Different associations of alcohol cue reactivity with negative alcohol expectancies in mandated and inpatient samples of young adults.

Alcohol cue reactivity, operationalized as a classically conditioned response to an alcohol related stimulus, can be assessed by changes in physiological functions such as heart rate variability (HRV), which reflect real time regulation of emotional and cognitive processes. Although ample evidence links drinking histories to cue reactivity, it is unclear whether in-the-moment cue reactivity becomes coupled to a set of consolidated beliefs about the effects of alcohol (i.e., expectancies) and whether treatment helps dissociate the relation of positive versus negative expectancies to cue reactivity. This study examined the relationship between reactivity to alcohol picture cues and alcohol expectancies in two groups of emerging adults: an inpatient sample with alcohol use disorders (n=28) and a college student sample who previously were mandated to a brief intervention for violating university policies about alcohol use in residence halls (n=43). Sequential regression analysis was conducted using several HRV indices and self-report arousal ratings as cue reactivity measures. Results indicated that the relationship between cue reactivity and negative alcohol outcome expectancies differed for the two groups. Greater cue reactivity, assessed using HRV indices, was associated with more negative expectancies in the inpatient sample but with less negative expectancies in the mandated student sample, while an opposite trend was found for subjective arousal. The present findings highlight the importance of characterizing cue reactivity through multi-dimensional assessment modalities that include physiological markers such as HRV.

Spontaneous changes in mitochondrial membrane potential in cultured neurons.

Using the mitochondrial membrane potential (DeltaPsi(m))-sensitive fluorescent dyes 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM), we have observed spontaneous changes in the DeltaPsi(m) of cultured forebrain neurons. These fluctuations in DeltaPsi(m) appear to represent partial, transient depolarizations of individual mitochondria. The frequency of these DeltaPsi(m) fluctuations can be significantly lowered by exposure to a photo-induced oxidant burden, an ATP synthase inhibitor, or a glutamate-induced sodium load, without changing overall JC-1 fluorescence intensity. These spontaneous fluctuations in JC-1 signal were not inhibited by altering plasma membrane activity with tetrodotoxin or MK-801 or by blocking the mitochondrial permeability transition pore (PTP) with cyclosporin A. Neurons loaded with TMRM showed similar, low-amplitude, spontaneous fluctuations in DeltaPsi(m). We hypothesize that these DeltaPsi(m) fluctuations are dependent on the proper functioning of the mitochondria and reflect mitochondria alternating between the active and inactive states of oxidative phosphorylation.

MitoTracker labeling in primary neuronal and astrocytic cultures: influence of mitochondrial membrane potential and oxidants.

MitoTracker dyes are fluorescent mitochondrial markers that covalently bind free sulfhydryls. The impact of alterations in mitochondrial membrane potential (Delta Psi(m)) and oxidant stress on MitoTracker staining in mitochondria in cultured neurons and astrocytes has been investigated. p-(Trifluoromethoxy) phenyl-hydrazone (FCCP) significantly decreased MitoTracker loading, except with MitoTracker Green in neurons and MitoTracker Red in astrocytes. Treatment with FCCP after loading increased fluorescence intensity and caused a relocalization of the dyes. The magnitude of these effects was contingent on which MitoTracker, cell type and dye concentration were used. H(2)O(2) pretreatment led to a consistent increase in neuronal MitoTracker Orange and Red and astrocytic MitoTracker Green and Orange fluorescence intensity. H(2)O(2) exposure following loading increased MitoTracker Red fluorescence in astrocytes. In rat brain mitochondria, high concentrations of MitoTracker dyes uncoupled respiration in state 4 and inhibited maximal respiration. Thus, loading and mitochondrial localization of the MitoTracker dyes can be influenced by loss of Delta Psi(m) and increased oxidant burden. These dyes can also directly inhibit respiration. Care must be taken in interpreting data collected using MitoTrackers dyes as these dyes have several potential limitations. Although MitoTrackers may have some value in identifying the location of mitochondria within cultured neurons and astrocytes, their sensitivity to Delta Psi(m) and oxidation negates their use as markers of mitochondrial dynamics in healthy cultures.

Glutamate uptake in mice bred for ethanol withdrawal severity.

RATIONALE: Withdrawal seizure-prone and withdrawal seizure-resistant mice were selectively bred to exhibit differences in handling-induced convulsion severity during ethanol withdrawal. The glutamatergic system has been implicated in seizure activity as well as ethanol withdrawal symptoms. OBJECTIVE: This study assessed L-[3H]glutamate uptake into hippocampal synaptosomes prepared from withdrawal seizure-prone and- resistant mice. METHODS: Glutamate uptake was characterized following repeated handling-induced convulsions, during acute intoxication, and during peak withdrawal following chronic ethanol exposure. RESULTS: Hippocampal synaptosomal L-[3H]glutamate uptake did not differ between convulsion- and ethanol-naive withdrawal seizure-prone and- resistant mice. Furthermore, exposure to convulsions or to a hypnotic dose of ethanol (4 g/kg) did not alter L-[3H]glutamate uptake. However, withdrawal from 72 h of ethanol exposure significantly increased L-[3H]glutamate uptake in both mouse lines as compared to their respective ethanol-naive controls. CONCLUSIONS: These data suggest that glutamate uptake is influenced by chronic ethanol exposure similarly in both withdrawal seizure-prone and- resistant mice. The observed increases in glutamate uptake during withdrawal may be associated with compensatory mechanisms triggered by chronic intoxication and are independent of the selected differences for withdrawal severity.

Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion.

The goal of this study was to investigate changes in glutamatergic synapses in the striatum of rats at two different time-points following a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. One month following this lesion of the nigrostriatal pathway, there was an increase (70%) in the mean percentage of asymmetrical synapses within the dorsolateral striatum containing a discontinuous, or perforated, postsynaptic density, possibly suggesting an increase in glutamatergic activity. This was correlated, in the same brain region, with a decrease (44%) in the density of glutamate immunoreactivity within nerve terminals associated with all asymmetrical synapses and also with those terminals associated with a perforated postsynaptic density. These morphological changes were consistent with an increase (>two-fold) in the basal extracellular level of striatal glutamate, as measured by in vivo microdialysis. The density of GABA immunolabeling within symmetrical nerve terminals was increased (25%) at this one month time-period. Dopamine levels within the lesioned striatum were >99% depleted. However, at three months, while an increase in the mean percentage of striatal perforated synapses was maintained, a significant increase (50%) in the density of striatal nerve terminal glutamate immunolabeling within all asymmetrical synapses and those associated with a perforated postsynaptic density was observed. This was correlated with a small, but significant, decrease (32%) in the basal extracellular level of striatal glutamate. The density of GABA immunolabeling within nerve terminals associated with a symmetrical contact remained elevated at this three month time-period, while striatal dopamine levels remained depleted. While the density of nerve terminal GABA immunolabeling remained elevated at both the one and three month time-periods, there appeared to be a differential effect on glutamatergic synapses. The in vivo microdialysis data suggest that glutamate synapses were more active at a basal level at one month and become less active compared to the control group at the three month time-period. These data suggest that there are compensatory changes in glutamatergic synapses within the striatum following a 6-hydroxydopamine lesion that appear to be independent of the level of striatal dopamine or GABA. We propose that changes in the activity of the thalamo-cortico-striatal pathway may help to explain the differential time-course change in striatal glutamatergic synaptic activity.

Glial differences between naïve withdrawal seizure-prone and -resistant mice.

BACKGROUND: Withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice were bred in replicate (i.e., WSP-1 and WSP-2) to exhibit differences in handling-induced convulsion severity during ethanol withdrawal. METHODS: We examined the role of the glutamatergic system in susceptibility to ethanol-withdrawal convulsions in naive mice by measuring the density of immunolabeling for several glutamate transporters and the glutamate-metabolizing enzyme, glutamine synthetase. The density of glial fibrillary acidic protein immunolabeling (a marker of glial structure) and cytochrome oxidase activity (a marker of neuronal activity) were also characterized in naive mice. RESULTS: We observed a significantly greater density of immunolabeling for the glial transporter, glutamate/aspartate transporter, in CA1 subfield of the hippocampus (CA1) of naive WSP-2 mice as compared to WSR-2 mice. No other significant differences were observed. However, as compared to WSR mice, naive WSP mice exhibited a trend toward (a) greater immunolabeling for the glial glutamate transporter, glutamate transporter-1, in CA3, (b) greater immunolabeling for glial-specific glutamate-metabolizing enzyme, glutamine synthetase, in CA1 (replicate-2 only), and (c) less immunolabeling for the glial structural protein, glial fibrillary acidic protein, in all brain regions tested. In contrast, no trends or significant differences in the labeling density for the neuronal transporter, excitatory amino acid carrier 1, or the neuronal activity marker, cytochrome oxidase, were observed between the selected lines. CONCLUSIONS: These data suggest that the glutamatergic system and glia may play a pivotal role in the increased susceptibility to handling-induced convulsions observed in WSP mice.

Immunocytochemical analysis of glutamate and GABA in selectively bred mice.

Withdrawal Seizure Prone (WSP) and Withdrawal Seizure Resistant (WSR) mice have been selectively bred for differential ethanol withdrawal handling-induced convulsions (HICs). In addition, it has been observed that WSP mice exhibit drug-naive HICs. This latter finding suggests that WSP and WSR mice differ in their susceptibility to HICs. Alterations in the glutamate and gamma-aminobutyric acid (GABA) systems have been implicated in convulsive activity and have been proposed to underlie the manifestation of ethanol withdrawal symptoms. It is therefore possible that WSP and WSR mice are genetically different with respect to their glutamatergic and/or GABAergic systems. To test this hypothesis, we have analyzed WSP and WSR mice that are both drug- and HIC-naive for differences in the density of nerve terminal glutamate and GABA immunoreactivity within the CA1 subfield of the hippocampus (CA1) and layer II of the somatosensory cortex (SSC). The major finding of this study is that drug- and HIC-naive WSP mice exhibit a significantly greater density of presynaptic glutamate immunoreactivity associated with asymmetric synapses within the CA1, but not the SSC, when compared to WSR mice. The density of GABA immunoreactivity within nerve terminals associated with symmetric synapses does not differ between the selected lines in either brain region. Since prior drug exposure and HICs cannot account for the observed differences in these naive mice, the results strongly suggest that the density of nerve terminal glutamate immunoreactivity within the CA1 is a reflection of inherent genetic differences between WSP and WSR mice. Furthermore, an elevated density of presynaptic glutamate immunoreactivity may be an underlying neurochemical correlate to increased susceptibility to drug-naive and ethanol withdrawal convulsions.

Activation of corticostriatal pathway leads to similar morphological changes observed following haloperidol treatment.

Treatment with haloperidol, a dopamine receptor D-2 antagonist, for one month resulted in an increase in the mean percentage of asymmetric synapses containing a discontinuous, or perforated, postsynaptic density (PSD) [Meshul et al. (1994) Brain Res., 648:181-195] and a change in the density of striatal glutamate immunoreactivity within those presynaptic terminals [Meshul and Tan (1994) Synapse, 18:205-217]. We speculated that this haloperidol-induced change in glutamate density might be due to an activation of the corticostriatal pathway. To determine if activation of this pathway leads to similar morphological changes previously described following haloperidol treatment, GABA (10(-5) M, 0.5 microliters) was injected into the thalamic motor (VL/VM) nuclei daily for 3 weeks. This treatment resulted in an increase in the mean percentage of striatal asymmetric synapses containing a perforated PSD and an increase in the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated or non-perforated PSD. Subchronic injections of GABA into the thalamic somatosensory nuclei (VPM/VPL) had no effect on the mean percentage of synapses with perforated PSDs but resulted in a small, but significant, increase in density of glutamate immunoreactivity. Using in vivo microdialysis, an acute injection of GABA (10(-5) M, 15 microliters) into VL/VM resulted in a prolonged rise in the extracellular level of striatal glutamate. The increase in asymmetric synapses with perforated PSDs and in glutamate immunoreactivity within nerve terminals of the striatum following either subchronic haloperidol treatment or GABA injections into VL/VM suggest that an increase in glutamate release may be a common factor in these two experiments. It is possible that the extrapyramidal side effects associated with haloperidol treatment may be due, in part, to an increase in release of glutamate within the corticostriatal pathway.