Rescue of PB-resistant neonatal seizures with single-dose of small-molecule TrkB antagonist show long-term benefits.

A recently characterized CD-1 mouse model of phenobarbital (PB)-resistant neonatal ischemic-seizures (i.e.; unilateral carotid ligation) was shown to be associated with age-dependent (P7 vs. P10) acute seizure severity and PB-efficacy (i.e.; PB-resistant vs. PB-responsive). ANA12, a novel small-molecule TrkB antagonist, rescued the PB-resistance at P7 in a dose-dependent manner and prevented the post-ischemic downregulation of KCC2, the chief Cl- extruder in neurons. The long-term consequences of this novel rescue-intervention with ANA12 + PB in P7 and P10 ligated pups was investigated and compared to the standard first-line protocol of PB-alone loading dose. The mice underwent neurobehavioral testing, 24 h video-EEG-EMG monitoring, and immunohistochemistry in ipsi- and contralateral cortices as adults following the neonatal interventions. ANA12 + PB rescued the emergence of hyperactivity in post-ischemic P7, but not in P10 pups as adults. ANA12 + PB administration at neither P7 nor P10 significantly altered 24 h macro-sleep architecture in adults when compared to PB-alone. Behavioral state-dependent gamma (35-50 Hz) power homeostasis showed the most significant between-group differences that were age-dependent. ANA12 + PB treatment, but not PB-alone, rescued the loss of gamma power homeostasis present in P7 ligate-control but absent in P10 ligate group, highlighting the age-dependence. In contrast, PB-alone treatment, but not ANA12+PB, significantly reduced the elevated delta-AUC observed in P10 ligate-controls, when PB is efficacious by itself. These results indicate that the rescue of acute PB-resistant neonatal seizures using a novel intervention positively modulates the long-term outcomes at P7 when the seizures are refractory.

Sleep dysfunction following neonatal ischemic seizures are differential by neonatal age of insult as determined by qEEG in a mouse model.

Neonatal seizures associated with hypoxic-ischemic encephalopathy (HIE) pose a challenge in their acute clinical management and are often followed by long-term neurological consequences. We used a newly characterized CD-1 mouse model of neonatal ischemic seizures associated with age-dependent (P7 vs. P10) seizure severity and phenobarbital efficacy (i.e.; PB-resistant vs. PB-efficacious respectively) following unilateral carotid ligation. The long-term consequences following untreated neonatal seizures in P7 vs. P10 ligated pups were investigated using neurobehavioral testing, 24 h v- quantitative EEG -EMG (qEEG, qEMG), and western blot analyses in adult mice. Significant hyperactivity emerged in a small sub-set of mice in both age-groups associated with a failure to habituate during open-field (OF) testing. 24 h continuous qEEGs detected significantly altered sleep architecture due to long-wake cycles in both age-groups. Delta power (0.5-4 Hz) quantification during slow-wave-sleep (SWS) revealed significant SWS compensation in P10 ligates following periods of increased sleep pressure which the P7 ligate group failed to show. Theta/beta ratios deemed as negative correlation markers of attentional control were significantly higher only in the P10 ligates. These results indicate that neonatal age-dependent differences in the characteristics of ischemic neonatal seizures in CD-1 pups differentially modulate long-term outcomes, when evaluated with v-qEEG/EMG as adults.

Circadian cycle-dependent EEG biomarkers of pathogenicity in adult mice following prenatal exposure to in utero inflammation.

Intrauterine infection or inflammation in preterm neonates is a known risk for adverse neurological outcomes, including cognitive, motor and behavioral disabilities. Our previous data suggest that there is acute fetal brain inflammation in a mouse model of intrauterine exposure to lipopolysaccharides (LPS). We hypothesized that the in utero inflammation induced by LPS produces long-term electroencephalogram (EEG) biomarkers of neurodegeneration in the exposed mice that could be determined by using continuous quantitative video/EEG/electromyogram (EMG) analyses. A single LPS injection at E17 was performed in pregnant CD1 dams. Control dams were injected with same volumes of saline (LPS n=10, Control n=8). At postnatal age of P90-100, 24-h synchronous video/EEG/EMG recordings were done using a tethered recording system and implanted subdural electrodes. Behavioral state scoring was performed blind to treatment group, on each 10s EEG epoch using synchronous video, EMG and EEG trace signatures to generate individual hypnograms. Automated EEG power spectrums were analyzed for delta and theta-beta power ratios during wake vs. sleep cycles. Both control and LPS hypnograms showed an ultradian wake/sleep cycling. Since rodents are nocturnal animals, control mice showed the expected diurnal variation with significantly longer time spent in wake states during the dark cycle phase. In contrast, the LPS-treated mice lost this circadian rhythm. Sleep microstructure also showed significant alteration in the LPS mice specifically during the dark cycle, caused by significantly longer average non-rapid eye movement (NREM) cycle durations. No significance was found between treatment groups for the delta power data; however, significant activity-dependent changes in theta-beta power ratios seen in controls were absent in the LPS-exposed mice. In conclusion, exposure to in utero inflammation in CD1 mice resulted in significantly altered sleep architecture as adults that were circadian cycle and activity state dependent.

Diagnostic Accuracy of the Primary Care Screener for Affective Disorder (PC-SAD) in Primary Care.

BACKGROUND: Depression goes often unrecognised and untreated in non-psychiatric medical settings. Screening has recently gained acceptance as a first step towards improving depression recognition and management. The Primary Care Screener for Affective Disorders (PC-SAD) is a self-administered questionnaire to screen for Major Depressive Disorder (MDD) and Dysthymic Disorder (Dys) which has a sophisticated scoring algorithm that confers several advantages. This study tested its performance against a 'gold standard' diagnostic interview in primary care. METHODS: A total of 416 adults attending 13 urban general internal medicine primary care practices completed the PC-SAD. Of 409 who returned a valid PC-SAD, all those scoring positive (N=151) and a random sample (N=106) of those scoring negative were selected for a 3-month telephone follow-up assessment including the administration of the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I) by a psychiatrist who was masked to PC-SAD results. RESULTS: Most selected patients (N=212) took part in the follow-up assessment. After adjustment for partial verification bias the sensitivity, specificity, positive and negative predictive value for MDD were 90%, 83%, 51%, and 98%. For Dys, the corresponding figures were 78%, 79%, 8%, and 88%. CONCLUSIONS: While some study limitations suggest caution in interpreting our results, this study corroborated the diagnostic validity of the PC-SAD, although the low PPV may limit its usefulness with regard to Dys. Given its good psychometric properties and the short average administration time, the PC-SAD might be the screening instrument of choice in settings where the technology for computer automated scoring is available.

Development and preliminary validation of the PC-SAD5, a screener-derived short depression severity measure.

BACKGROUND: The prevalence of depressive disorders is high among patients with skin disease. The PC-SAD is a 37-item self-administered depression screening questionnaire that has been validated in dermatological patients. OBJECTIVE: The aim of this study was to develop and validate a brief depression severity instrument derived from the PC-SAD that can be used to assess severity and monitor ongoing clinical course. METHODS: Two patient samples participated in the study: 72 adult dermatological inpatients and 73 adults attending six primary care practices. Psychiatric assessment included the Structured Clinical Interview for DSM-IV and an 18-item version of the PC-SAD; moreover, dermatological patients completed the Patient Health Questionnaire depression scale (PHQ-9), while primary care patients were administered the Montgomery-Asberg Depression Rating Scale (MADRS). A subset of five PC-SAD items showing the best psychometric properties were selected, and the reliability and validity of the resulting instrument (PC-SAD5) were examined. RESULTS: The PC-SAD5 showed satisfactory internal consistency in both samples. There was a high correlation between PC-SAD5 and PHQ-9 and MADRS scores. Multiple regression analysis revealed a gradient of PC-SAD5 scores from patients with no mental disorder, those with milder forms of depression, to those with Major Depressive Disorder. Similar results were observed for the 18-item version of the PC-SAD. CONCLUSION: The availability of valid and reliable continuous measures of depression severity derived from the PC-SAD extends its field of application from depression screening to use as a follow-up measure of depression severity in routine clinical practice. A validated very short instrument such as the PC-SAD5 may have substantial clinical value.

Patient-based health status assessments in an outpatient psychiatry setting.

OBJECTIVE: The reliability, validity, and feasibility of the routine use of a generic health status instrument, the Short-Form-36 Health Survey (SF-36), were examined in a psychiatric outpatient clinic of a general hospital. METHODS: The sample comprised 411 patients referred to an outpatient psychiatry department between April 1994 and March 1995. They filled out the SF-36 along with their admission forms. Scores and reports were generated, and the results were returned to the charts and used at weekly clinical conference discussions. Feasibility was evaluated using subjective and objective data on administration of the instrument, its psychometric properties, and costs. Results from the outpatient psychiatry patients were compared with those from patients scheduled for elective surgery and a healthy normative sample. RESULTS: Routine administration of the SF-36 was successfully achieved with minimal resistance from staff and patients. The SF-36 provided reliable and valid data. As predicted, patients with emotional disorders scored lower, indicating more impairment, on scales measuring mental health than did the elective surgery patients and the normative sample. However, the psychiatric patients' scores on the physical health scale were lower than clinicians expected. Compared with the elective surgery patients, the psychiatric patients were less impaired on only the physical functioning and bodily pain scales; no difference was found between the two groups in role functioning due to physical problems. CONCLUSIONS: Routine use of the SF-36 in a general hospital psychiatric outpatient clinic was feasible, and the results were reliable, valid, and helpful to clinicians. Psychiatric patients' significantly lower scores in physical health and social and role functioning provided additional information about their difficulties.

The human acid ceramidase gene (ASAH): structure, chromosomal location, mutation analysis, and expression.

Acid ceramidase (AC) is the lysosomal enzyme that degrades ceramide into sphingosine and fatty acid. A deficiency in human AC activity leads to the lysosomal storage disorder, Farber disease (FD). The human AC gene (HGMW-approved symbol ASAH) was cloned and characterized, revealing an organization similar to that of the murine AC gene. The human gene spans about 30 kb in length and contains 14 exons ranging in size from 46 to 1201 bp. The exon/intron junctions were determined and found to follow the GT-AG rule. The putative promoter region had a GC content over 60%, lacked a TATA box, and contained several sequences matching transcription factor binding sites, including nine SP-1 sites, one AP-1 site, and three CACC boxes. The promoter activity of a 475-bp fragment from within this region was demonstrated by chloramphenicol acyltransferase assays. Northern blotting revealed variable expression of the human AC RNA; i.e., expression of the major 2.4-kb transcript was high in heart and kidney, followed by lung and placenta, but low in pancreas, liver, brain, and skeletal muscle. Two minor AC transcripts of 1.7 and 1.2 kb also were detected in heart and skeletal muscle. The human AC gene was mapped to the chromosomal region 8p21.3-p22 by in situ hybridization and FISH analyses, syntenic with the mouse chromosomal location. Finally, three new missense mutations, E138V, R254G, and P362R, were identified in the human AC gene from FD patients. Mutant AC cDNAs containing these point mutations were constructed and examined using the FLAG-tagged expression system. Although the levels of protein expression for these mutant ACs were about equivalent to that of the controls, their enzymatic activity was markedly reduced, confirming their authenticity.

Evidence of evolutionary up-regulation of the single active X chromosome in mammals based on Clc4 expression levels in Mus spretus and Mus musculus.

Previous studies have shown that the chloride channel gene Clc4 is X-linked and subject to X inactivation in Mus spretus, but that the same gene is autosomal in laboratory strains of mice. This exception to the conservation of linkage of the X chromosome in one of two interfertile mouse species was exploited to compare expression of Clc4 from the X chromosome to that from the autosome. Clc4 was found to be highly expressed in brain tissues of both mouse species. Quantitative analyses of species-specific expression of Clc4 in brain tissues from mice resulting from M. spretus x laboratory strain crosses, demonstrate that each autosomal locus has half the level of Clc4 expression as compared with the single active X-linked locus. In contrast expression of another chloride channel gene, Clc3, which is autosomal in both mouse species is equal between alleles in F1 animals. There is no evidence of imprinting of the Clc4 autosomal locus. These results are consistent with Ohno's hypothesis of an evolutionary requirement for a higher expression of genes on the single active X chromosome to maintain balance with autosomal gene expression [Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes (Springer, Berlin)].

Possible paroxetine-induced bruxism.

OBJECTIVE: To report the case of a patient with possible paroxetine-induced bruxism that was effectively treated with buspirone. CASE SUMMARY: A 20-year-old woman with no active medical conditions besides acne and no history of dental problems was seen in an outpatient psychiatry clinic for the evaluation of ongoing depression. The patient was prescribed paroxetine 10 mg every morning. After 5 days of therapy the patient reported no adverse effects, and the paroxetine dosage was increased to 20 mg every morning. Due to increased somnolence, the dosing schedule was subsequently changed to 20 mg hs. Two months later during a dental visit for a tooth extraction, the dentist noted that the patient's teeth appeared damaged in what he believed to be a pattern consistent with the grinding and clenching of teeth. Prior to this time, dental examinations had not revealed any tooth damage. The patient was thought to have paroxetine-induced bruxism and, based on earlier case reports, was treated with buspirone 5 mg hs. On day 4 of buspirone therapy the patient reported a significant reduction in the extent of gritting, tooth pain, and jaw tenderness. DISCUSSION: The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline have been associated with bruxism in previous reports. This case suggests paroxetine-induced bruxism. The exact mechanism of SSRI-induced bruxism remains unclear. Many theories have been proposed, including sleep disturbance, serotonergic-mediated inhibition of dopamine manifesting as akathisia, and SSRI-induced anxiety. According to published reports, SSRI-induced bruxism may respond to therapy with buspirone. Consistent with these reports, this patient responded favorably to buspirone therapy. CONCLUSIONS: Clinicians should be aware that the potential for paroxetine-induced bruxism exists and that buspirone may be an appropriate therapeutic intervention.

The entire genomic sequence and cDNA expression of mouse alpha-galactosidase A.

The full-length cDNA and genomic sequences encoding mouse alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22), a lysosomal galactohydrolase, were isolated and characterized. The cDNA's open reading frame encoded 419 amino acids and had 82% nucleotide (nt) and 78% amino acid identity with the human sequence, although the carboxy terminus of the mouse alpha-Gal A polypeptide was 10 amino acids shorter. The functional integrity of the mouse cDNA was demonstrated by transient expression in COS-1 cells. Northern analysis revealed two mRNA species of about 1.6 and 3.4 kb due to alternative polyadenylation signals. The entire 14.4-kb mouse genomic sequence was determined; each of its seven exons was interrupted by intronic sequence at the identical positions as the exons in the human gene. The mouse 5' flanking region (250 nt) had one Sp1, site, five CAAT boxes, and no TATA box and had 67% identity with the human promoter region. The gene contained 18 complete or partial Alu-repetitive elements (13 type 1 and 5 type 2 repeats), and three putative functional AATAAA consensus polyadenylation signals were identified 72, 1668, and 1682 nt after the TAA termination codon. Use of the 72-nt site and the 1866 and/or 1682 sites were consistent with the shorter and longer transcripts. The availability of the full-length cDNA and genomic sequence encoding mouse alpha-Gal A should facilitate structure/function studies of this lysosomal glycosidase and the construction of alpha-Gal A-deficient mice by targeted gene disruption.

Different chromosomal localization of the Clcn4 gene in Mus spretus and C57BL/6J mice.

We report the unprecedented finding of a gene with a different map position in two mouse strains. The Clcn4 gene was found to map to the X chromosome in the wild Mediterrean mouse, Mus spretus but to chromosome 7 in the inbred strain of laboratory mouse C57BL/6J. These data indicate that a recent evolutionary rearrangement occurred on the mouse sex chromosomes, very close to the pseudoautosomal region. Our data provide molecular evidence for a major divergence near the pseudoautosomal region, consistent with the hypothesis that hybrid sterility in these species results from abnormal pairing of sex chromosomes during male meiosis.

The X-linked methylated DNA binding protein, Mecp2, is subject to X inactivation in the mouse.

DNA methylation at the promoter region of X-linked genes is associated with the maintenance of X inactivation in mammals. One of the methylated DNA binding proteins, MECP2, that binds to methylated bases in DNA is encoded by a gene (Mecp2) located on the mouse X Chromosome (Chr). To determine whether this gene was expressed from the inactive X Chr, and X-autosome translocation (T(X;16)16H) system in which expression from the Mecp2 allele on the inactive X Chr could be assayed was used. Results from these experiments indicate that Mecp2 is subject to X inactivation in mouse.

Schizophrenia and the life cycle.

We reframe the longitudinal treatment of persons with schizophrenia from the perspective of phases in adult development. This approach articulates the need for different interventions of varying intensities over the person's lifetime. The paper discusses the implications of an adult developmental perspective in managing pharmacologic treatment and psychosocial interventions, and in reallocating financial resources for improved long-term outcomes. This perspective is especially useful in the context of a comprehensive community mental health program permitting access to a continuum of services throughout the lifecycle.

The mouse Sb1.8 gene located at the distal end of the X chromosome is subject to X inactivation.

The mouse homolog of the human DXS423E (SB1.8) gene has been isolated by screening a mouse cDNA library. Like its human counterpart, the mouse Sb1.8 gene is X-linked, as shown by Southern blot analysis and by in situ hybridization to metaphase chromosomes. Sb1.8 was sublocalized to band F of the mouse X chromosome, distal to Alas2 and proximal to DXPas1, which confirms a region of conservation between band Xp11.21-p11.22 in human and band XF in mouse. In situ hybridization also showed that the Smcx (Xe169) gene maps near Sb1.8 in band F. The Sb1.8 gene was shown to be highly conserved in mammals; partial DNA sequence analysis indicates 92% identity between the mouse and human genes. In contrast to the human DXS423E gene, the mouse Sb1.8 gene is subject to X inactivation, as shown by restriction enzyme and sequence analysis of mRNA from mice with Searle's translocation (T(X;16)16H). Absence of Sb1.8 expression from the inactive mouse X chromosome in vitro was confirmed by analysis of a cell line (Hobmski) in which the M. spretus X chromosome is inactivated. The Sb1.8 gene is a new member of a group of genes that escape X inactivation in human, but are inactivated in mouse.

Chromosomal mapping and expression levels of a mouse soluble epoxide hydrolase gene.

The chromosomal location of a murine soluble epoxide hydrolase gene was determined using in situ mapping, restriction fragment length polymorphism (RFLP) and simple sequence length polymorphism (SSLP) analysis. In situ hybridization to mouse metaphase chromosomes using a soluble epoxide hydrolase cDNA probe showed that soluble epoxide hydrolase maps at band D of chromosome 14. An RFLP found between Mus castaneus (CAST) and Mus musculus (MEV) was used to map the soluble epoxide hydrolase gene in CAST x MEV intersubspecific testcross progeny to 14 cM from the Np-1 locus on mouse chromosome 14. SSLP markers were then used to confirm the location of soluble epoxide hydrolase at 14.0 +/- 3.7 cM distal to Np-1 and 19.2 +/- 4.3 cM proximal to D14Mit7. This region of mouse chromosome 14 is homologous with human chromosomes 8, 13 and 14. Enzyme assays and immunoblotting results suggest significant quantitative differences in expression of soluble epoxide hydrolase among three mouse strains. Northern blotting analysis showed that soluble epoxide hydrolase mRNA levels were correlated with the relative level of soluble epoxide hydrolase enzyme activity and soluble epoxide hydrolase protein in all three mouse strains.

The case for a services-based approach to payment for mental illness under national health care reform.

In this position paper drafted by the committee on psychopathology of the Group for the Advancement of Psychiatry, the authors discuss merits and disadvantages of three different approaches to equitable coverage of mental illness: coverage for selected psychiatric diagnoses, coverage based on severity of impairment, and coverage of services. They believe that coverage of selected disorders has political appeal but is discriminatory and arbitrary; it is also impractical because clinicians may overdiagnose conditions covered by insurance and underdiagnose excluded conditions. Coverage based on severity of impairment, or disability, has similar limitations. The authors believe services should be the principal basis for coverage, as under general medical insurance. The approach is nondiscriminatory, and costs can be controlled through such means as managed care, changes in the payment system, or benefit design.

Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification.

Human and rabbit paraoxonases/arylesterases were purified to homogeneity by chromatographic and gel electrophoretic/isofocusing procedures coupled with activity stains. N-terminal and peptide sequence analysis suggested retention of the secretion signal sequence and allowed design of oligonucleotide probes. The probes were used to isolate a 1294-bp rabbit paraoxonase cDNA clone, which, in turn, was used to isolate three human cDNA clones. Comparison of rabbit and human protein and cDNA sequences indicated a high degree of sequence conservation (approximately 85% identity) and verified that paraoxonase retains its signal sequence (except for the N-terminal Met). The rabbit cDNA encodes a protein of 359 amino acids and the human a protein of 355 amino acids. In situ hybridization demonstrated, as expected, that the paraoxonase gene maps to the long arm of human chromosome 7. Arginine at position 192 specifies high activity paraoxonase and glutamine low activity human paraoxonase. Variation in protein levels explains the variation of enzyme activity observed within a genetic class. Toxicity studies showed that raising rat plasma paraoxonase levels by i.v. administration of partially purified rabbit paraoxonase protected animals against cholinesterase inhibition by paraoxon and chlorpyrifos oxon. Protection correlated with the relative rates of hydrolysis of these two compounds.

Maintenance of X inactivation of the Rps4, Zfx, and Ube1 genes in a mouse in vitro system.

Several genes, including RPS4X (ribosomal protein subunit 4), ZFX (zinc finger on the X chromosome), and UBE1 (ubiquitin-activating enzyme), have been shown to be expressed from the inactive X chromosome of cultured human cells. By contrast, these genes are subject to X-chromosome inactivation in tissues from adult mice. We have now examined the inactivation status of these genes in cultured mouse cells to determine whether the differences in X-chromosome inactivation between species is due to an intrinsic difference between human and mouse X-chromosome genes or whether it is a function of gene reactivation in cell culture per se. The expression of three mouse X-chromosome genes, Rps4, Zfx, and Ube1 was examined by reverse transcriptase polymerase chain reaction (RT-PCR) in heterozygous cultured cells from a cross of a laboratory mouse by Mus spretus, which were selected to uniformly express the X chromosome from the laboratory mouse parent. No expression of the M. spretus alleles of these genes was observed in the cell line (Hobmski), which is consistent with the patterns of expression previously observed in mouse in vivo and indicates that these genes remain stably inactivated in an immortalized mouse cell line. By cytogenetic and RT-PCR analyses the Hobmski cell line was shown to retain a late-replicating X chromosome from M. spretus, which expressed the M. spretus allele of the X (inactive) specific transcript (Xist). The Hobmski cell line will be a useful resource for studying the features that maintain X-chromosome genes inactive.

The molecular basis of the human serum paraoxonase activity polymorphism.

The organophosphate cholinesterase inhibitor paraoxon is hydrolysed by serum paraoxonase/arylesterase. A genetic polymorphism of paraoxonase (PON) activity which determines high versus low paraoxon hydrolysis in human populations, may determine sensitivity to parathion poisoning. We demonstrate that arginine at position 192 specifies high activity PON whereas a glutamine specifies the low activity variant. Allele-specific probes or restriction enzyme analysis of amplified DNA allow for the genotyping of individuals. PON maps to chromosome 7q21-22, proximal to the cystic fibrosis gene, in agreement with previous genetic linkage studies.