Gsx1 expression defines neurons required for prepulse inhibition.

In schizophrenia, cognitive overload is thought to reflect an inability to suppress non-salient information, a process which is studied using prepulse inhibition (PPI) of the startle response. PPI is reduced in schizophrenia and routinely tested in animal models and preclinical trials of antipsychotic drugs. However, the underlying neuronal circuitry is not well understood. We used a novel genetic screen in larval zebrafish to reveal the molecular identity of neurons that are required for PPI in fish and mice. Ablation or optogenetic silencing of neurons with developmental expression of the transcription factor genomic screen homeobox 1 (gsx1) produced profound defects in PPI in zebrafish, and PPI was similarly impaired in Gsx1 knockout mice. Gsx1-expressing neurons reside in the dorsal brainstem and form synapses closely apposed to neurons that initiate the startle response. Surprisingly, brainstem Gsx1 neurons are primarily glutamatergic despite their role in a functionally inhibitory pathway. As Gsx1 has an important role in regulating interneuron development in the forebrain, these findings reveal a molecular link between control of interneuron specification and circuits that gate sensory information across brain regions.

Unidirectional startle responses and disrupted left-right co-ordination of motor behaviors in robo3 mutant zebrafish.

The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. Although significant progress has been made toward understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. In this study, we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3. We show that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice/robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice/robo3 in sensory-guided behavior.

Cleavage of doublecortin-like kinase by calpain releases an active kinase fragment from a microtubule anchorage domain.

Doublecortin-like kinase (DCLK) is widely expressed in postmitotic neurons throughout the embryonic nervous system. DCLK consists of an N-terminal doublecortin domain, responsible for its localization to microtubules, and a C-terminal serine-threonine kinase domain. Here we report that DCLK is a physiological substrate for the cysteine protease calpain. Cleavage of DCLK by calpain severs the kinase domain from its microtubule anchorage domain and releases it into the cytoplasm. The isolated kinase domain retains catalytic activity and is structurally similar to CPG16, a second product of the DCLK gene expressed in the adult brain that lacks the doublecortin domain. We propose that in neurons cleavage of DCLK by calpain represents a calcium responsive mechanism to regulate localization of the DCLK kinase domain.

Doublecortin-like kinase is associated with microtubules in neuronal growth cones.

Doublecortin-like kinase (DCLK) shares sequence similarity to Doublecortin (DCX) in its N-terminal region. It contains the evolutionary conserved DC repeat motif as well a C-terminal kinase domain. Ectopic expression of DCLK in COS cells results in colocalization with microtubules, and phosphorylated DCLK copurifies with microtubules during assembly from embryonic brain extract. During brain development DCLK is expressed mainly in postmigratory neurons in a similar pattern to DCX. We demonstrate that DCLK is a microtubule-associated active protein kinase expressed in growth cones of postmitotic neurons.

Doublecortin mutations cluster in evolutionarily conserved functional domains.

Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.

Analysis of lissencephaly-causing LIS1 mutations.

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.

KIAA0369, doublecortin-like kinase, is expressed during brain development.

During embryonic development, the cerebral cortex attains its characteristic adult laminated structure. The finding that X-linked lissencephaly patients harbor mutations in the doublecortin gene implicated this gene product in the process of corticogenesis. An autosomal human gene, KIAA0369, with a high level of similarity to doublecortin, has been cloned from human adult brain. This gene product contains a kinase domain in addition to a doublecortin-like domain. In order to evaluate whether this doublecortin-like kinase also plays a role during brain development, we cloned and studied the expression pattern of the mouse homolog. Three cDNA products of this gene were cloned: one, doublecortin-like kinase, the second containing only the doublecortin-like region, and the third containing only the kinase domain, a homolog of the previously cloned rat CPG16 gene. We studied doublecortin-like kinase expression in mouse using Northern blot analysis, in situ hybridization, and Western blot analysis, and conclude that doublecortin-like kinase is expressed in multiple regions of embryonic brain including the developing cerebral cortex.

Parameters of normotensive women and women with pregnancy induced hypertension (PIH) in Lusaka.

This paper reports results of a survey of mean arterial pressures (MAP) in a population of 125 control patients and 30 cases who developed pregnancy induced hypertension. The investigation was carried out to see if MAP would be a useful addition to methods used to screen for PIH. More of the cases had elevated MAP than did the controls, suggesting that use of this easily obtainable information may be helpful in identifying patients at risk for pregnancy--induced hypertension. Addition baseline information on the 155 patients and their pregnancy outcome is also presented.

PIP: 35 cases of pregnancy-induced-hypertension (PIH) and 125 controls taken on nonconsecutive days from 792 deliveries during the period June-August 1988 at the University Teaching Hospital, Lusaka, Zambia, were analyzed for mean arterial pressure (MAP), and contributing factors. Data were taken from delivery logs and patients' antenatal cards. The cases were women diagnosed with PIH or a history of seizures. Controls were the 5 preceding deliveries with adequate data. Results for MAP were presented as a matrix showing good outcome controls, poor outcome controls, and cases, by MAP at antenatal visits 1-4, with numbers of women in groupings by MAP ranges. There were more high MAP values for cases than for controls. Over 25% of both groups were women aged 17-19. 64.7% of cases were primigravida, compared to 43% of controls. Data on weight gain were not consistently available, but a higher proportion of cases gained 20 pounds than controls. Most women gained 0 or 1-10 pounds. 44% of cases and 41% of controls had hemoglobin 10 mg/dl. There were 4 positive VDRLs among 34 women tested. 4% of the control infants were stillborn; 7% died in hospital. Among the cases there were 1 fetal death, 2 stillborns, 4 hospital deaths, 20% pregnancy loss overall. Apgar scores were lower among cases, with 25% 4-6, compared to 3.3% of controls. These results did not indicate that MAP would be useful in comparison with blood pressure and other risk factors in monitoring pregnant women for PIH.

Use of the labor graph in Malawi.

PIP: A facsimile of the 1-page labor chart provided by the Ministry of Health and used at all maternity clinics in Malawi is described. Malawi has 350 maternity units which take care of 45% of deliveries. Personnel range from nonprofessional to registered nurse-midwives to physicians. The most notable feature of the chart is a partogram marked with "alert" and "action" lines, for comparing cervical dilation per hour. The alert line is a diagonal line starting at 3 cm dilated and fully effaced, drawn at an angle representing the slowest 10% of normal African primigravidas. Normally vaginal delivery would be expected at the time of the end of the action line. The action line is drawn arbitrarily 4 hours to the right, allowing time for normal delivery or transfer of the patient. Fetal descent is also charted in fifths of head palpable above the pelvic brim. There is room to the left of the page to chart progress in latent phase. One side of the chart contains demographic and admission data and the progress graph. The reverse side has space for pelvic assessment, results of 2nd and 3rd stage of labor and charting of up to 6 days' postpartum stay for mother and infant. There are blanks to record whether the woman has had sleep, food or homemade medicine, and room for making referrals. In practice fetal descent is followed by abdominal palpation hourly, fetal heart rate every 30 minutes, and charted as type 1, 2, or 3. Vaginal examinations are done every 4 hours for primigravidas and every 3 in multiparas. Fetal head molding is charted as -or 1+, 2+ or 3+.^ieng

The relative bioavailability of paracetamol after rectal administration of suppositories containing a mixture of paracetamol, codeine phosphate and buclizine hydrochloride in healthy volunteers.

'New' and 'old' suppositories (6 months and 30 months since manufacture) containing 800 mg paracetamol, 16 mg codeine phosphate and 12.5 mg buclizine hydrochloride in an identical base were administered to 10 normal volunteers at an interval of 2 weeks. Blood samples were taken at intervals up to 300 minutes after administration for estimation of paracetamol plasma concentrations using high pressure liquid chromatography. Mean peak concentrations were obtained of 4.75 +/- 0.74 mg/ml at 1.75 hours with the new suppositories and of 4.6 +/- 0.67 mg/ml at 2.0 hours with the old suppositories. The difference was not significant. Mean elimination half-life was 4.4 +/- 0.42 hours and 3.73 +/- 0.28 hours, respectively. Again, the difference was not significant, indicating that the absorption characteristics for the suppositories did not appear to deteriorate with ageing for 24 months. Bioavailability data for paracetamol derived from the results were similar to those reported by other workers who studied suppositories containing paracetamol as the only active ingredient. This indicates that the inclusion of codeine phosphate and buclizine hydrochloride in the suppository formulation investigated in the present study did not affect adversely the absorption of paracetamol.