Mark A. Prendergast

Molecular basis for interactions of HIV and drugs of abuse.

Summary: In certain populations around the world, the HIV pandemic is being driven by drug‐abusing populations. Mounting evidence suggests that these patient populations have accelerated and more severe neurocognitive dysfunction compared with non‐drug‐abusing HIV‐infected populations. Because most drugs of abuse are central nervous system stimulants, it stands to reason that these drugs may synergize with neurotoxic substances released during the course of HIV infection. Clinical and laboratory evidence suggests that the dopaminergic systems are most vulnerable to such combined neurotoxicity. Identifying common mechanisms of neuronal injury is critical to developing therapeutic strategies for drug‐abusing HIV‐infected populations. This article reviews 1) the current evidence for neurodegeneration in the setting of combined HIV infection and use of methamphetamine, cocaine, heroin or alcohol; 2) the proposed underlying mechanisms involved in this combined neurotoxicity; and 3) future directions for research. This article also suggests therapeutic approaches based on our current understanding of the neuropathogenesis of dementia due to HIV infection and drugs of abuse.

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

Lesions of the nucleus paragigantocellularis : effects on mating behavior in male rats

We evaluated the effects of bilateral radio-frequency lesions of the paragigantocellular (PGi) reticular nucleus in the ventral medulla on male rat copulatory behavior. In Experiment 1, sexually naive male rats with such lesions were more likely than sham-operated controls to copulate to ejaculation during their first exposure to an estrous female. Additionally, among the rats that copulated to ejaculation, those with lesions demonstrated a reduction in mount frequency (MF), intromission frequency (IF), and ejaculation latency (EL), and an increase in copulatory efficiency (CE). In Expt. 2, sexually experienced male rats were allowed to mate to sexual exhaustion. Males with PGi lesions showed an increased latency to sexual exhaustion and an increased number of ejaculations prior to exhaustion. Additionally, rats with PGi lesions displayed reductions in IF, EL, and post-ejaculatory interval (PEI) as they approached sexual exhaustion. Our results provide further evidence that the PGi is a supraspinal locus of descending inhibitory influence on spinal nuclei mediating ejaculatory reflexes in the male rat.

Fluoxetine-induced inhibition of male rat copulatory behavior: Modification by lesions of the nucleus paragigantocellularis

In Experiment 1, the 5-HT uptake blocker fluoxetine (FLX; 20 mg/kg) reduced the proportion of sexually experienced male rats displaying ejaculations. Among those animals that did ejaculate there was an increase in intromission frequency (IF), ejaculation latency (EL), and postejaculatory interval (PEI) and a reduction in copulatory efficiency (CE) during the final copulatory sequence prior to sexual exhaustion. In Experiment 2, we found similar inhibitory effects of FLX as well as facilitating effects of lesions of the nucleus paragigantocellularis (PGi) on male rat copulatory behavior. Males with PGi lesions displayed more ejaculations and a longer latency to sexual exhaustion compared to intact animals. When FLX was given to rats with PGi lesions, it did not influence the proportion of rats ejaculating nor did it alter IF, EL, or PEI during the final copulatory series prior to exhaustion. These findings suggest that the inhibitory influences of FLX on male rat copulatory behavior are mediated in part by the interaction of FLX with neurons originating in the PGi.

The neurotoxicity induced by ethanol withdrawal in mature organotypic hippocampal slices might involve cross-talk between metabotropic glutamate type 5 receptors and N-methyl-D-aspartate receptors

BACKGROUND We recently reported that the sodium salt of acamprosate (Na-acamprosate) demonstrates the characteristics of an antagonist at metabotropic glutamate type 5 receptors (mGluR5s) rather than at N-methyl-d-aspartate receptors (NMDARs). Because mGluR5s are able to enhance the function of NMDARs, this interplay may be involved in the dysregulation of glutamatergic transmission during ethanol withdrawal. The following studies use organotypic hippocampal slice cultures at a mature age to investigate the potential for this interplay in the neurotoxicity associated with withdrawal from long-term ethanol exposure. METHODS At 25 days in vitro, organotypic hippocampal slice cultures prepared from male and female 8-day-old rats were exposed to an initial concentration of 100 mM ethanol for 10 days before undergoing a 24-hr period of withdrawal. The effects of Na-acamprosate; 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), a noncompetitive antagonist at mGluR5s; 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester, a noncompetitive antagonist at mGluR1s; dizocilpine (MK-801), a noncompetitive NMDAR antagonist; and staurosporine on the neurotoxicity induced by ethanol withdrawal were assessed by determining differences in propidium iodide uptake. Polypeptide levels of mGluR5s and the NR1 and NR2B subunits of NMDARs were also determined via Western blot analyses after 10 days of ethanol exposure. RESULTS Significant neurotoxicity was always evident in the CA1 hippocampal region after a 24-hr withdrawal period. This spontaneous neurotoxicity resulted from intrinsic changes induced by the long-term presence of ethanol. Na-acamprosate (200-1000 microM), SIB-1893 (200-500 microM), MK-801 (20 microM), and staurosporine (200 nM) were all neuroprotective. The polypeptide levels of mGluR5s and NR1 and NR2B subunits of NMDARs were all increased after ethanol exposure; however, the increase in mGluR5s did not achieve statistical significance. CONCLUSIONS From this model of long-term ethanol exposure and withdrawal, the functional interplay between mGluR5s and NMDARs might represent a novel target for the prevention of neurotoxicity associated with ethanol withdrawal.

Nitric oxide synthase inhibition impairs spatial navigation learning and induces conditioned taste aversion

The free radical gas nitric oxide (NO) is formed from the amino acid precursor L-arginine in brain regions which are associated with learning and the formation of memory. We have previously reported that administration of the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-Name) impairs delayed recall in non-human primates but that, at higher doses, impairment is associated with aversive gastrointestinal side effects. The purpose of the present study was to examine the effects of L-Name on learning in a rat spatial navigation task and to assess the ability of L-Name to induce a conditioned taste aversion (CTA) to a novel sucrose solution in a two-bottle choice paradigm. In the Morris water maze. L-Name (5, 20, and 50 mg/kg) markedly impaired cued spatial learning required to locate a hidden platform on three consecutive days of testing, but did not affect general activity levels. These data also demonstrated the ability of L-Name to induce a potent CTA, though only with the 20 and 50 mg/kg doses. Both the impairment of learning and CTA were blocked by administration of a mole equivalent dose of L-arginine, indicating that attenuated NO activity was associated with both behavioral effects. These data demonstrate that inhibition of NO activity by L-Name induces significant and selective impairment of cognitive performance at low pharmacologic doses (< 20 mg/kg). However, with higher doses of NOS inhibitors, impairment may be a secondary effect of drug-induced malaise, possibly related to peristaltic dysregulation of gastrointestinal musculature. Therefore, conclusions as to the mediation of learning and memory processes by CNS NO may be difficult to interpret without the use of selective, centrally-acting compounds.

Enhanced delayed matching performance in younger and older macaques administered the 5-HT4 receptor agonist, RS 17017.

Abstract Recent evidence indicates that the 5-HT4 subtype of serotonin receptor may modulate central cholinergic activity in regions of the mammalian CNS important to memory such as the frontal cortex, hippocampus and amygdala. These receptors could represent targets for drugs designed for the symptomatic therapy of Alzheimer’s disease (AD) and other disorders of memory. In the present study, the binding activity of RS 17017 (previously described as a selective 5-HT4 agonist) was assessed across a number of neurotransmitter receptors and binding sites, pharmacokinetic data were obtained, and the compound was evaluated in macaques for mnemonic effects via a computer-assisted delayed matching-to-sample task (DMTS). Binding data confirmed the 5-HT4 selectivity of the compound, while pharmacokinetic results revealed low oral bioavailability, but a large volume of distribution of the compound. Significant and reproducible improvements in DMTS accuracy were observed after oral administration of the compound across a dose-effect series in both younger and older monkeys. The results suggest that RS 17017 offers a potential for memory enhancement in disorders involving cognitive decline, and are consistent with a role for central 5-HT4 receptors in memory. Improvements in DMTS performance in aged monkeys may have particular implications for neurodegenerative conditions such as AD, whereas positive results in the younger monkeys indicate that RS 17017 (or similar compounds) may have additional potential in the therapeutics of memory disorders not necessarily associated with advanced age.

Chronic, intermittent exposure to chlorpyrifos in rats: Protracted effects on axonal transport, neurotrophin receptors, cholinergic markers, and information processing

Persistent behavioral abnormalities have been commonly associated with acute organophosphate (OP) pesticide poisoning; however, relatively little is known about the consequences of chronic OP exposures that are not associated with acute cholinergic symptoms. In this study, the behavioral and neurochemical effects of chronic, intermittent, and subthreshold exposures to the OP pesticide, chlorpyrifos (CPF), were investigated. Rats were injected with CPF s.c. (dose range, 2.5–18.0 mg/kg) every other day over the course of 30 days and then were given a 2-week CPF-free washout period. In behavioral experiments conducted during the washout period, dose-dependent decrements in a water-maze hidden platform task and a prepulse inhibition procedure were observed, without significant effects on open-field activity, Rotorod performance, grip strength, or a spontaneous novel object recognition task. After washout, levels of CPF and its metabolite 3,5,6-trichloro-2-pyridinol were minimal in plasma and brain; however, cholinesterase inhibition was still detectable. Furthermore, the 18.0 mg/kg dose of CPF was associated with (brain region-dependent) decreases in nerve growth factor receptors and cholinergic proteins including the vesicular acetylcholine transporter, the high-affinity choline transporter, and the α7-nicotinic acetylcholine receptor. These deficits were accompanied by decreases in anterograde and retrograde axonal transport measured in sciatic nerves ex vivo. Thus, low-level (intermittent) exposure to CPF has persistent effects on neurotrophin receptors and cholinergic proteins, possibly through inhibition of fast axonal transport. Such neurochemical changes may lead to deficits in information processing and cognitive function.

Neurotoxic effects of the human immunodeficiency virus type-1 transcription factor Tat require function of a polyamine sensitive-site on the N-methyl-d-aspartate receptor

Human immunodeficiency virus type-I (HIV-1) infection is often associated with neuronal loss in cortical and subcortical regions that may manifest as motor dysfunction and dementia. The function of the HIV-1 transcription protein Tat and subsequent activation of N-methyl-D-aspartate receptors (NMDAr) have been implicated in this form of neurodegeneration. However, it is unclear if Tat interacts directly with the NMDAr and the role of specific NMDAr subunit composition in mediating effects of Tat is also unclear. The present studies examined the ability of HIV-1 Tat1-72 protein (10 pM-1.0 microM) to displace [3H]MK-801 binding and to attenuate spermidine-induced potentiation of this binding in rat brain homogenate comprised of cerebellum, hippocampus, and cerebral cortex. The role of NMDAr polyamine-site function in the neurotoxic effects of Tat was determined using organotypic hippocampal slice cultures. Binding of [3H]MK-801 in adult rat brain homogenate was not reduced by Tat at concentrations below 1 microM. Tat potently inhibited the potentiation of [3H]MK-801 binding produced by co-exposure of membranes to the NMDAr co-agonist spermidine (IC(50)=3.74 nM). In hippocampal explants, Tat produced neurotoxicity in the CA3 and CA1 pyramidal cell layers, as well as in the dentate gyrus, that was significantly reduced by co-exposure to MK-801 (20 microM) and the NMDAr polyamine-site antagonist arcaine (10 microM). Exposure to the HIV-1 Tat deletion mutant (Tatdelta31-61) did not produce neurotoxicity in hippocampal explants. These data suggest that the neurotoxic effects of HIV-1 Tat are mediated, in part, by direct interactions with a polyamine-sensitive site on the NMDAr that positively modulates the function of this receptor.

Microtubule-associated targets in chlorpyrifos oxon hippocampal neurotoxicity.

Prolonged exposure to organophosphate (OP) pesticides may produce cognitive deficits reflective of hippocampal injury in both humans and rodents. Recent work has indicated that microtubule trafficking is also adversely affected by exposure to the OP pesticide chlorpyrifos, suggesting a novel mode of OP-induced neurotoxicity. The present studies examined effects of prolonged exposure to chlorpyrifos oxon (CPO) on acetylcholinesterase (AChE) activity, immunoreactivity (IR) of microtubule-associated proteins, neuronal injury, and tubulin polymerization using in vitro organotypic slice cultures of rat hippocampus and bovine tubulin. Cultures were exposed to CPO (0.1-10 microM) in cell culture medium for 1-7 days, a regimen producing progressive reductions in AChE activity of 15-60%. Cytotoxicity (somatic uptake of the non-vital marker propidium iodide), as well as IR of alpha-tubulin and microtubule-associated protein-2 (a/b) [MAP-2], was assessed 1, 3, and 7 days after the start of CPO exposure. As early as 24 h after the start of exposure, CPO-induced deficits in MAP-2 IR were evident and progressive in each region of slice cultures at concentrations as low as 0.1 microM. CPO exposure did not alter alpha-tubulin IR at any time point. Concentration-dependent injury in the cornu ammonis (CA)1 pyramidal cell layer and to a lesser extent, CA3 and dentate cells, was evident 3 days after the start of CPO exposure (>or=0.1 microM) and was greatest after 7 days. Tubulin polymerization assays indicated that CPO (>or=0.1 microM) markedly inhibited the polymerization of purified tubulin and MAP-rich tubulin, though effects on MAP-rich tubulin were more pronounced. These data suggest that exposure to CPO produces a progressive decrease in neuronal viability that may be associated with impaired microtubule synthesis and/or function.