even though severe hyperthermia and the behavior of retrograde propulsion occurred during AMPH exposure. Neurodegeneration was not detected in any of the previously mentioned brain regions in controls and weanlings made hyperthermic by a warm environment. However, signs of spontaneous neurodegeneration were seen in the posterior piriform cortex (Pir), posteriolateral cortical amygdaloid nucleus (PLCo), and the amygdalopiriform transition area (APir) of control weanlings. The doses of AMPH and the degree of hyperthermia necessary to induce seizures were substantially lower in weanlings compared to those previously observed in adult rats. Further studies will be necessary to determine if the susceptibility of weanlings to AMPH-induced seizures is related to or dependent on the same processes involved in producing degeneration in the posterior limbic system of saline controls.

Methamphetamine exposure can produce neuronal degeneration in mouse hippocampal remnants.

Neuronal cell death in hippocampal remnants was seen after methamphetamine (METH) exposure. Two techniques (Fluoro-Jade labeling and argyrophylia) showed that neuronal degeneration occurred in the indusium griseum, tenia tecta and fasciola cinerea within 5 days post-METH exposure in 70% of the mice. Neurodegeneration also occasionally occurred in the piriform cortex, hippocampus and frontal/parietal cortex. This cell death, unlike striatal neurotoxicity, was not dependent on magnitude of hyperthermia occurring but did correlate with behavioral seizure activity during METH exposure. Excitotoxic mechanisms may be underlying the neuronal degeneration since co-administration of phenobarbital blocked cell death.

Methamphetamine-stimulated striatal dopamine release declines rapidly over time following microdialysis probe insertion.

To investigate changes in striatal dopamine release over a series of brief methamphetamine (METH) exposures, METH was pulsed three times at 2-h intervals, with the first exposure occurring 2 h after microdialysis probe insertion. Whether METH was administered directly into the striatum via the microdialysate (20 microM of METH for 10 min), or via peripheral intraperitoneal (i.p.) injection (1 mg/kg METH, i.p.), the dopamine (DA) peak elicited by the third METH exposure was only 50% as large as that elicited by the first exposure, 4 h earlier. This decline in the magnitude of METH-induced DA release probably continued over at least 24 h, since the magnitude of a single peak 26 h after probe implantation was only one-seventh of that at 2 h. This reduction in the response to METH was a function of time post-probe insertion, and not of prior METH exposure. Thus, peak size was the same at 6 h post-implantation in animals which received two prior METH pulses or no prior METH pulses, and in both cases this 6-h peak was substantially lower than that at 2 h post-implantation. Circadian influences were also excluded as a factor, because size of the initial METH-induced DA peak did not vary as a function of time of probe implantation. It is concluded that METH-stimulated striatal DA release declines rapidly over time post-probe insertion. When METH exposures occur repeatedly at short intervals, this decline can mimic, but is not caused by, desensitization or depletion in response to prior METH exposure.

Low environmental temperatures or pharmacologic agents that produce hypothermia decrease methamphetamine neurotoxicity in mice.

Recently we have reported that methamphetamine (METH) neurotoxicity in rats depends on the environmental temperature. Here, we evaluate whether a cold environment (4 degrees C) or drugs which chloride and glutamate ion channel function block METH neurotoxicity in mice. Adult male CD mice received METH i.p. (4 x 10 mg/kg METH at 23 degrees C along with saline. 2.5 mg/kg (+)-MK-801, 40 mg/kg phenobarbital or 2.5 mg/kg diazepam and either 4 x 10 or 4 x 20 mg/kg METH at 4 degrees C). Multiple injections of METH (4 x 10 mg/kg i.p.) at room temperature (23 degrees C) produced a significant depletion of dopamine (DA) in striatum at 24, 72 h, 1 and 2 weeks. Three days post 4 x 10 mg/kg METH at 23 degrees C, an 80% decrease in striatal dopamine (DA) occurred while the same dose at 4 degrees C produced only a 20% DA decrease, and 4 x 20 mg/kg METH at 4 degrees C produced a 54% DA decrease. At 23 degrees C (+)MK-801 completely blocked while phenobarbital (40% decrease) and diazepam (65% decrease) partially blocked decreases in striatal DA produced by 4 x 10 mg/kg METH. Decreases in DOPAC and HVA were similar to the decreases in DA after METH and antagonists. Multiple injections of METH (4 x 10 mg/kg, i.p.) at room temperature also produced a significant depletion of serotonin (5-HT) in striatum at 24, 72 h, 1 and 2 weeks. This depletion of 5-HT at room temperature was blocked either by changing the environmental temperature to 4 degrees C, or by pretreatment with MK-801, diazepam and phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenobarbital and dizocilpine can block methamphetamine-induced neurotoxicity in mice by mechanisms that are independent of thermoregulation.

Body temperature profiles observed during methamphetamine (METH) exposure are known to affect dopamine and tyrosine hydroxylase (TH) levels in the striatum of mice; hyperthermia potentiates depletion while hypothermia is protective against depletions. In the current study, the doses of METH were sufficiently great that significant dopamine and TH depletions occurred even though hypothermia occurred. Four doses, administered at 2 h intervals, of 15 mg/kg (4x15 mg/kg) D-METH significantly decreased TH and dopamine levels to 50% of control in mice becoming hypothermic during dosing in a 13 degrees C environment. Phenobarbital or dizocilpine during METH exposure blocked the depletions while diazepam did not. Phenobarbital and dizocilpine did not block depletions by altering the hypothermic profiles from that observed during METH only exposure. Here we show that phenobarbital and dizocilpine can block measures of METH neurotoxicity by non-thermoregulatory mechanisms.

Nitric oxide regulation of methamphetamine-induced dopamine release in caudate/putamen.

A possible role for NO modulation of dopamine (DA) release in the caudate/putamen (CPU) during methamphetamine (METH) exposure was investigated using in vivo microdialysis in rats. Inclusion of the nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine (NOARG), NG-nitro-L-arginine methyl ester (L-NAME) or D-NAME (less potent inhibitor) in the microdialysis buffer prior to METH minimally affected basal levels of DA, DOPAC or HVA in CPU microdialysate. However, L-NAME and NOARG produced concentration-dependent decreases of up to 64% (100 microM) in CPU DA levels in microdialysate during exposure to four doses of METH (5 mg/kg i.p./2 h), with lesser effects on DOPAC or HVA. Reversal of the NOARG inhibition was produced by inclusion of 500 microM of either L-arginine or L-citrulline in the microdialysate. D-NAME (100 microM) minimally affected levels of DA or metabolites. Paradoxically, inclusion of from 20 to 2 microM of the NOx generators isosorbide dinitrate (ISON) or sodium nitroprusside (SNP) in the microdialysis buffer decreased DA and DOPAC levels in microdialysate during METH exposure. This paradox might result from the concentrations of NOx produced by SNP or ISON being great and not regionally specific resulting in inhibition of DA release and/or synthesis while the NO generated endogenously during METH exposure may have localized and site-specific actions. Alternatively, NOx may inhibit NOS or other enzymes in the NO synthesis pathway, thereby reducing levels of an intermediate (other than NO) which potentiates DA release. In their entirety, our results indicate that NO generation in the CPU may augment the release of DA during METH exposure.