Cyclooxygenase-2 contributes to VX-induced cell death in cultured cortical neurons.

The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat cortical neurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE(2) and PGF(2α)) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat cortical neuron.

Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

A sensitizing regimen of amphetamine impairs visual attention in the 5-choice serial reaction time test: reversal by a D1 receptor agonist injected into the medial prefrontal cortex.

Exposure to repeated, intermittent, escalating doses of amphetamine in rats disrupts information processing in several tasks. Some of these deficits, notably impaired attentional set shifting, may reflect altered prefrontal cortex function. This study examined the effects of repeated treatment with amphetamine on performance in the 5-choice serial reaction time test. This test measures sustained visual attention, a behavior that is known to require the prefrontal cortex. Rats were trained to respond to a brief light stimulus presented randomly in one of five spatial locations, with 100 trials per session. Once performance had stabilized rats were treated with escalating doses of amphetamine (three injections per week for 5 weeks at 1-5 mg/kg per week); testing was continued on nondrug days, and for several weeks of withdrawal. During the amphetamine-treatment and withdrawal phases accuracy of responding was unaffected, but errors of omission increased. Lengthening the stimulus duration abolished this effect. Reducing the stimulus duration also reduced response accuracy and this effect was more marked in amphetamine-treated rats. Both reduced accuracy, and increased omissions, seen in amphetamine-treated rats were reversed by injecting the D1 receptor agonist SKF38393 into the medial prefrontal cortex. This treatment also prevented the decline in accuracy in control animals that resulted from reducing the stimulus duration. These results, indicating that exposure to amphetamine induces a long-lasting deficit in visual attention, add to a growing list of deficits suggesting that amphetamine-sensitized state may model the cognitive deficit state in schizophrenia. The reversal of these deficits by a D1 receptor agonist provides further evidence that prefrontal D1 dopamine receptors are involved in cognition, and may be a potential target for treatment of impaired cognition in schizophrenia.

Physiological neuroprotection by melatonin in a 6-hydroxydopamine model of Parkinson's disease.

There is considerable evidence that pharmacological doses of the pineal hormone, melatonin, are neuroprotective in diverse models of neurodegeneration including Parkinson's disease. However, there is limited information about the effects of physiological doses of this hormone in similar models. In this study, rats were chronically treated with melatonin via drinking water following partial 6-hydroxydopamine lesioning in the striatum. The two doses of melatonin (0.4 microg/ml and 4.0 microg/ml) were within the reported physiological concentrations present in the serum and cerebrospinal fluid respectively. At 2 weeks after surgery, the higher dose of melatonin significantly attenuated rotational behavior in hemi-parkinsonian rats compared to similarly lesioned animals receiving either vehicle (P < 0.001) or the lower dose of melatonin (P < 0.01). Animals were perfused or sacrificed 10 weeks after commencing melatonin treatment for immunohistochemical or mRNA studies. Animals treated with 4.0 microg/ml melatonin exhibited normal tyrosine hydroxylase (TH) immunoreactivity in the lesioned striatum, whereas little or no TH immunofluorescence was visible in similarly lesioned animals receiving vehicle. In contrast, semiquantitative RT-PCR analysis revealed no group differences in TH mRNA, suggesting spontaneous recovery of this transcript as observed previously in partially lesioned animals. There were no significant differences in striatal GDNF mRNA levels between sham and lesioned animals. However, there was a significant (P < 0.01) increase in GDNF mRNA expression in the intact contralateral striata of lesioned animals treated with vehicle. Interestingly, melatonin treatment attenuated this novel compensatory contralateral increase in striatal GDNF expression, presumably due to its neuroprotective effect. These findings support a physiological role for melatonin in protecting against parkinsonian neurodegeneration in the nigrostriatal system.

Sensitization to amphetamine, but not PCP, impairs attentional set shifting: reversal by a D1 receptor agonist injected into the medial prefrontal cortex.

RATIONALE: Repeated exposure to psychomotor stimulants can lead to sensitization to their effects, and sensitization has been implicated in the pathophysiology of schizophrenia and drug abuse. These disorders are characterized by cognitive deficits, particularly in prefrontally mediated executive function. OBJECTIVE: The present experiments were conducted to investigate the effects of sensitizing regimens of amphetamine and phencyclidine (PCP) on attentional set shifting. METHODS: Rats received injections of amphetamine, PCP or saline three times per week for 5 weeks. Four weeks later, rats were trained to dig for food in one of two bowls, each bowl having an odour and a texture. Only one dimension (odour or texture) correctly predicted which bowl was baited. Rats were then tested on a series of discriminations including those requiring an intra-dimensional shift (IDS), an extra-dimensional shift (EDS) or a reversal of previously relevant and irrelevant stimuli. RESULTS: Rats sensitized to amphetamine performed normally on the IDS, but were impaired on the EDS, as well as on reversal discriminations. PCP-sensitized rats were unaffected on any of the discriminations. In amphetamine-sensitized rats the deficit at the EDS stage was reversed by infusion of the D(1) receptor agonist SKF38393 into the medial prefrontal cortex (mPFC). CONCLUSIONS: Results show that the amphetamine-sensitized state impairs prefrontally mediated attentional set shifting. This is consistent with cognitive deficits in schizophrenia and addiction, and with the evidence that amphetamine sensitization is accompanied by functional changes in the mPFC. These results further add to a growing literature showing that activating D(1) receptors in the mPFC improves aspects of cognition.

Sensitization to amphetamine, but not phencyclidine, disrupts prepulse inhibition and latent inhibition.

RATIONALE: Schizophrenia has been linked to dysregulation of dopamine and glutamate transmitter systems. Attempts to model aspects of schizophrenia in animals have made use of treatments that primarily affect dopaminergic (e.g., amphetamine, Amp) and glutamatergic (e.g., phencyclidine, PCP) function. In addition to exerting short-term acute effects, these agents also induce long-term effects, as seen, for example, in neurochemical and behavioural sensitization. OBJECTIVES: The goal of this work was to compare Amp- and PCP-sensitized states on two measures of information processing that are impaired in schizophrenia, prepulse inhibition (PPI) of the acoustic startle reflex and latent inhibition (LI). METHODS: Rats received injections of Amp, PCP or saline 3 days per week for 3 weeks. The Amp dose increased from 1 to 3 mg/kg, at the rate of 1 mg/kg each week. The PCP dose was 3 mg/kg throughout. After various periods of withdrawal rats were tested for PPI and LI. RESULTS: Repeated intermittent treatment with Amp or PCP resulted in augmented locomotor responses to challenge with each drug, providing an operational index that sensitization had occurred. Rats sensitized to Amp showed disrupted PPI when tested drug free at 3, 21 and 60 days of withdrawal. Amp-sensitized rats also showed abolition of the LI effect. Rats sensitized to PCP did not show deficits in any of these behaviours when tested drug free. CONCLUSIONS: Because disrupted PPI and LI have both been reported in schizophrenic patients, these results suggest that the Amp-sensitized state may represent a useful model for investigating the neural bases of information processing deficits in schizophrenia.

A putative animal model of the "prodromal" state of schizophrenia.

BACKGROUND: There is growing interest in detecting and treating schizophrenia during the "prodrome," before the symptoms are fully manifested. The objective of this study was to develop a putative model of the prodrome and study the effects of medications on it. METHODS: Rats were treated with different regimens of amphetamine to produce full sensitization (full syndrome) and partial sensitization (to model the prodromal state) and were then treated with typical and atypical antipsychotics and a D1 antagonist to mimic early intervention. After several weeks of withdrawal, locomotor activity in response to amphetamine and behavioral deficits (prepulse inhibition [PPI] and latent inhibition [LI]) were examined. RESULTS: Animals that received the full sensitization showed significant increase in locomotor activity and a disruption in both PPI and LI. Animals treated with a partial regimen showed only a muted phenotype. The animals that received "early intervention" did not show progression from the prodromal to the full-blown phenotype. CONCLUSIONS: The partial regimen of amphetamine injections provided a modified phenotype that could be regarded as a representative of the "prodromal" state. Early intervention, instituted once the prodromal state was already developed, prevented further progression into the full phenotype analogous to schizophrenia.

Amphetamine-sensitized animals show a sensorimotor gating and neurochemical abnormality similar to that of schizophrenia.

The aim of these studies was to examine whether amphetamine-induced sensitization in rats could be used as an animal model to study the basis of certain abnormalities seen in schizophrenia. Specifically, these experiments examined whether rats subjected to a sensitizing regimen of amphetamine would show the sensorimotor gating and greater amphetamine-induced displacement of radio-raclopride binding deficit that is observed in schizophrenia. In the first experiment, animals were divided into two groups with each rat receiving an intraperitoneal injection of amphetamine (AMPH) or saline (SAL) (1 ml/kg) three times per week for 3 weeks for a total of nine injections. AMPH dose was increased weekly from 1 mg/kg in the first week to 3 mg/kg in the third. Twenty-two days after the last injection, prepulse inhibition (PPI) of the acoustic startle response was tested. In addition, rats were tested for the effects of a challenge dose of 0.5 mg/kg AMPH on locomotor activity and [3H]raclopride (RAC) binding potential (BP) in the striatum. The tests for PPI confirmed that sensorimotor gating was disrupted in the AMPH-induced sensitized-state rats at baseline. The AMPH-sensitized rats also exhibited higher locomotor response to AMPH and a lower binding of striatal [3H]raclopride when challenged with the drug. The results were replicated and even more pronounced in rats that were treated with AMPH for 5 weeks, with doses ranging from 1mg/kg in the first week to 5 mg/kg in the fifth. These sensorimotor gating deficits and neurochemical (greater AMPH-induced displacement of radio-raclopride binding) abnormalities show similarities with the pathophysiology of schizophrenia and suggest that the AMPH-sensitized-state rats could be used to model certain aspects of schizophrenia.