Katherine J. Smith

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.

Fast dopamine release events in the nucleus accumbens of early adolescent rats

Subsecond fluctuations in dopamine (dopamine transients) in the nucleus accumbens are often time-locked to rewards and cues and provide an important learning signal during reward processing. As the mesolimbic dopamine system undergoes dynamic changes during adolescence in the rat, it is possible that dopamine transients encode reward and stimulus presentations differently in adolescents. However, to date no measurements of dopamine transients in awake adolescents have been made. Thus, we used fast scan cyclic voltammetry to measure dopamine transients in the nucleus accumbens core of male rats (29-30 days of age) at baseline and with the presentation of various stimuli that have been shown to trigger dopamine release in adult rats. We found that dopamine transients were detectable in adolescent rats and occurred at a baseline rate similar to adult rats (71-72 days of age). However, unlike adults, adolescent rats did not reliably exhibit dopamine transients at the unexpected presentation of visual, audible and odorous stimuli. In contrast, brief interaction with another rat increased dopamine transients in both adolescent and adult rats. While this effect habituated in adults at a second interaction, it persisted in the adolescents. These data are the first demonstration of dopamine transients in adolescent rats and reveal an important divergence from adults in the occurrence of these transients that may result in differential learning about rewards.

Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl-D-asparate-type glutamate receptors

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl-D-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [(125)I]MK-801 binding) and spermidine (100 microM)-potentiated [(125)I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1-10 microM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 microM) or ifenprodil (100 microM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [(125)I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 microM) exposure produced marked neurodegeneration (approximately 350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or DL-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [(125)I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

Sex Differences in Caffeine Neurotoxicity Following Chronic Ethanol Exposure and Withdrawal

AIMS Caffeine is a central nervous system stimulant that produces its primary effects via antagonism of the A(1) and A(2A) adenosine receptor subtypes. Previous work demonstrated a sex difference in neurotoxicity produced by specific adenosine A(1) receptor antagonism during ethanol withdrawal (EWD) in vitro that was attributable to effects downstream of A(1) receptors at NMDA receptors. The current studies were designed to examine the effect of non-specific adenosine receptor antagonism with caffeine during ethanol withdrawal on hippocampal toxicity in cultures derived from male and female rats. METHODS At 5 days in vitro (DIV), half of the male and female organotypic hippocampal slice cultures were exposed to 50 mM ethanol (EtOH) in culture media for 10 days before exposure to caffeine (5, 20 and 100 microM) for the duration of a 24 h EWD period. In keeping with this timeline, the remaining ethanol-naïve cultures were given media changes at 10 and 15 DIV and exposed to caffeine (5, 20 and 100 microM) for 24 h at 15 DIV. Cytotoxicity was assessed by fluorescent microscopy and quantification of propidium iodide (PI) uptake in the pyramidal cell layers of the CA1 and CA3 regions and the granule cell layer of the dentate gyrus (DG). A two-way (sex x treatment) ANOVA was conducted within each hippocampal region. RESULTS Twenty-four-hour withdrawal from 10-day exposure to 50 mM ethanol did not produce increased PI uptake in any hippocampal region. Caffeine exposure (5, 20 and 100 microM) in ethanol-naïve cultures did not produce toxicity in the DG or CA1 region, but 20 microM caffeine produced modest toxicity in the CA3 region. Exposure to 20 microM caffeine during EWD produced cytotoxicity in all hippocampal regions, though toxicity was sex-dependent in the DG and CA1 region. In the DG, both 5 and 20 microM caffeine produced significantly greater PI uptake in ethanol-exposed female cultures compared to ethanol-naïve female cultures and all male cultures. Similarly, 20 microM caffeine caused markedly greater toxicity in female cultures as compared to male cultures in the CA1 region. CONCLUSIONS Twenty-four-hour exposure to caffeine during EWD produced significant toxicity in the pyramidal cell layer of the CA3 region in male and female cultures, though toxicity in the granule cell layer of the DG and pyramidal cell layer of the CA1 region was observed only in female cultures. Greater sensitivity of the female slice cultures to toxicity upon caffeine exposure after prolonged ethanol exposure is consistent with previous studies of effects of a specific A(1) receptor antagonism during EWD on toxicity and indicate that this effect is independent of the hormonal milieu. Together, these data suggest that the A(1) receptor subtype is predominant in mediating caffeines neurotoxic effects during EWD. These findings demonstrate the importance of considering gender/sex when examining neuroadaptive changes in response to ethanol exposure and withdrawal.

Sex Differences in the Neurotoxic Effects of Adenosine A1 Receptor Antagonism During Ethanol Withdrawal: Reversal With an A1 Receptor Agonist or an NMDA Receptor Antagonist

BACKGROUND Neuronal adaptations that occur during chronic ethanol (EtOH) exposure have been observed to sensitize the brain to excitotoxic insult during withdrawal. The adenosine receptor system warrants further examination in this regard, as recent evidence has implicated adenosine receptor involvement in the behavioral effects of both EtOH exposure and withdrawal. METHODS The current studies examined effects of adenosine A(1) receptor manipulation on neuronal injury in EtOH-naive and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naive and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A(1) receptor agonist 2-Chloro-N(6)-cyclopentyladenosine (CCPA; 10 nM), the A(1) receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX;10 nM), or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L,-2-amino-5-phosphovalerate (APV; 20 microM) at 15 days in vitro (DIV). Cytotoxicity was measured in the primary neuronal layers of the dentate gyrus, CA3 and CA1 hippocampal regions by quantification of propidium iodide (PI) fluorescence after 24 hours. Immunohistochemical analysis of A(1) receptor abundance was conducted in EtOH-naive and EtOH pretreated slice cultures at 15 DIV. RESULTS Twenty-four hour exposure to DPCPX in EtOH-naive slice cultures did not produced neurotoxicity in any region of slice cultures. Though withdrawal from 10 day EtOH exposure produced no toxicity in either male or female slice cultures, exposure to DPCPX during 24 hours of EtOH withdrawal produced a marked increase in PI uptake in all hippocampal culture subregions in female cultures (to approximately 160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A(1) antagonist during withdrawal was greater than that observed in male cultures. These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co-exposure to either the A(1) agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A(1) receptors were observed in male and female EtOH-naive or EtOH pretreated cultures. CONCLUSIONS The current findings suggest that the female hippocampus possesses an innate sensitivity to effects of EtOH exposure and withdrawal on neuronal excitability that is independent of hormonal influences. Further, this sex difference is not related to effects of EtOH exposure on A(1) receptor abundance, but likely reflects increased NMDA receptor-mediated signaling downstream of A(1) inhibition in females.

Inhibition of sigma-1 receptor reduces N-methyl-D-aspartate induced neuronal injury in methamphetamine-exposed and -naive hippocampi.

Acute and prolonged methamphetamine (METH) exposure has been reported to moderate the function of N-methyl-d-aspartate type glutamate receptors (NMDAr) in the hippocampus. These effects have been found to be associated with enhanced NMDAr-dependent release of Ca(2+) from IP(3)-sensitive intracellular stores. The present studies were designed to extend these findings and examine the role of the endoplasmic membrane (ER) bound orphan receptor, the sigma-1 receptor, in NMDA-induced neuronal injury and METH withdrawal-potentiated NMDA-induced neuronal injury. Organotypic hippocampal slice cultures were exposed to METH (0 or 100microM) for 6 days and withdrawn for 7 days, then exposed to NMDA (0 or 5microM) for 24h. Additional cultures were also exposed to this regimen and were co-incubated with BD1047 (100microM), a specific inhibitor of ER-bound sigma-1 receptors, for the 24h NMDA exposure. Cytotoxicity was assessed by analysis of propidium iodide uptake. These studies demonstrated that protracted METH exposure and withdrawal significantly potentiated the neuronal injury produced by NMDA exposure. Further, co-exposure to BD1047 with NMDA markedly attenuated neuronal injury in METH-naïve and METH-withdrawn organotypic cultures. As a whole, these data demonstrate that prolonged METH exposure, even at non-toxic concentrations, significantly alters glutamate receptor signaling. Inhibition of sigma-1 receptor-dependent Ca(2+) release from the ER entirely prevented NMDA-induced toxicity in METH-naïve cultures and markedly reduced METH-potentiated toxicity. These findings demonstrate the importance of Ca(2+)-induced intracellular Ca(2+) release in excitotoxic insult and suggest that blockade of glutamatergic overactivity may represent a therapeutic target in the treatment of METH withdrawal.

Intra-cornu ammonis 1 administration of the human immunodeficiency virus-1 protein trans-activator of transcription exacerbates the ethanol withdrawal syndrome in rodents and activates N-methyl-d-aspartate glutamate receptors to produce persisting spatial learning deficits

Human immunodeficiency virus-1 (HIV-1) infection may produce neurological deficits, such as cognitive decline, that may be worsened by concurrent ethanol (EtOH) abuse. Among the many biochemical cascades likely mediating HIV-1-associated neuronal injury is enhancement of N-methyl-d-aspartate (NMDA) receptor function and progression to excitotoxicity, an effect that may be directly or indirectly related to accumulation in brain of the HIV-1 trans-activator of transcription (Tat) factor. The present studies were designed to examine the hypothesis that binge-like EtOH pre-exposure would enhance effects of Tat on NMDA receptor function. These studies employed a modified in vivo binge EtOH exposure regimen designed to produce peak blood EtOH levels (BEL) of <200 mg/dl in adult male rats and were designed to examine effects of intra-hippocampal injection of Tat (0.5 microl/500 pM/2 min) on EtOH withdrawal-related behavior, spatial learning, and histological measures. Unilateral cannulae were implanted into the cornu ammonis 1 (CA1) pyramidal cell layer of animals prior to beginning a 4-day binge EtOH regimen. EtOH was administered via intragastric intubation ( approximately 3.0-5.0 g/kg) with dose determined by behavioral ratings of intoxication daily for 4 days (at 08:00, 16:00, and 24:00 h). EtOH withdrawal behaviors were monitored 12 h after the last administration of EtOH. Morris water maze learning was assessed during the following 4 days, at which times brains were harvested for autoradiographic measurement of NMDA receptor density and neuroinflammation. Maximal BELs of 187.69 mg/dl were observed 60 min after EtOH administration on day 2 of the regimen. In contrast, peak BELs of approximately 100 mg/dl were observed 60 min after EtOH administration on day 4 of the regimen, suggesting development of metabolic tolerance. Significant behavioral abnormalities were observed in EtOH withdrawn animals, including tremor and seizures. Intra-CA1 region injection of Tat significantly potentiated EtOH withdrawal behavioral abnormalities, an effect that was reduced by MK-801 pre-exposure. While EtOH withdrawn animals showed learning similar to control animals, EtOH withdrawn animals that received intra-CA1 Tat injection demonstrated persisting deficits in spatial learning on days 3 and 4 of training, effects that were markedly reduced by administration of the competitive NMDA receptor antagonist MK-801 30 min prior to Tat injection. No changes in [(3)H]MK-801 binding were observed. Binding density of [(3)H]PK11195, a ligand for peripheral benzodiazepine receptors expressed on activated microglia, was elevated proximal to cannula tracks in all animals, but was not altered by EtOH or Tat exposure. These findings suggest that EtOH abuse and/or dependence in HIV-positive individuals may promote HIV-1-associated cognitive deficits by altering NMDA receptor function in the absence of microglial activation or neuroinflammation.

Methamphetamine exposure antagonizes N-methyl-D-aspartate receptor-mediated neurotoxicity in organotypic hippocampal slice cultures.

Glutamatergic systems have been increasingly recognized as mediators of methamphetamines (METH) pharmacological effects though little is known about the means by which METH interacts with glutamate receptors. The present studies examined effects of METH (0.1-100 microM) on [3H]MK-801 binding to membranes prepared from adult rat cortex, hippocampus and cerebellum, as well as the neurotoxicity produced by 24-h exposure to N-methyl-D-aspartate (5-10 microM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [3H]MK-801 with METH (0.1-100 microM) did not reduce dextromethorphan (1 mM)-displaceable ligand binding. Exposure of slice cultures to NMDA for 24-h produced increases in uptake of the non-vital fluorescent marker propidium iodide (PI) of 150-500% above control levels, most notably, in the CA1 region pyramidal cell layer. Co-exposure to METH (>1.0 microM) with NMDA (5 microM) reduced PI uptake by approximately 50% in each subregion, though the CA1 pyramidal cell layer was markedly more sensitive to the protective effects of METH exposure. In contrast, METH exposure did not reduce PI uptake stimulated by 24-h exposure to 10 microM NMDA. Co-exposure to the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM) prevented toxicity produced by exposure to 5 or 10 microM NMDA. These findings indicate that the pharmacological effects of short-term METH exposure involve inhibition of NMDA receptor-mediated neuronal signaling, not reflective of direct channel inhibition at an MK-801-sensitive site.

Potentiation of N-Methyl-d-Aspartate receptor-mediated neuronal injury during methamphetamine withdrawal in vitro requires co-activation of IP3 receptors

Recent findings suggest that methamphetamine (METH) functions acutely to inhibit N-methyl-d-aspartate (NMDA) receptor function. Protracted withdrawal from METH exposure may increase the sensitivity of NMDA receptors to agonist exposure, promoting neuronal excitability. However, the relevance of METH effects on NMDA receptor activity with regard to neuronal viability has not been fully studied. The present studies examined the effects of protracted METH exposure (6 or 7 days; 1.0-100 microM) and withdrawal (1 or 7 days) on NMDA receptor-dependent neurotoxicity, determined with use of the non-vital fluorescent marker propidium iodide, in organotypic slice cultures of male and female rats. Prolonged exposure to METH (100 microM) produced only modest toxicity in the granule cell layer of the dentate gyrus. Withdrawal from METH exposure (1 or 7 days) did not produce overt neuronal injury in any region of slice cultures. Exposure to NMDA (5 microM) produced marked neurotoxicity in the CA1 pyramidal cell layer. Neither co-exposure to METH nor 1 day of METH withdrawal in combination with NMDA exposure altered NMDA-induced neurotoxicity. In contrast, protracted withdrawal from METH exposure (7 days) was associated with a marked (approximately 400%) increase in NMDA-induced neurotoxicity in CA1 region pyramidal cells. This potentiation of neurotoxicity was prevented by co-exposure to the selective NMDA receptor antagonist 5-2-amino-5-phosphonovaleric acid (20 microM) and was markedly attenuated by co-exposure of slices to xestospongin C (1 microM), an antagonist of IP(3) receptors. The results of the present studies suggest that long-term METH withdrawal functionally sensitizes the NMDA receptor to agonist exposure and requires the co-activation of NMDA and IP(3) receptors.

HIV Immune Complexes Prevent Excitotoxicity by Interaction with NMDA Receptors

PURPOSE Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenerative disease for which there is no available neuroprotective therapy. Viral proteins, such as Tat, have been implicated as agents of neurotoxicity via multiple mechanisms, including effects by directly binding to the NMDA receptor. We evaluated the ability of the immune response against Tat to modulate neurotoxicity at glutamate receptors. METHODS Neurotoxicity was measured in primary neuronal-glial cultures and in hippocampal slice cultures. We used immunoprecipitation experiments to demonstrate interaction between Tat, NMDA receptor, and anti-Tat antibody. Using known structures of Tat and NMDA receptors, we developed a model of their interactions. RESULTS Antibodies to Tat attenuated Tat-mediated neurotoxicity. Interestingly, Tat immune complexes also blocked neurotoxicity caused by NMDA receptor agonists but not kainate/AMPA receptor agonists. Neither Tat nor antibody alone blocked the excitotoxic effect, nor did an unrelated antigen-antibody complex. The protective effect of the Tat immune complexes was also lost when Tat was modified by nitrosylation or by using a deletion mutant of Tat. CONCLUSIONS The ability of viral immune complexes to interact with NMDA receptors and prevent excitotoxicity represents a novel host defense mechanism. Host immune responses may influence host susceptibility to various effects of viral proteins, modulating HIV complications, such as onset of HAND. These observations provide rationale for development of vaccine therapies targeting Tat for prevention of HAND.