Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain.

Phosphatidate, a neuronal phospholipid, stimulated the uptake of calcium by nerve terminals isolated from the striatum of rat brain. This effect was not produced by other phospholipids or glycolipids. Phosphatidate, but not other phospholipids, evoked the release of [3H] dopamine from striatal synaptosomes. The magnitude of both effects was similar to that observed after chemical depolarization of the nerve terminals. These results show that phosphatidate is the only membrane lipid component that acts as a functionally competent ionophore and support the suggestion that phosphatidate may serve as a link between depolarization and neurotransmitter release in the brain.

Effects of selection for ethanol preference on gene expression in the nucleus accumbens of HS-CC mice.

Previous studies on changes in murine brain gene expression associated with the selection for ethanol preference have used F2 intercross or heterogeneous stock (HS) founders, derived from standard laboratory strains. However, these populations represent only a small proportion of the genetic variance available in Mus musculus. To investigate a wider range of genetic diversity, we selected mice for ethanol preference using an HS derived from the eight strains of the collaborative cross. These HS mice were selectively bred (four generations) for high and low ethanol preference. The nucleus accumbens shell of naive S4 mice was interrogated using RNA sequencing (RNA-Seq). Gene networks were constructed using the weighted gene coexpression network analysis assessing both coexpression and cosplicing. Selection targeted one of the network coexpression modules (greenyellow) that was significantly enriched in genes associated with receptor signaling activity including Chrna7, Grin2a, Htr2a and Oprd1. Connectivity in the module as measured by changes in the hub nodes was significantly reduced in the low preference line. Of particular interest was the observation that selection had marked effects on a large number of cell adhesion molecules, including cadherins and protocadherins. In addition, the coexpression data showed that selection had marked effects on long non-coding RNA hub nodes. Analysis of the cosplicing network data showed a significant effect of selection on a large cluster of Ras GTPase-binding genes including Cdkl5, Cyfip1, Ndrg1, Sod1 and Stxbp5. These data in part support the earlier observation that preference is linked to Ras/Mapk pathways.

Effects of non-electrolyte molecules with anesthetic activity on the physical properties of DMPC multilamellar liposomes.

The effects of 13 non-electrolytes with moderate anesthetic potency on the order of DMPC liposomes were examined. Changes in order were monitored by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPG). At 30 degrees C, all of the compounds tested decreased the DPH steady-state anisotropy (rs), with potencies highly correlated to their oil/water partition coefficients. However, only the most hydrophobic anesthetics decreased TMA-DPH RS. Some of the most hydrophilic compounds, including ethanol and urethane, actually increased TMA-DPH rs, suggestive of an increase in membrane order. The concept of selectivity was borrowed from partitioning theory and used to explain some effects on anesthetic potency of decreasing temperature to 18 degrees C. In the gel as opposed to the liquid crystalline phase, selectivity for decreasing membrane order (as monitored by DPH) markedly increased, suggesting that anesthetic partitioning and/or the site of anesthetic action was occurring in a more hydrophobic domain. The solute-independent difference (or capacity) between two membranes for perturbation was defined as membrane sensitivity. Sensitivity appeared to also decrease with decreasing temperature, despite the decrease in membrane partitioning. This effect is thought to result from the selective delivery of the anesthetic solute to the membrane interior and away from more hydrophilic domains where anesthetics may order membrane structure.

Ethanol-induced changes in neuronal membrane order. An NMR study.

The effects of ethanol-d6 on the lipid matrix of rat brain neuronal membranes were investigated by delayed Fourier transform 1H-NMR techniques. At 24 degrees C, neither 0.1 nor 0.2% (v/v) ethanol-d6 measurably affected the methylene resonance intensity. However, 0.4 and 1.0% ethanol-d6 increased resonance intensity, 35 and 51%, respectively. With increasing temperature, a decrease in resonance intensity for 0.1% ethanol-d6 was observed reaching a maximum of 20% at 42 degrees C. Furthermore, increasing temperature attenuated the increases in resonance intensity seen with 0.4 and 1.0% ethanol-d6. At 24 degrees C, no concentration of ethanol-d6 had a significant effect on the choline methyl resonance. However, with increasing temperature both 0.1 and 0.2% ethanol-d6 decreased this resonance's intensity. The intensity of the terminal methyl resonance was increased in a dose related fashion by ethanol-d6, reaching a maximum of +41% at 1.0% (24 degrees C). Increasing temperature attenuated this effect, but no concentration of ethanol-d6 significantly decreased resonance intensity. The increases and decreases in resonance intensity induced by ethanol-d6 are interpreted in terms of a decrease and an increase in membrane order, respectively. It is proposed that ethanol-d6 exerts two effects on neuronal membranes, an ordering effect on the membrane surface and a disordering effect in the membrane interior. A higher enthalpy of ethanol binding to the surface as compared to the interior of the membrane leads to an attenuation of the ethanol disordering effect with increasing temperature.

Harnessing the mouse to unravel the genetics of human disease.

Complex traits, i.e. those with multiple genetic and environmental determinants, represent the greatest challenge for genetic analysis, largely due to the difficulty of isolating the effects of any one gene amid the noise of other genetic and environmental influences. Methods exist for detecting and mapping the Quantitative Trait Loci (QTLs) that influence complex traits. However, once mapped, gene identification commonly involves reduction of focus to single candidate genes or isolated chromosomal regions. To reach the next level in unraveling the genetics of human disease will require moving beyond the focus on one gene at a time, to explorations of pleiotropism, epistasis and environment-dependency of genetic effects. Genetic interactions and unique environmental features must be as carefully scrutinized as are single gene effects. No one genetic approach is likely to possess all the necessary features for comprehensive analysis of a complex disease. Rather, the entire arsenal of behavioral genomic and other approaches will be needed, such as random mutagenesis, QTL analyses, transgenic and knockout models, viral mediated gene transfer, pharmacological analyses, gene expression assays, antisense approaches and importantly, revitalization of classical genetic methods. In our view, classical breeding designs are currently underutilized, and will shorten the distance to the target of understanding the complex genetic and environmental interactions associated with disease. We assert that unique combinations of classical approaches with current behavioral and molecular genomic approaches will more rapidly advance the field.

Growth hormone response to apomorphine and diagnosis: a comparison of three diagnostic systems.

Our study takes a further look at the apomorphine test in the psychoses and affective disorders, with special reference to the use of different diagnostic systems. Patients meeting Research Diagnostic Criteria (RDC) for schizophrenia, schizoaffective disorder, or manic disorder were included. In addition to the RDC, diagnosis was also made using the DSM-III and ICD-9. All patients underwent an evaluation of peak GH response to apomorphine administration. The results show that RDC and ICD-9 are similar, in that for both systems, a high GH response correlates with a schizoaffective disorder and distinguishes those patients significantly from manic patients. The DMS-III brings in some new dimensions, in that schizophreniform disorder (6-month cut-off) is distinguished from schizophrenia. In addition, patients with affective symptoms and mood-incongruent psychoses are more closely related to schizophreniform disorder than to classical manic disorder.

Down-regulation of central dopamine receptors in schizophrenia.

CSF homovanillic acid (HVA) levels reflecting central dopamine release and apomorphine-stimulated human growth hormone (HGH) secretion reflecting central dopamine receptor activity were concomitantly determined in 20 schizophrenic patients. There was a strong negative correlation between HVA and HGH levels: high dopamine release was associated with lower HGH responses to dopamine receptor activation by apomorphine. Studies are reviewed which suggest that the presently observed relationship reflects release-mediated down-regulation of central D2 receptors, the dopamine receptor subtype associated with the antipsychotic properties of neuroleptic medication.

Dopamine D2 receptor availability in opiate-dependent subjects before and after naloxone-precipitated withdrawal.

Dopamine may play a role in opiate withdrawal and dependence. We measured dopamine D2 receptor availability in 11 opiate-dependent subjects using PFT and [11C]raclopride at baseline and during naloxone-precipitated withdrawal. Because [11C]raclopride is sensitive to endogenous dopamine, this strategy enabled us to test whether we could document in humans the DA reductions reported in animal models of opiate withdrawal. Results were compared with values from 11 controls, two of which also received naloxone. The ratio of the distribution volume in striatum to that in cerebellum (Bmax/Kd + 1) was used as model parameter for D2 receptor availability. Baseline measures for Bmax/Kd were lower in opiate-dependent subjects (2.44 +/- 0.4) than in controls (2.97 +/- 0.45 P < or = .009). Naloxone precipitated an intense withdrawal in the abusers but did not change the Bmax/Kd ratio. This study documents decreases in D2 receptors in opiate-dependent subjects but does not document significant changes in striatal DA concentration during acute withdrawal.

Intersubject variability of brain glucose metabolic measurements in young normal males.

UNLABELLED: This study evaluates intersubject variability on regional glucose metabolic values in a group of 50 healthy right-handed males between 20 and 40 yr of age. METHODS: Brain glucose metabolism was measured using PET and 2-deoxy-2[18F]fluoro-D-glucose under resting conditions and was separately assessed for subjects in their twenties (n = 34) and those in their thirties (n = 16). RESULTS: Regional brain metabolic values showed significant intersubject variability with coefficients of variation (CV) that ranged between 11.1% to 15.2% (twenties) and 7.2% to 12.6% (thirties). Relative measures (regional/global) were less variable than absolute measures and the CV ranged between 4.1% to 8.3% (twenties) and 3.9% to 10% (thirties). Whereas global brain metabolic rate for subjects in their twenties was not significantly different from that of subjects in their thirties, the metabolic rate in left frontal regions was significantly lower in the older subjects. CONCLUSION: The correlations between age and absolute and relative metabolism in the left frontal region were r = 0.438, p < 0.002 and r = 0.447, p < 0.001, respectively. This study shows significant intersubject variability for regional brain metabolic values in normal controls and documents age-related decreases in frontal metabolism that occur even in relatively young adults.

Reproducibility of repeated measures of endogenous dopamine competition with [11C]raclopride in the human brain in response to methylphenidate.

UNLABELLED: The measure of changes in synaptic dopamine (DA) concentration in response to the psychostimulant drug methylphenidate (MP) has been used as an indicator of responsiveness of the DA system. The purpose of this study was to assess the reproducibility of these measures. METHODS: Seven healthy subjects were scanned with PET and [11C]raclopride twice in the same day: 7 min after placebo or methylphenidate (0.5 mg/kg) administration. In parallel we also measured the physiologic and behavioral responses to placebo and to methylphenidate. The same procedures were repeated 1-2 wk later to assess test-retest reproducibility. RESULTS: Measures of plasma to brain transfer constant (K1), striatal distribution volume (DVstr) and DA D2 receptor availability (Bmax/Kd), for the placebo condition were similar for the first (E1) and second (E2) evaluations (Bmax/Kd, E1: 2.77+/-0.44; E2: 2.97+/-0.44). MP administration did not change K1, but it significantly decreased DVstr (E1: -25.9%+/-8.7%, P < or = 0.0002; E2: -20.7%+/-11.7%, P < or = 0.007) and Bmax/Kd (E1: -18.4%+/-8.7%, P < or = 0.002; E2: -13.4%+/-9.2%, P < or = 0.008), and the magnitude of these changes, though lower for E2, did not differ significantly. MP increased pulse rate (E1: +64%+/-43%, P < or = 0.002; E2: +69%+/-33%, P < or = 0.001), systolic pressure (E1: +37%+/-19%, P < or = 0.0006; E2: +29%+/-15%, P < or = 0.0009), self reports for drug effects (0: nothing to 10: extreme) of "rush" (E1: +8+/-3, P < or = 0.0004; E2: +6+/-4, P < or = 0.01) and "high" (E1: +8+/-3, P < or = 0.0001, E2: +8+/-3, P < or = 0.0003), anxiety (E1: +5+/-4, P < or = 0.02; E2: +4+/-4, P = 0.1) and restlessness (E1: +4+/-4, P < or = 0.04; E2: +4+/-5, P = 0.1). The magnitude of the cardiovascular and behavioral effects did not differ between E1 and E2. CONCLUSION: MP-induced changes in striatal DV and in Bmax/Kd, as well as the behavioral and cardiovascular effects, were reproducible with repeated administration.

Reproducibility of regional brain metabolic responses to lorazepam.

UNLABELLED: Changes in regional brain glucose metabolism in response to benzodiazepine agonists have been used as indicators of benzodiazepine-GABA receptor function. The purpose of this study was to assess the reproducibility of these responses. METHODS: Sixteen healthy right-handed men underwent scanning with PET and [18F]fluorodeoxyglucose (FDG) twice: before placebo and before lorazepam (30 micrograms/kg). The same double FDG procedure was repeated 6-8 wk later on the men to assess test-retest reproducibility. RESULTS: The regional absolute brain metabolic values obtained during the second evaluation were significantly lower than those obtained from the first evaluation regardless of condition (p < or = 0.001). Lorazepam significantly and consistently decreased both whole-brain metabolism and the magnitude. The regional pattern of the changes were comparable for both studies (12.3% +/- 6.9% and 13.7% +/- 7.4%). Lorazepam effects were the largest in the thalamus (22.2% +/- 8.6% and 22.4% +/- 6.9%) and occipital cortex (19% +/- 8.9% and 21.8% +/- 8.9%). Relative metabolic measures were highly reproducible both for pharmacologic and replication condition. CONCLUSION: This study measured the test-retest reproducibility in regional brain metabolic responses, and although the global and regional metabolic values were significantly lower for the repeated evaluation, the response to lorazepam was highly reproducible.

Measuring reproducibility of regional brain metabolic responses to lorazepam using statistical parametric maps.

UNLABELLED: Statistical parametric mapping (SPM) is a method for localizing differences in brain activation patterns without the need for anatomic predefined constraints. The purpose of this study was to assess the reproducibility of the patterns of activation obtained with SPM for baseline measures and for metabolic changes in response to lorazepam on a test-retest design. The results were compared with those we previously published using region-of-interest (ROI) methods. METHODS: Sixteen healthy right-handed men were scanned twice with PET and [18F]fluorodeoxyglucose (FDG): before placebo and before lorazepam (30 microg/kg). The same double FDG procedure was repeated 6-8 wk later to assess test-retest reproducibility. Image datasets were analyzed by using SPM95 software. Difference images between baseline and lorazepam were compared for the first and second evaluations, both for relative decreases as well as increases in metabolism. Significance level was systematically varied to P < 0.001, P < 0.01 and P < 0.05. RESULTS: There were no differences in the baseline SPM maps obtained for the first and second evaluations. SPM showed similar, although not identical, differences in response to lorazepam between the two evaluations. Both evaluations showed significant decreases in occipital cortex (9.7% and 10%) and significant relative increases in left temporal pole (6.8% and 10.4%). However, the second evaluation showed a decrease in the left frontal cortex (areas 6 and 8), which was not present in the first evaluation. The results were very similar to those we had obtained with ROI methods, except for the activation in the left temporal pole, which we had not observed with ROI analyses. CONCLUSION: Although the overall pattern of lorazepam-induced activation depicted by SPM was reproducible in pattern and magnitude, there were some differences that included a left frontal area of deactivation during the second but not the first evaluation. Results with SPM are similar to those with the ROI method, and, because it systematically analyses the whole brain, SPM can uncover patterns not seen with the ROI method.

Further evidence against the coupling of dopamine receptors to phosphoinositide hydrolysis in rat striatum.

The effects of D1 and D2 dopamine receptor agonists on phosphoinositide hydrolysis were studied by measuring the accumulation of radioactive inositol phosphates in slices of rat corpus striatum prelabelled with [3H]inositol. All assays were performed in the presence of lithium. Neither the D1 receptor agonist SKF 38393 nor the D2 receptor agonist quinpirole, alone or in combination, had an effect on basal accumulation of inositol phosphates. The muscarinic receptor agonist carbachol produced a robust increase in the accumulation of inositol monophosphate and a smaller increase in the accumulation of inositol bisphosphate. These effects were not altered by the presence of quinpirole. Additionally, quinpirole also had no effect when assays were conducted in the presence of the muscarinic receptor antagonist scopolamine, the glutamic acid receptor antagonist kynurenic acid, and the antioxidant glutathione. These results are discussed in relation to recent contradictory reports and lend support to the position that D2 dopamine receptors are not coupled to phosphoinositide hydrolysis in rat striatum.

Specific opioid-amphetamine interactions in the caudate putamen.

Bilateral microinjection of morphine (0.003-3 micrograms/side) into the caudate putamen enhances the behavior induced by the IP injection of 1 mg/kg d-amphetamine phosphate in a dose-related manner. The duration of activity was prolonged and ambulation was changed to d-amphetamine stereotypy, a behavior normally associated with higher doses of d-amphetamine. The opioid activity was stereospecific in that levorphanol was active, whereas dextrorphan was not. The enhancement of d-amphetamine-induced behavior by the opioids was blocked by naloxone. D-ala2-met-Enkephalin also enhanced the amphetamine-induced behavior. This enhancement appears to be specific to the caudate putamen because the oral stereotypy observed appears to be a unique action of amphetamine in this region of the brain.

Effects of withdrawal from chronic amphetamine intoxication on exploratory and stereotyped behaviors in the rat.

Rats were administered 3, 6, and 12 mg/kg of d-amphetamine s.c. twice daily on a weekly increasing staircase schedule. On days 1, 7, 14, and 28 after the last injection of amphetamine the animals were challenged with 1 and 3 mg/kg of d-amphetamine and their behavior was observed. The 7-, 14-, and 28-day withdrawn animals required less amphetamine than controls to induce stereotyped behaviors. However, it was found that withdrawn animals and control animals were equally sensitive to the effects of apomorphine. Reserpine pretreatment eliminated the differences between control and withdrawn animals. alpha-Methyl tyrosine pretreatment blocked the effects of 1 but not 3 mg/kg of d-amphetamine in the withdrawn animals. Possible chemical mechanisms underlying the change in amphetamine sensitivity in the withdrawn animals are discussed.

Fluphenazine activity and antipsychotic response.

Plasma fluphenazine levels and plasma total neuroleptic activity (as quantitated by the neuroleptic receptor binding assay) were related to therapeutic response in 15 DSM-III schizophrenic patients who received a predetermined, fixed dose of fluphenazine for 14 days. Mean neuroleptic activity of the plasma was 84% greater than can be accounted for by the parent fluphenazine alone, and varied widely between patients. A sigmoidal relationship between total neuroleptic activity of plasma and response was found, with a continued plateau of response at higher total neuroleptic levels. Furthermore, the RBA data suggested (P less than 0.002) that two populations of drug-responsive schizophrenics exist which may be discriminated by the total D2 binding activity of plasma required for response.

Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.

Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.

Electroconvulsive shock activates endogenous opioid systems: behavioral and biochemical correlates.

From the evidence reviewed above, there is little doubt that ECS activates endogenous opioids and modifies their receptors. Thus, this form of SIA is accompanied by many other corollaries of opioid-like actions, including catalepsy, similar EEG patterns, common autonomic effects, and increases in opioid receptor binding sites. Investigations have further indicated that the amnestic effects of ECS can also be attenuated by naloxone, and that pituitary-derived opioids may play an important role as a predominant source of opioids that contribute to these opioid-like effects following ECS. It is hoped that these many attempts to correlate SIA with other behavioral and physiological endpoints following ECS will provide a more global perspective on the role of endogenous opioid systems in ECS. From these results, it is suggested that other forms of SIA may also share many of these properties in common with ECS-induced SIA. Nonetheless, ECS and other forms of SIA, such as cold water exposure and restraint, share with ECS a common history of clinical use in the treatment of human depression. It is possible that the common thread linking these experimental observations to endogenous opioid systems may provide new insights into the cause and treatment of mental disorders as well as the perception of pain.

Developmental changes in synaptic membrane fluidity: a comparison of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH).

Cortical synaptic plasma membranes were prepared from rats 3, 7, 10, 14, 21, 28 and 120 days of age. Developmental changes in the fluidity of these membranes were assessed using fluorescence polarization techniques. 1,6-Diphenyl-1,3,5-hexatriene (DPH), a probe of the membrane interior, showed a marked developmental increase in polarization, suggesting a developmental decrease in fluidity. The magnitude of the change from day 3 to the adult was the equivalent of lowering the temperature 7 degrees C. The developmental change in DPH polarization was maintained in liposomes (multilamellar vesicles) prepared from membrane total lipid extracts. In contrast to DPH, 1-[4-(trimethylamino)phenyl]6-phenyl-1,3,5-hexatriene (TMA-DPH), a probe of the membrane surface reported no significant developmental effect on polarization for intact membranes; however, TMA-DPH did report a significant increase in polarization for the total lipid extract liposomes. For the intact membranes, both cis- and trans-parinarate, fluorescent probes of the mid-region of the acyl chains, reported significant developmental increases in polarization. The role of gangliosides in the developmental regulation of fluidity was examined. Gangliosides did not appear to play a role in the developmental changes, but they do have a significant effect (increased polarization) on the membrane surface as reported by TMA-DPH. Fluorescence lifetime and heterogeneity analyses were performed for DPH. There was a small but significant increase in probe lifetime during development. Thus, polarization measurements alone underestimated the increases in membrane order. In an attempt to amplify the differences in membrane organization between the developing and adult membranes, we examined the effects of the membrane perturbant ethanol, on DPH polarization at the different ages. No developmental effect on the ethanol-induced fluidization of synaptic membranes was observed.