Shared functional defect in IP3R-mediated calcium signaling in diverse monogenic autism syndromes.

Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca(2+) signaling in ASD. Here, we report that patient-derived fibroblasts from three monogenic models of ASD-fragile X and tuberous sclerosis TSC1 and TSC2 syndromes-display depressed Ca(2+) release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca(2+) signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca(2+) events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca(2+) signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca(2+) screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD.

Blood, brain, and hair GHB concentrations following fatal ingestion.

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.

Striatal dopaminergic and serotonergic markers in human heroin users.

To establish whether chronic opiate exposure might impair brain dopaminergic or serotonergic function in humans, we assessed biochemical indices of monoaminergic neurotransmitter activity and integrity in post mortem striatum of nine chronic heroin users and 14 control subjects. Striatal levels of the vesicular monoamine transporter were normal, suggesting that the density of dopamine nerve terminals is not reduced in heroin users. In nucleus accumbens, levels of tyrosine hydroxylase protein (-25%) and those of the dopamine metabolite homovanillic acid (-33%) were reduced significantly together with a trend for decreased dopamine (-32%) concentration. These changes could reflect either a compensatory downregulation of dopamine biosynthesis in response to prolonged dopaminergic stimulation caused by heroin, or reduced axoplasmic transport of tyrosine hydroxylase. Striatal levels of serotonin were either normal or elevated whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were decreased by 27-38%. Our data suggest that chronic heroin exposure might produce a modest reduction in dopaminergic and serotonergic activity that could affect motivational state and impulse control, respectively.

Regional distribution of methamphetamine in autopsied brain of chronic human methamphetamine users.

We measured levels of methamphetamine and those of its metabolite amphetamine in 15 autopsied brain regions of 14 human methamphetamine users. Only slight regional differences were observed in drug concentrations among the brain areas. Although, some redistribution of the drugs probably occurred postmortem, these data suggest that methamphetamine might not be preferentially retained in dopamine-rich brain areas but is heterogenously distributed in brain of chronic human users of the drug. The possible pharmacological actions of methamphetamine in both dopamine-rich and poor brain areas of chronic drug users need to be considered.

Dopamine D1 receptor protein is elevated in nucleus accumbens of human, chronic methamphetamine users.

Animal data have long suggested that an adaptive upregulation of nucleus accumbens dopamine D1 receptor function might underlie part of the dependency on drugs of abuse. We measured by quantitative immunoblotting protein levels of dopamine D1 and, for comparison, D2 receptors in brain of chronic users of methamphetamine, cocaine, and heroin. As compared with the controls, brain dopamine D1 receptor concentrations were selectively increased (by 44%) in the nucleus accumbens of the methamphetamine users, whereas a trend was observed in this brain area for reduced protein levels of the dopamine D2 receptor in all three drug groups (-25 to -37%; P < 0.05 for heroin group only). Our data support the hypothesis that aspects of the drug-dependent state in human methamphetamine users might be related to increased dopamine D1 receptor function in limbic brain.

Regional distribution of cocaine in postmortem brain of chronic human cocaine users.

We measured concentrations of cocaine and its major metabolites (benzoylecgonine, ecgonine methylester, norcocaine, and cocaethylene) in 15 autopsied brain regions of 14 human chronic cocaine users. Only slight differences were observed in concentrations of cocaine and its metabolites amongst the examined brain areas. Although it is likely that some postmortem redistribution of the drug must have occurred, our data are consistent with the possibility that behaviorally relevant doses of cocaine are widely distributed throughout the brain of humans who use the drug on a chronic basis. Consideration should therefore be given to the possible pharmacological and toxicological actions of cocaine in both striatal and extra-striatal brain areas in human users of the drug.

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin.

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.

Fatal hypernatremic dehydration in exclusively breast-fed newborn infants due to maternal lactation failure.

Infants who die of hypernatremic dehydration usually demonstrate at autopsy an underlying condition or disease process that predisposes to increased water loss. In the absence of such findings, forensic concerns may focus sharply on parental or caretaker neglect as an underlying cause of death. In this case report, we describe unrecognized fatal hypernatremic dehydration in two exclusively breast-fed neonates due solely to failure of maternal lactation. We further describe epidemiologic and etiologic features of such deaths and discuss forensic difficulties encountered in their certification.

Striatal dopamine nerve terminal markers in human, chronic methamphetamine users.

Methamphetamine is a drug that is significantly abused worldwide, Although long-lasting depletion of dopamine and other dopamine nerve terminal markers has been reported in striatum of nonhuman primates receiving very high doses of the psychostimulant, no information is available for humans. We found reduced levels of three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase and the dopamine transporter) in post-mortem striatum (nucleus accumbens, caudate, putamen) of chronic methamphetamine users. However, levels of DOPA decarboxylase and the vesicular monoamine transporter, known to be reduced in Parkinson's disease, were normal. This suggests that chronic exposure to methamphetamine does not cause permanent degeneration of striatal dopamine nerve terminals at the doses used by the young subjects in our study. However, the dopamine reduction might explain some of the dysphoric effects of the drug, whereas the decreased dopamine transporter could provide the basis for dose escalation occurring in some methamphetamine users.

Morphology and lytic mechanisms of interleukin 3-dependent natural cytotoxic cells: tumor necrosis factor as a possible mediator.

Populations of interleukin 3 (IL 3)-dependent cells can be derived from mouse bone marrow that display natural cytotoxicity (NC) against Wehi-164 target cells but do not display natural killing against YAC-1 cells. These bone marrow-derived NC cells cultured up to 2 mo in IL 3 do not contain rearranged T cell receptor beta-chain genes. They appear to be mast-like cells by electron microscopy and contain heterogeneous type granules. The molecules that mediate NC appear to be contained in these granules and are preformed because protein synthesis inhibitors have no effect on the capacity of IL 3-dependent NC cells to lyse Wehi-164 target cells. In addition to the IL 3-dependent bone marrow-derived cells, the basophilic leukemia cells, RBL-1, but not P815 mastocytoma cells were found to mediate NC against Wehi-164 cells. Both bone marrow-derived NC and RBL-1 cells can lyse L929 cells in 18 hr, suggesting that the putative NC mediator may be related to lymphotoxin/tumor necrosis factor (TNF). Recombinant human TNF displayed identical properties as NC cells; both entities possessed the same target cell specificity and had similar kinetics of target cell killing. The use of polyclonal rabbit antimouse TNF antibody blocked the actions of NC cells. Thus we believe that the mediation of NC is through the actions of a TNF-like molecule.

Prostaglandin E1 responsive ductus at 11 months of age.

An 11-month-old boy with congestive heart failure and an intermittently closing patent ductus arteriosus (PDA) is presented. During cardiac catheterization, the ductus proved responsive to prostaglandin E1. Permanent closure of the PDA could not be attained with indomethacin. The infant underwent surgical ligation of the PDA.

Anti-aggregatory actions of calcium channel blockers in cat platelets.

Four calcium channel blockers, verapamil, nifedipine, nisoldipine and nimodipine were studied in terms of their ability to inhibit platelet aggregation in cat platelet rich plasma by optical aggregometry. Only verapamil and nisoldipine significantly inhibited aggregation in cat platelets to ADP (2 microM). None of the calcium channel blockers exerted marked inhibition of aggregation induced by 1mM arachidonic acid. However, verapamil produced a moderate but significant inhibition of aggregation to arachidonic acid. Increasing the calcium ion concentration could reverse this inhibition. Thus, there are significant differences in the anti-aggregatory activity of calcium channel blockers in cat platelet rich plasma.

Infarct size restriction in cats by the beta-adrenergic blocker timolol.

The beta-adrenergic antagonist, timolol, was previously shown to exert a protective effect in cats subjected to 5 h of myocardial ischemia. The present study was designed to determine the effect of timolol on infarct size in cats 24 h after coronary occlusion. Timolol (25 microgram/kg) or vehicle (0.9% NaCl) was administered 0.5, 5, 10, 15 and 20 h after acute ligation of the left anterior descending coronary artery. There was no significant difference in arterial blood pressure or heart rate in MI cats receiving timolol or vehicle. Timolol markedly decreased S-T segment elevation at 2-12 h (P less than 0.05). Left ventricular weights in MI + vehicle cats (8.5 +/- 0.7 g, n = 6) were similar to timolol-treated MI cats (8.9 +/- 0.4 g, n = 7). However, the percent of the left ventricular myocardium infarcted, determined by nitroblue tetrazolium staining, was significantly less (P less than 0.001) in timolol MI cats compared to saline-treated cats, 9.8 +/- 1.2% (n = 7) vs. 18.9 +/- 1.8% (n = 6), respectively. Hemodynamic or cytoprotective actions of timolol do not appear to explain these results. Rather, the mechanism of infarct size reduction by timolol is probably explained by antagonism of beta-receptor-mediated metabolic effects.

Antagonism of thromboxane analog-induced vasoconstriction by non-steroidal anti-inflammatory agents.

The ability of six non-steroidal anti-inflammatory agents--meclofenamate, indomethacin, flurbiprofen, naproxen, ibuprofen, and acetylsalicylic acid--to inhibit coronary constriction induced by the thromboxane agonist carbocyclic thromboxane A2 (CTA2) was studied in isolated perfused cat coronary arteries. At constant flow, 7.5 nM CTA2 increased coronary artery perfusion pressure by 33 +/- 3 mm Hg (n = 20). Sodium meclofenamate reduced 7.5 nM CTA2-induced vasoconstriction by 2% at 0.34 microM (ns), 38% at 3.4 microM (ns), and 99% at 34 microM (p less than 0.025). The IC50 for meclofenamate was 5.4 microM; the IC50 for indomethacin, flurbiprofen, naproxen, ibuprofen, and acetylsalicylic acid was 42, 150, 250, 485, and 610 microM, respectively. Increasing the external calcium concentration to 10 mM completely reversed the inhibition of CTA2-induced coronary vasoconstriction. Furthermore, the data suggest that inhibition of CTA2-induced coronary vasoconstriction by anti-inflammatory agents may be explained either by thromboxane receptor antagonism or calcium channel blockade.