Reduced cortical expression of a newly identified splicing variant of the DLG1 gene in patients with early-onset schizophrenia.

The human discs, large homolog 1 gene (DLG1) is mapped to the schizophrenia-susceptibility locus 3q29, and it encodes a scaffold protein that interacts with the N-methyl-D-aspartate receptor presumably dysregulated in schizophrenia. In the current study, we have newly identified a splicing variant of DLG1, which is transcribed from an unreported 95-base-pair exon (exon 3b) and is labeled 3b(+). We investigated the mRNA expression of 3b(+) in the post-mortem dorsolateral prefrontal cortices of patients with psychiatric disorders, obtained from The Stanley Medical Research Institute, and examined the potential association of the expression with the genotype of the single-nucleotide polymorphism (SNP) rs3915512 located within exon 3b. A real-time quantitative reverse transcriptase-polymerase chain reaction revealed that the mRNA levels of 3b(+) were significantly reduced in patients with early-onset schizophrenia (onset at <18 years old, P=0.0003) but not in those with non-early-onset schizophrenia, early-onset or non-early-onset bipolar disorder or in the controls. Furthermore, the genotype at the rs3915512 SNP was closely associated with the levels of 3b(+) mRNA expression. It is inferred that the T allele fails to meet the exonic splicing enhancer consensus, thus resulting in skipping of exon 3b, leading to the expression of 3b(-) (the previously known DLG1 variant) but not 3b(+). Because all the subjects with early-onset schizophrenia in the current study possess the T/T genotype, the reduced level of the DLG1 3b(+) transcript may be involved in the susceptibility and/or pathophysiology of early-onset schizophrenia.

A prolonged course of Group A streptococcus-associated nephritis: a mild case of dense deposit disease (DDD)?

We herein report the case of a 12-year-old boy with dense deposit disease (DDD) evoked by streptococcal infection. He had been diagnosed to have asymptomatic hematuria syndrome at the age of 6 during school screening. At 12 years of age, he was found to have macrohematuria and overt proteinuria with hypocomplementemia 2 months after streptococcal pharyngitis. Renal biopsy showed endocapillary proliferative glomerulonephritis with double contours of the glomerular basement membrane. Hypocomplementemia and proteinuria were sustained for over 8 weeks. He was suspected to have dense deposit disease due to intramembranous deposits in the first and the second biopsies. 1 month after treatment with methylprednisolone pulse therapy, proteinuria decreased to a normal level. Microscopic hematuria disappeared 2 years later, but mild hypocomplementemia persisted for more than 7 years. Nephritis-associated plasmin receptor (NAPlr), a nephritic antigen for acute poststreptococcal glomerulonephritis, was found to be positive in the glomeruli for more than 8 weeks. DDD is suggested to be caused by dysgeneration of the alternative pathway due to C3NeF and impaired Factor H activity. A persistent deposition of NAPlr might be one of the factors which lead to complement dysgeneration. A close relationship was suggested to exist between the streptococcal infection and dense deposit disease in this case.

Evidence for involvement of glial cell activity in the control of extracellular D-serine contents in the rat brain.

The continuous intra-cortical infusion of a glia toxin, fluorocitrate, at the concentration of 1 mM caused a decrease in the cortical extracellular contents of an intrinsic coagonist for the N-methyl-D-aspartate (NMDA) type glutamate receptor, D-serine, by peaking at 40 min by -25% but produced an increase in those of glycine and L-serine. The attenuated glial activity by fluorocitrate was verified by a marked reduction in the extracellular glutamine contents. The present findings suggest that a group of glial cells such as a population of the protoplasmic astrocytes could, at least in part, participate differently in the regulation of the extracellular release of D-serine and another NMDA coagonist glycine in the medial frontal cortex of the rat.

Distribution of anxiogenic-induced c-Fos in the forebrain regions of developing rats.

An anxiogenic or a pharmacological stressor, N-methyl-beta-carboline-3-carboxamide (FG-7142), (20 mg/kg, intraperitoneally injected) induced a dense nuclear c-Fos-like immunoreactivity in the pyriform cortex, cingulate and retrosplenial cortex, layers II-VI of the neocortex, lateral habenula, lateral septum, paraventricular nucleus of the thalamus, striatum, central and medial nucleus of the amygdala, but a sparse c-Fos immunostaining in the hippocampus and layer I of the neocortex in the forebrain of 56-day-old rats. Among these regions, the 8-day-old rats expressed much fewer c-Fos-positive cells in the neocortex, lateral habenula, lateral septum and medial nucleus of the amygdala than the young adult rats following the FG-7142 injection. These differences in the regional distribution of a neuronal activity marker, c-Fos, could reflect the postnatal development of neuronal populations or neuron circuits involved in stress and/or emotional response in the forebrain.

A developmentally regulated and psychostimulant-inducible novel rat gene mrt1 encoding PDZ-PX proteins isolated in the neocortex.

Single or repeated exposure to psychostimulants such as amphetamines and cocaine after postnatal week 3 leads to an enduring enhancement in the psychotomimetic responses elicited by a subsequent challenge of a stimulant in rodents. This behavioral sensitization phenomenon has been considered to be the neural consequences of stimulant-induced alterations in gene expression in the brain after a critical period of postnatal development. Using a differential cloning technique, RNA arbitrarily primed PCR, we have now identified from the rat neocortex a novel and developmentally regulated methamphetamine (MAP)-inducible gene mrt1 (MAP responsive transcript 1). mrt1 encodes two major types of PDZ- and PX-domains containing proteins of approximately 62 kDa in size with different carboxy termini, Mrt1a and Mrt1b. The mrt1 mRNAs for Mrt1a, mrt1a, and for Mrt1b, mrt1b, are predominantly expressed in various brain regions and the testes, respectively. Acute MAP injection upregulated mrt1b expression in the neocortex after postnatal week 3 in a D1 receptor antagonist-sensitive manner without affecting mrt1a expression. This upregulation was mimicked by another stimulant, cocaine, whereas pentobarbital and D1 antagonist failed to change the mrt1b transcript levels. Moreover, repeated daily treatment of MAP, but not MAP plus D1 antagonist, for 5 days caused an augmentation of the basal expression of mrt1b 2 and 3 weeks after the drug discontinuation. These late-developing, cocaine-crossreactive, D1 antagonist-sensitive and long-term regulations of mrt1b by MAP are similar to the pharmacological profiles of stimulant-induced behavioral sensitization, and therefore may be associated with the initiation and/or maintenance of the long-term neuronal adaptation.

Uptake of D-serine by synaptosomal P2 fraction isolated from rat brain.

We have demonstrated that the crude synaptosome P2 fraction prepared from the rat brain is able to accumulate [(3)H]D-serine in a saturable, temperature-dependent and partially sodium- and potassium-dependent manner with an affinity of a hundred micromolar range. The inhibition profile of D-serine accumulation by various amino acids is different from those of uptake systems reported for glycine and other amino acids. The present data suggest that the endogenous D-serine may be taken up mainly through a carrier-mediated transport system to regulate its extracellular concentration in the mammalian brain.

Nomifensine-induced c-fos mRNA expression in discrete brain areas of the developing rat.

In rats, the subcutaneous injection of a dopamine uptake inhibitor, nomifensine (40 mg/kg), induced a significant increase in the c-fos mRNA levels in the neocortex on postnatal days 23 and 49, in the striatum on days 8, 14, 23 and 49, and in the hippocampus on day 23, when compared with saline administration. The repeated injection with nomifensine (40 mg/kg once daily) from postnatal days 49 to 53 and from 23 to 27, but not from days 8 to 12 and 14 to 18, resulted in an enhanced stereotypy response elicited by a subsequent challenge of the drug (5 mg/kg) 21 days after the last injection (behavioral sensitization). The present results suggest that the neuronal circuits regulated by nomifensine might undergo regionally-different developmental changes, which could be implicated in the development of behavioral expressions including nomifensine-induced stereotypy sensitization.

Thickness of the retinal nerve fiber layer in amblyopic and normal eyes: a scanning laser polarimetry study.

PURPOSE: To compare scanning laser polarimeter (GDx, Laser Diagnostic Technologies, San Diego, Calif) measurements of the peripapillar retinal nerve fiber layer in amblyopic and normal eyes. METHODS: Scanning laser polarimetry was performed on 21 patients with unilateral strabismic amblyopia who had an absence of neurologic diseases or glaucoma and a minimum age of 7 years. A mean retardation map was calculated from separate scans or was considered to be the best scan obtained for each eye. Polarimetric indices were analyzed comparing amblyopic and contralateral normal eyes. RESULTS: The mean age was 15 +/- 9 years (7-35 years) and the male:female ratio was 13:8. There were 6 right and 15 left amblyopic eyes, with the amblyopic group having a mean visual acuity of 0.3 +/- 0.1. The mean (+/- SD) indices did not differ significantly between normal and amblyopic eyes, except the number that summates information from the individual parameters, which was higher in normal (20.71 +/- 11.98) than in amblyopic (15.14 +/- 6.81) eyes, P =.02. CONCLUSION: There was no statistical difference in thickness of the nerve fiber layer between amblyopic and normal eyes. A previous study found similar results in adults with strabismic amblyopia.

Cloning of a D-serine-regulated transcript dsr-1 from the rat cerebral cortex.

To obtain insight into the molecular mechanisms underlying the metabolism and functions of endogenous d-serine, we have explored d-serine-regulated transcripts in the neocortex of the infant rat treated with acute d-serine administration by using an RNA fingerprinting technique. Cloning and sequence analysis of the corresponding cDNAs to the identified transcripts have revealed that the dsr-1 (d-serine responsive transcript-1) mRNA is presumed to contain a novel sequence at the 5'-region, while the 631-base nucleotide sequence of its 3'-end is identical with that of rat M9.2 mRNA encoding a subunit of vacuolar type proton-ATPase. The predicted two open reading frames and their deduced amino acid sequences suggest that the dsr-1 product has a membrane spanning domain. The dsr-1 transcript was detected as a single band around 2.1 kb on the Northern blot. RT-PCR analyses have indicated that the dsr-1 transcript is expressed predominantly in the brain, lung, and testis, and that acute intraperitoneal injection of d-serine significantly upregulates dsr-1 expression in the neocortex 3 and 15 h later without affecting the levels of the M9.2 gene transcript. These results suggest that dsr-1 products may be involved in the d-serine-related metabolic or signaling pathways in mammalian brains.

[Endogenous D-serine in mammalian brains].

It is well established that, like glycine and D-alanine, D-serine potentiates glutamate neurotransmission via the N-methyl-D-aspartate (NMDA) receptor by selective stimulation of its strychnine-insensitive glycine site and acts as a co-agonist of the glutamate receptor. D-Serine has been found to modify behavioral changes associated with higher brain functions such as memory, convulsion, anxiety, psychotomimetic-induced abnormal behavior and cerebellar ataxia. Interestingly, a substantial amount of free D-serine has been demonstrated in mammalian brains, although it has long been presumed that D-amino acids are uncommon in mammals. Free D-serine is predominantly concentrated in the brain with a persistent high content throughout life. The patterns of the regional variations and the postnatal changes in brain D-serine are closely correlated with those of the R2B subunit of the N-methyl-D-aspartate (NMDA) type excitatory amino acid receptor. Moreover, D-serine is released to the extracellular space and taken up into the brain homogenates, C6 glioma cells and primary culture of astrocytes of the rat cerebral cortex. Recently, the conversion of L-serine to its D-form by serine racemase has been suggested by in vivo and in vitro experiments. These data are consistent with the view that D-serine might be an intrinsic positive modulator of the brain NMDA receptor containing the R2B subunit and play a pivotal role in controlling behavioral expression in mammals.

Characterization of the phencyclidine-induced increase in prefrontal cortical dopamine metabolism in the rat.

1. We have investigated the effects of a schizophrenomimetic drug phencyclidine (PCP) and N-methyl-D-aspartate (NMDA)-related agents alone or in combination on dopamine metabolism in the medial prefrontal cortex and striatum of the rats by measuring the tissue concentrations of dopamine and its metabolite, 3,4-dihyroxyphenylacetic acid (DOPAC), and the rate of dopamine disappearance (dopamine utilization) after its synthesis inhibition. 2. Systemic injection of PCP and selective, non-competitive, NMDA antagonists caused an increase of both tissue concentrations of DOPAC and dopamine utilization in the prefrontal cortex but not in the striatum. The PCP-induced augmentation of cortical dopamine metabolism was not influenced by selective lesion of ascending noradrenergic neurones. 3. Intra-prefrontal cortical infusion of PCP or selective competitive or non-competitive antagonists of the NMDA receptor mimicked the ability of systemic PCP injection to enhance DOPAC levels and dopamine utilization in the prefrontal cortex. However, an NMDA antagonist injected into the cell body area of the mesocortical dopaminergic neurones failed to affect dopamine metabolism in the prefrontal cortex. 4. The increasing effects of PCP and selective NMDA antagonists on cortical dopamine utilization were not additive, although a dopamine receptor antagonist, haloperidol, still accelerated the disappearance of dopamine, even in the presence of PCP. 5. Intra-cortical or intra-ventricular infusion of NMDA or D-alanine but not L-alanine, attenuated the ability of systemic PCP administration to facilitate prefrontal dopamine utilization. 6. These data suggest that PCP might activate prefrontal cortical dopaminergic neurones, at least in part, by blocking the NMDA receptor in the prefrontal cortex which participates in a tonic inhibitory control of the mesoprefrontal dopaminergic projections.

Evidence for N-methyl-D-aspartate receptor-mediated increase in norepinephrine utilization in the prefrontal cortex of unanesthetized rats.

Local injection of N-methyl-D-aspartate (NMDA; 10-20 nmol/rat) into the prefrontal cortex of conscious rats caused a dose-related and an NMDA antagonist-reversible facilitation of norepinephrine (NE) disappearance in the cortical region during 35 min after inhibition of tyrosine hydroxylase. Destruction of ascending NE neurons by bilateral application of 6-hydroxydopamine into the superior cerebellar peduncle failed to affect [3H]N-(1-[2-thienyl]cyclohexyl)piperidine binding to the NMDA receptor-associated ion channel in the prefrontal cortex. These results indicate that, under unanesthetized conditions, the prefrontal NE neurons may be under glutamatergic facilitatory control mediated by the NMDA receptors which are located on the non-NE systems in the frontal cortex.

Developmental changes in distribution patterns of phencyclidine-induced c-Fos in rat forebrain.

In the forebrain of 56-day-old rats, histochemical studies revealed that the subcutaneous injection of a psychotomimetic phencyclidine (PCP; 1 and 10 mg/kg) induced a dose-related and dense nuclear c-Fos-like immunoreactivity in the pyriform cortex, layers IV-VI of the neocortex and septum, but a sparse c-Fos immunostaining in the olfactory tubercle and mid-lateral striatum. Infant rats at postnatal day 8 expressed much fewer and more confined c-Fos-positive cells in the neocortex than young adult rats following PCP injection. However, a similar expression pattern of PCP-induced c-Fos was observed in the pyriform cortex, mid-lateral striatum, olfactory tubercle and septum between the infant and adult periods. These developmental changes in the regional distribution of a neuronal activity marker, c-Fos, suggest that neuronal populations involved in PCP-induced abnormal behavior are influenced by postnatal development, at least, in the neocortex.

Differential effects of phencyclidine and methamphetamine on dopamine metabolism in rat frontal cortex and striatum as revealed by in vivo dialysis.

We have examined the effects of schizophrenomimetic drugs including phencyclidine (PCP) and methamphetamine (MAP) on cortical and striatal dopamine (DA) metabolism using an in vivo dialysis technique in the rat. An acute systemic injection of PCP (2.5-10 mg/kg, intraperitoneally (i.p.)) dramatically increased concentrations of DA, 3,4-dihydroxy-phenylacetic acid, and homovanillic acid in the dialysates from the medial frontal cortex in a dose-dependent fashion. However, PCP (2.5-10 mg/kg, i.p.) caused a much lower augmentation of extracellular DA release, with a significant decrease in dialysate DOPAC levels in the striatum. Moreover, continuous infusion of tetrodotoxin (TTX, 10(-5) M) into the prefrontal or striatal region through the microdialysis tube completely blocked the ability of PCP (10 mg/kg, i.p.) to alter the extracellular release of DA and its metabolites in the respective areas. In contrast, MAP (4.8 mg/kg, i.p.) elicited a marked and tetrodotoxin-resistant increase in DA levels with a significant loss of DOPAC contents in the extracellular space of both the frontal cortex and the striatum. The present results clearly demonstrate the differential effects of PCP on cortical and striatal DA transmission, suggesting that PCP may facilitate DA release in the medial frontal cortex by increasing impulse flow in the DA neurons projecting to the cortical area, whereas PCP-induced elevation of extracellular DA in the striatum may be caused mainly by reuptake inhibition of DA liberated by basal activity of the striatal DA neurons. The regional variation in PCP-induced DA release would be due to the combination of NMDA (N-methyl-D-aspartate) receptor blocking and DA reuptake inhibition by the drug. The uniform and TTX-resistant nature of MAP-induced changes in brain DA metabolism may result from the direct actions of MAP at DA nerve terminals.

Dizocilpine (MK-801) elicits a tetrodotoxin-sensitive increase in extracellular release of dopamine in rat medial frontal cortex.

We have examined in the rat the effects of a selective non-competitive antagonist for the N-methyl-D-aspartate (NMDA) type excitatory amino acid receptor, dizocilpine (MK-801), on cortical dopamine (DA) metabolism using an in vivo dialysis technique. An acute intraperitoneal injection of MK-801 (0.4-1.25 mg/kg) dramatically increased the concentrations of dopamine, 3,4-dihydroxy-phenylacetic acid and homovanillic acid in the dialysates from the medial frontal cortex in a dose-dependent fashion. Moreover, MK-801 caused a delayed and small augmentation of the cortical extracellular release of 5-hydroxyindoleacetic acid. Continuous infusion of tetrodotoxin into the prefrontal region via the microdialysis tube completely blocked the ability of MK-801 (1.25 mg/kg, intraperitoneally) to augment the extracellular release of DA, its metabolites and the serotonin metabolite in the frontal cortex. The present results suggest that MK-801 facilitates DA release in the medial frontal cortex by increasing impulse flow in the DA neurons projecting to the cortical area adding further support to the view that the NMDA receptor may be involved in the tonic inhibition of frontal cortical DA neurons. It is also proposed that frontal serotonin neurons might be under regulation by excitatory amino acidergic transmission via the NMDA receptor.

Methamphetamine-induced nuclear c-Fos in rat brain regions.

To explore the possible robust changes in neuronal activity in dopamine-poor brain regions after an indirect dopamine agonist, methamphetamine, we have investigated its effects on c-fos expression in rat brain using immunocytochemistry of c-Fos. Intraperitoneal injection of methamphetamine (1.6-4.8 mg/kg), but not of saline, induced a widespread nuclear c-Fos-like immunoreactivity in the pyriform cortex and olfactory tubercle with greatest density followed by the II-VI layers of the neocortex, amygdala, hypothalamus, thalamus, nucleus accumbens and striatum. These expression patterns resemble those elicited by amphetamine and suggest that not only the dopamine-rich subcortical regions but also the cerebral cortex may play a crucial role in behavioral abnormality induced by methamphetamine.

p-chlorophenylalanine-reversible reduction of sigma binding sites by chronic imipramine treatment in rat brain.

Repeated treatment with imipramine (10 mg/kg intraperitoneally (i.p.), once daily for 14 days) caused a decrease in the Bmax, without affecting the Kd, of [3H]DTG (1,3-di-o-tolylguanidine) binding to the haloperidol-sensitive sigma sites in the striatum, hippocampus and cerebral cortex of the rat. A similar reduction was observed after chronic administration of a selective serotonin uptake inhibitor, fluoxetine (10 mg/kg i.p., twice daily for 14 days), but not of a selective norepinephrine uptake inhibitor, desipramine (10 mg/kg i.p., once daily for 14 days). Neither a single injection of imipramine (10 mg/kg i.p.) nor addition of imipramine or fluoxetine into the binding assay medium mimicked the changes in the maximal binding of brain sigma sites induced by chronic treatment with these drugs. Finally, depletion of brain serotonin by means of repeated administration of p-chlorophenylalanine, which produces inhibition of the amine synthesis, blocked the ability of repeated imipramine treatment to reduce the maximal number of [3H]DTG binding sites in the striatum and hippocampus. The present results suggest that cerebral serotonergic transmission may play a role in the regulation of cerebral sigma binding sites in the rat.

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat.

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpenduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.