Peter Clausing

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.

Parenterally administered 3-nitropropionic acid and amphetamine can combine to produce damage to terminals and cell bodies in the striatum

The combined effects of amphetamine (AMPH) and 3-nitropropionic acid (3-NPA) were investigated to determine how the energy depletion proposed to be produced by AMPH interacts with an inhibitor of mitochondrial respiration to produce striatal neurotoxicity. Neither two doses (2 h apart) of 3.75 mg/kg AMPH alone nor a single dose of 30 mg/kg 3-NPA i.p. produced neurotoxicity in the striatum or lowered striatal dopamine content in rat. Administration of 40 mg/kg of 3-NPA alone almost invariably produced either lethality or did not produce neurotoxicity in the striatum of surviving animals. However, 30 mg/kg of 3-NPA administered along with 2 doses of 3.75 mg/kg AMPH to 47 animals produced striatal damage in the 31 survivors with 15 of the surviving rats showing muscle rigidity/catatonia for several days after dosing, along with decreased food consumption. Thirteen of these 15 rats showed degeneration of axons and cell bodies in the medial caudate-putamen with minimal damage to the globus pallidus. However, two rats exhibited hindlimb paralysis and signs of axonal and neuronal soma degeneration in the thalamus and cerebellar nuclei as well as striatum. Sixteen of the rats given both AMPH and 3-NPA exhibited only torpidity and loss of muscle tone 1-3 h after dosing. Such rats showed no signs of neuronal cell degeneration in the striatum, but did show significant dopamine depletions (60% of control) and reductions in tyrosine hydroxylase immunoreactivity at 14 days postexposure. The mitochondrial dysfunction produced by 3-NPA combined with activation of neuronal pathways by AMPH may have predisposed terminals, axons and cell bodies in striatum to degeneration.

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.

Time Course of Brain Temperature and Caudate/Putamen Microdialysate Levels of Amphetamine and Dopamine in Rats after Multiple Doses of d‐Amphetamine

ABSTRACT: 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°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.

Determination of D-fenfluramine, D-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of D-fenfluramine (FEN), D-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/microl), plasma (2 pmol/ microl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/microl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.

Prenatal Ethanol Exposure in Rats: Long-Lasting Effects on Learning

Pregnant Sprague-Dawley rats were fed a liquid diet containing either 0% (group C), 18% (group L), or 36% (group H) ethanol-derived calories (EDC) from gestational day 1 to 20. Male offspring were assessed under a conditioned taste aversion paradigm (PND 35-45), in a complex maze (PND 68-80), and for operant behavior (temporal response differentiation and motivation to work for food, PND 140-198). Although conditioned taste aversion was fully acquired by all groups, retention of the conditioned taste aversion response was impaired in group H animals. Importantly, deficits in the acquisition of timing behavior were found in group H (group L not tested), confirming that this operant task is quite sensitive in detecting prenatal drug effects and demonstrating that neurological effects of prenatal ethanol exposure persist into late adulthood.

Differential effects of communal rearing and preweaning handling on open-field behavior and hot-plate latencies in mice

On day 2 after delivery, dams of the DBA/1 mouse inbred strain (n = 20/group) with their litter were allocated to one of the following groups: NH21, nonhandling, housed 1 litter/cage, weaned on postnatal day (PND) 21;H21, handling, housed 1 litter/cage, weaned on PND 21; NH30, nonhandling, group-housed (5 litters/cage), weaned on PND 30; H30, handling, group-housed (5 litters/cage), weaned on PND 30. Two male pups of each litter were color marked on PND 2. From PND 8-21 they were removed from their cage, gently held in the experimenters hand for 5 min/day. The two marked males of each litter were housed together after weaning, and tested in the open-field on PNDs 51-53, and one of each of these siblings was tested for hot-plate latencies on PND 54. Being raised in group-housing and weaned on PND 30 resulted in offspring exhibiting shorter latencies to initiate behavior and higher percentages of centerfield entries in the open field, hot-plate latencies, however, remained unaffected. Preweaning handling increased hot-plate latencies and the number of grooming episodes in the open field, and it decreased defecation, percent centerfield entries and open-field activity in general. It is concluded that the two forms of early experience have different effects on neurobehavioral endpoints 8 weeks after birth.

Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid

An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method.

Preweaning experience as a modifier of prenatal drug effects in rats and mice--a review.

The effects of preweaning experience in rats and mice on neuroendocrine and behavioral end points and their implications for prenatal drug effects are reviewed. The hypothalamo-pituitary-adrenal axis and the dopaminergic system were shown to be affected. Behavior related to hippocampal, adrenocortical functions and to the benzodiazepine receptor system was also modified. Other paradigms (nociception, conditioned taste aversion) exhibited susceptibility to such preweaning manipulations also. The effects of these early experiences seem to be mediated through complex factors including neuroendocrine responses of the pup to hypothermia and a permanent alteration of mother-infant interactions, with subsequent effects on neuroendocrine functions that are important for postnatal brain organization. Studies of interactions between prenatal drug effects and preweaning manipulations have been performed only with ethanol. When extending this work to other compounds, the systems and functions described above may provide some guidance in looking for possible interactions. In most cases the preweaning manipulations alleviated the effects of prenatal ethanol exposure. These findings may have important implications regarding the controversy about environmental influences affecting the outcome of exposure to neurobehavioral teratogens.

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 × 5 mg/kg AMPH and 3) high versus low rotators differed neither in their AMPH nor in their dopamine extracellular striatal concentrations.