Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity inhibits N-methyl-D-aspartate- (NMDA-) induced excitotoxic (20 μM NMDA) cell death and changes in synaptophysin immunoreactivity. Significant NMDA-induced cytotoxicity (as measured by propidium iodide [PI] uptake) was found in the CA1 region of the hippocampus at all timepoints examined (24, 72, 120 hours), an effect significantly attenuated by co-exposure to the selective NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (APV), but not MDL-28170, a potent cysteine protease inhibitor. Results indicated sparing of NMDA-induced loss of the synaptic vesicular protein synaptophysin in all regions of the hippocampus by MDL-28170, though only at early timepoints after injury. These results suggest calcium-dependent recruitment of cysteine proteases within 24 hours of excitotoxic insult, but activation of alternative cellular degrading mechanisms after 24 hours. Further, these data suggest that synaptophysin may be a substrate for calpains and related proteases.
NMDA receptors; excitotoxicity; synaptophysin; cysteine protease; calpain
Ethanol affects the function of neurotransmitter systems, resulting in neuroadaptations that alter neural excitability. Adenosine is one such receptor system that is changed by ethanol exposure. The current review is focused on the A1 and the A2A receptor subtypes in the context of ethanol-related neuroadaptations and ethanol withdrawal because these subtypes (i) are activated by basal levels of adenosine, (ii) have been most well-studied for their role in neuroprotection and ethanol-related phenomena, and (iii) are the primary site of action for caffeine in the brain, a substance commonly ingested with ethanol. It is clear that alterations in adenosinergic signaling mediate many of the effects of acute ethanol administration, particularly with regard to motor function and sedation. Further, prolonged ethanol exposure has been shown to produce adaptations in the cell surface expression or function of both A1 and the A2A receptor subtypes, effects that likely promote neuronal excitability during ethanol withdrawal. As a whole, these findings demonstrate a significant role for ethanol-induced adaptations in adenosine receptor signaling that likely influence neuronal function, viability, and relapse to ethanol intake following abstinence.
Ethanol Withdrawal; A1 Receptor; A2A Receptor; Caffeine
Depression in patients with temporal lobe epilepsy (TLE) is highly prevalent and carries significant morbidity and mortality. Its neural basis is poorly understood. We used quantitative, surface-based MRI analysis to correlate brain morphometry with severity of depressive symptoms in 38 TLE patients and 45 controls. Increasing severity of depressive symptoms was associated with orbitofrontal cortex (OFC) thinning in controls, but with OFC thickening in TLE patients. These results demonstrate distinct neuroanatomical substrates for depression with and without TLE, and suggest a unique role for OFC – a limbic region for emotional processing strongly interconnected with medial temporal structures – in TLE-related depressive symptoms.
Septal nuclei, components of basal forebrain, are strongly and reciprocally connected with hippocampus, and have been shown in animals to play a critical role in memory. In humans, the septal forebrain has received little attention. To examine the role of human septal forebrain in memory, we acquired high-resolution magnetic resonance imaging scans from 25 healthy subjects and calculated septal forebrain volume using recently developed probabilistic cytoarchitectonic maps. We indexed memory with the California Verbal Learning Test-II. Linear regression showed that bilateral septal forebrain volume was a significant positive predictor of recognition memory accuracy. More specifically, larger septal forebrain volume was associated with the ability to recall item source/context accuracy. Results indicate specific involvement of septal forebrain in human source memory, and highlight the need for additional research into the role of septal nuclei in memory and other impairments associated with human diseases.
Septal nuclei; Basal forebrain; Episodic memory CVLT; MRI; Morphometry; Parahippocampal gyrus
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 Ca2+ from IP3-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 100 µM) for 6 days and withdrawn for 7 days, then exposed to NMDA (0 or 5 µM) for 24 hrs. Additional cultures were also exposed to this regimen and were co-incubated with BD1047 (100 µM), a specific inhibitor of ER-bound sigma 1 receptors, for the 24 hr 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 Ca2+ 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 Ca2+-induced intracellular Ca2+ release in excitotoxic insult and suggest that blockade of glutamatergic overactivity may represent a therapeutic target in the treatment of METH withdrawal.
NMDA; METHAMPHETAMINE; HIPPOCAMPUS; SIGMA-1 RECEPTOR
The human immunodeficiency virus 1 (HIV-1) protein Trans-activator of Transcription (Tat) is a nuclear regulatory protein that may contribute to the development of HIV-1 associated dementia by disrupting the neuronal cytoskeleton. The present studies examined effects of recombinant Tat(1-86; 1–100 nM) on microtubule-associated protein (MAP)-dependent and MAP-independent microtubule formation ex vivo and oxidative neuronal injury in rat organotypic hippocampal explants. Acute exposure to Tat(1-86) (≥1 nM) markedly reduced MAP-dependent and –independent microtubule formation ex vivo, as did vincristine sulfate (0.1–10 μM). Cytotoxicity, as measured by propidium iodide uptake, was observed in granule cells of the DG with exposure to 100 nM Tat(1-86) for 24 or 72 h, while significant reductions in MAP-2 immunoreactivity were observed in granule cells and pyramidal cells of the CA1 and CA3 regions at each timepoint. These effects were prevented by co-exposure to the soluble vitamin E analog Trolox (500 μM). Thus, effects of Tat(1-86) on the neuronal viability may be associated with direct interactions with microtubules and generation of oxidative stress.
Acquired immune deficiency syndrome; Neurotoxicity; Trolox; Tubulin
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. [125I]MK-801 binding) and spermidine (100 μM)-potentiated [125I]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 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), 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 [125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (~350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or APV. The addition of spermidine significantly potentiated [125I]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.
glutamate; calcium; head injury; amino acid; spermidine
Aims: Caffeine is a central nervous system stimulant that produces its primary effects via antagonism of the A1 and A2A adenosine receptor subtypes. Previous work demonstrated a sex difference in neurotoxicity produced by specific adenosine A1 receptor antagonism during ethanol withdrawal (EWD) in vitro that was attributable to effects downstream of A1 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 μM) 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 μM) 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 × 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 μM) in ethanol-naïve cultures did not produce toxicity in the DG or CA1 region, but 20 μM caffeine produced modest toxicity in the CA3 region. Exposure to 20 μM 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 μM caffeine produced significantly greater PI uptake in ethanol-exposed female cultures compared to ethanol-naïve female cultures and all male cultures. Similarly, 20 μM 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 A1 receptor antagonism during EWD on toxicity and indicate that this effect is independent of the hormonal milieu. Together, these data suggest that the A1 receptor subtype is predominant in mediating caffeine's neurotoxic effects during EWD. These findings demonstrate the importance of considering gender/sex when examining neuroadaptive changes in response to ethanol exposure and withdrawal.
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 transcription factor Tat. 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 (B.E.L.) of <200 mg/dl in adult male rats and were designed to examine effects of intra-hippocampal injection of Tat (0.5 µl/500 pM/2 min) on EtOH withdrawal-related behavior, spatial learning, and histological measures. Unilateral cannulae were implanted into the cornu ammonus 1 (CA1) pyramidal cell layer of animals prior to beginning a 4-day binge EtOH regimen. EtOH was administered via intragastric intubation (~3.0–5.0g/kg) with dose determined by behavioral ratings of intoxication daily for four days (at 0800, 1600, and 2400 hrs). EtOH withdrawal behaviors were monitored 12 hr 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 B.E.L.s of 187.69 mg/dl were observed 60 min after EtOH administration on Day 2 of the regimen. In contrast, peak B.E.L.s 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 [3H]MK-801 binding were observed. Binding density of [3H]PK11195, a ligand for peripheral benzodiazepine receptors expressed on activated microglia, was elevated proximal to cannulae tracts in all animals, but was not altered by EtOH or Tat exposure. These finding 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.
Alcoholism; AIDS; Memory; Substance Abuse; HIV-Associated Dementia; Hippocampus
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.
The current studies examined effects of adenosine A1 receptor manipulation on neuronal injury in EtOH-naïve and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naïve and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine (CCPA; 10 nM), the A1 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 μM) 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 A1 receptor abundance was conducted in EtOH-naïve and EtOH pretreated slice cultures at 15 DIV.
Twenty-four hour exposure to DPCPX in EtOH-naïve 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 ~160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A1 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 A1 agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A1 receptors were observed in male and female EtOH-naïve or EtOH pretreated cultures.
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 A1 receptor abundance, but likely reflects increased NMDA receptor-mediated signaling down-stream of A1 inhibition in females.
Alcoholism; Glutamate; Brain Injury; Gender
Glutamatergic systems have been increasingly recognized as mediators of methamphetamine’s (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 μM) 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 μM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [3H]MK-801 with METH (0.1–100 μM) 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 μM) with NMDA (5 μM) 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 μM NMDA. Co-exposure to the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (20 μM) prevented toxicity produced by exposure to 5 or 10 μM 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.
Stimulants; Glutamate; Hippocampus; Excitotoxicity; Amphetamine
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 studied. The present studies examined the effects of protracted METH exposure (6 or 7 days; 1.0-100 μM) 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 μM) 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 μM) 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 (~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-phoshonovaleric acid (20 μM) and was markedly attenuated by co-exposure of slices to xestospongin C (1 μM), an antagonist of IP3 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 IP3 receptors.
stimulants; drug abuse; amphetamine; glutamate; brain injury
Single photon emission computed tomography (SPECT) following injection of radiotracer during a seizure is known as ictal SPECT. Comparison of an ictal SPECT study to a baseline or interictal study can aid identification of a seizure focus.
A young woman with encephalitis and refractory seizures underwent brain SPECT during a period of frequent seizure-like episodes, and during a seizure-free period. A focal area of increased radiotracer uptake present only when she was experiencing frequent seizure-like episodes was originally localized to the brainstem, but with later computerized co-registration of SPECT to MRI, was found to lie outside the brain, in the region of the sphenoid sinus.
Low-resolution SPECT images present difficulties in interpretation, which can be overcome through co-registration to higher-resolution structural images.