[Localization and functions of the D-neuron: significance in pathogenesis of schizophrenia].

The author proposes the "D-cell hypothesis" for molecular basis of the mesolimbic dopamine (DA) hyperactivity of schizophrenia. D-neurons, which were defined as "non-monoaminergic aromatic L-amino acid decarboxylase (AADC)-containing cells", produce trace amines (TAs), such as tyramine, phenylethylamine (PEA) and tryptamine. D-neurons may also take up amine precursors, and may convert them to amines by decarboxylation. The author's preliminary report showed that the number of AADC-containing neurons, that is D-neurons, was reduced in the striatum and nucleus accumbens of patients with schizophrenia. TA-associated receptor type 1 (TAAR1) has been shown to have a number of ligands, such as tyramine, PEA, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and lysergic acid diethylamide (LSD), that may change human mental states. In patients with schizophrenia, the reduction of striatal D-neurons and possible decrease of striatal TA, is caused by neural stem cell dysfunction in the subventricular zone of the lateral ventricle. The reduced stimulation of TAAR1 on terminals of ventral tegmental area (VTA) DA neurons increases the firing frequency of VTA DA neurons, as recently published reports have shown, resulting in mesolimbic DA hyperactivity. In addition, increased DA D2 receptor stimulation, caused by striatal DA hyperactivity, may suppress forebrain neural stem cell proliferation, and would cause an additional decrease of D-neurons.

Pitfalls and practicalities in collecting and banking human brain tissues for research on psychiatric and neulogical disorders.

It is essential to examine brain materials for the understanding the cause and pathology of mental disorders. Recent methodological progress urges us to set up well qualified brain banks. Human tissue and Bio-banking is a complex field and the daily practice of brain banks needs to abide by several golden standards in order to avoid pitfalls in basic research: 1) A donor system in which informed consent is granted for the use of the samples for scientific research, including genetic analysis and access to medical records, 2) Rapid autopsy system, 3) Compatibility of protocols for procurement, management, handling and storage, 4) A generally accepted consensus on diagnostic criteria, 5) Quality control, 6) Abiding by local/international legal and ethical guidelines for work with human material, 7) Proper safety procedures. In the present review, the authors introduced the activities of European brain banks, and discussed on their current issues, and on the problems remain to be resolved.

Increased ratio of calcineurin immunoreactive neurons in the caudate nucleus of patients with schizophrenia.

Calcineurin (CaN) has been investigated extensively in numerous biochemical, behavioral, and genetic studies in schizophrenia because its function is closely related to dopamine-glutamate signal transduction, which is thought to be associated with pathophysiological changes in schizophrenia. Although evidence has suggested that dysfunction of CaN may be a risk factor for schizophrenia, there have been few reports focusing on the expression of CaN mRNA and CaN protein levels in the brains of schizophrenic patients. In addition, findings on CaN expression in postmortem brains from patients with schizophrenia have been inconsistent. Here, we conducted immunohistochemical examinations of several regions in postmortem brains, including the dorsolateral prefrontal cortex (DLPFC), hippocampus, caudate nucleus, and putamen, using specific antibodies, and compared the results from the brains of nine schizophrenic subjects to nine age- and sex-matched control subjects. There was no significant difference in the ratio of CaN immunoreactive (IR) neurons between schizophrenia and control groups in the DLPFC or hippocampus, and a significantly increased ratio of CaN-IR neurons was seen in the caudate nucleus in the brains from schizophrenia patients. As the striatum contains most of the brain dopamine, the results of the present study have critical implications and suggest that alterations in CaN signaling in the caudate contribute to the pathogenesis of schizophrenia. This is the first report of caudate CaN abnormalities in schizophrenia.

Detailed DARPP-32 expression profiles in postmortem brains from patients with schizophrenia: an immunohistochemical study.

The prevalence of dopamine and cAMP-regulated phosphoprotein 32kD (DARPP-32) is associated with the pathogenesis of schizophrenia. To date, the findings on DARPP-32 cellular expression and distribution in postmortem brains from patients with schizophrenia have been inconsistent. To clarify the detailed cellular expression of DARPP-32 in patients with schizophrenia, we immunohistochemically stained sections from postmortem brains using specific antibodies. We measured the density of immunopositive cells in various brain regions including the prefrontal cortex and compared the data from nine schizophrenia subjects with those of nine age- and sex-matched control subjects. The density of DARPP-32-immunoreactive (IR) neurons was significantly lower in layers II-V of the dorsolateral prefrontal cortex (DLPFC) from subjects with schizophrenia. In contrast, there were no marked differences in DARPP-32 expression in other brain regions. In addition, the density of threonine (Thr34)-phosphorylated DARPP-32-IR neurons was significantly higher in layer V of DLPFC from subjects with schizophrenia. These results suggest that the decrease in DARPP-32 in schizophrenia was more marked in neurons of DLPFC than in other cells or other brain regions, and that this decrease might be partly compensated for by an increase in expression of Thr34-phosphorylated DARPP-32 in DLPFC.

Profiles of DARPP-32 in the insular cortex with schizophrenia: a postmortem brain study.

In patients with schizophrenia, various physical disorders are sometimes discovered only when they have reached a later and more severe stage. This phenomenon is believed to be caused, at least in part, by an increase in pain threshold. The gamma-aminobutyric acid (GABA)-ergic and glutamatergic systems in the rostral agranular insular cortex (RAIC) are thought to be involved in the regulation of pain threshold. However, no postmortem studies of the cerebral cortex have previously been published. Dopamine and cAMP-regulated phosphoprotein 32 kD (DARPP-32), which is involved in the GABAergic and glutamatergic systems, is considered to be crucial for elucidating the pathogenesis of schizophrenia. Using specific antibodies, we conducted immunohistochemical examinations of the RAIC in 10 subjects from a healthy control group, and 11 subjects from a schizophrenia group. The sex, age, and postmortem interval (PMI) of the schizophrenia group were matched to those of the healthy control group. We revealed that the density of DARPP-32-immunoreactive (IR) neurons in the II and III layers of the RAIC was significantly decreased (p<0.05) in the schizophrenia group compared with the healthy control group. Our findings could partially explain the molecular basis of the pain threshold abnormalities found in patients with schizophrenia.

Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Schizophrenia is one of the major psychiatric disorders, and lipids have focused on the important roles in this disorder. In fact, lipids related to various functions in the brain. Previous studies have indicated that phospholipids, particularly ones containing polyunsaturated fatty acyl residues, are deficient in postmortem brains from patients with schizophrenia. However, due to the difficulties in handling human postmortem brains, particularly the large size and complex structures of the human brain, there is little agreement regarding the qualitative and quantitative abnormalities of phospholipids in brains from patients with schizophrenia, particularly if corresponding brain regions are not used. In this study, to overcome these problems, we employed matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS), enabling direct microregion analysis of phospholipids in the postmortem brain of a patient with schizophrenia via brain sections prepared on glass slides. With integration of traditional histochemical examination, we could analyze regions of interest in the brain at the micrometric level. We found abnormal phospholipid distributions within internal brain structures, namely, the frontal cortex and occipital cortex. IMS revealed abnormal distributions of phosphatidylcholine molecular species particularly in the cortical layer of frontal cortex region. In addition, the combined use of liquid chromatography/electrospray ionization tandem mass spectrometry strengthened the capability for identification of numerous lipid molecular species. Our results are expected to further elucidate various metabolic processes in the neural system.

Dopamine neurons in the ventral tegmental area: an autopsy case of disorganized type of schizophrenia.

The mesolimbic dopamine (DA) system has been associated with the pathogenesis of schizophrenia. Here, we examined DA-containing neuronal structures of the ventral tegmental area (VTA) of an autopsy case of disorganized type of schizophrenia (75-year-old female), using tyrosine hydroxylase (TH) immunohistochemistry. A free floating method using 50-µm cryostat sections and three-dimensional imaging analyzer AxioVision were applied to observe the wide range structures of TH-immunoreactive (-ir) neurons. TH-ir neuronal cell bodies in the VTA of the present case had irregular shape and various size, and TH-ir neuronal processes had irregular thickness and straightened shape or curved shape having many corners, when compared to a control autopsy case with no detectable neurological and psychiatric diseases (64-year-old male). The mechanisms underlying the morphological characteristics of DA neurons of the brains with schizophrenia should be elucidated epigenetically as well as genetically.

Aromatic L-amino acid decarboxylase-immunoreactive structures in human midbrain, pons, and medulla.

The objective of the present study was to determine with precision the localization of neurons and fibers immunoreactive (ir) for aromatic L-amino acid decarboxylase (AADC), the second-step enzyme responsible for conversion of L-dihydroxyphenylalanine (L-DOPA) to dopamine (DA) and 5-hydroxytryptophan (5-HTP) to serotonin (5-hydroxytryptamine: 5-HT) in the midbrain, pons, and medulla oblongata of the adult human brain. Intense AADC immunoreactivity was observed in a large number of presumptive 5-HT neuronal cell bodies distributed in all of the raphe nuclei, as well as in regions outside the raphe nuclei such as the ventral portions of the pons and medulla. Moderate to strong immunoreaction was observable in presumptive DA cells in the mesencephalic reticular formation, substantia nigra, and ventral tegmental area of Tsai, as well as in presumptive noradrenergic (NA) cells, which were aggregated in the locus coeruleus and dispersed in the subcoeruleus nuclei. In the medulla oblongata, immunoreaction of moderate intensity was distributed in the mid and ventrolateral portions of the intermediate reticular nucleus, which constitutes the oblique plate of A1/C1 presumptive adrenergic and/or NA neurons. The dorsal vagal AADC-ir neurons were fewer in number and stained more weakly than cells immunoreactive for tyrosine hydroxylase (TH). AADC immunoreactivity was not identified in an aggregate of TH-ir neurons lying in the gelatinous subnucleus of the solitary nucleus, a restricted region just rostroventral to the area postrema. Nonaminergic AADC-positive neurons (D neurons), which are abundant in the rat and cat midbrain, pons, and medulla, were hardly detectable in homologous regions in the human brain, although they were clearly distinguishable in the forebrain.

A study of monoamine neuronal systems of schizophrenic patients: using forensic autopsy brains.

Studies on autopsy brains of mental illnesses are essential for psychiatric research. Brain materials from forensic autopsy cases have been valuable research resource. We introduced some characteristic neuronal structures of schizophrenic post-mortem brains and normal controls detected using both forensic and pathological autopsy cases and immunohistochemical method. Dopamine-related neuronal structures in schizophrenic cases were investigated by using immunohistochemistry for tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and phenylethanolamine-N-methyltransferase (PNMT). At the level of the striatum, the number of AADC-positive, but TH-negative neurons (D-neurons) reduced in schizophrenics compared with controls. In the brainstem structures, we described some morphological findings characteristic to catecholaminergic neurons of the ventral tegmental area of schizophrenic cases and reduction of the number of medullary catecholaminergic neurons. These results might be in accordance with recent reports describing neural stem cell dysfunction in schizophrenic brains. The authors also introduced the first systematic psychiatric brain bank in Japan, the Fukushima Psychiatric Brain Bank, constructed in 1997, and mentioned the necessity for arrangement of brain bank systems to further promote the psychiatric brain research.

Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1.

PURPOSE: Stable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity. METHODS: The seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species. RESULTS: Except for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1. CONCLUSIONS: Results in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

Significance of human striatal D-neurons: implications in neuropsychiatric functions.

The human striatum, especially its ventral part, the nucleus accumbens (Acc), contains numerous nonmonoaminergic aromatic L-amino acid decarboxylase (AADC) [=dopa decarboxylase (DDC)] neurons (D-neurons). AADC is the second-step synthesizing enzyme for monoamines and is also the rate-limiting enzyme of phenylethylamine (PEA) synthesis. D-neurons may participate in the manifestation of efficacy of pharmacotherapy for Parkinson's disease by taking up monoamine precursors including L-dopa or droxidopa (L-threo-DOPS) and by converting them to dopamine or noradrenaline, respectively. Although previous studies have shown that AADC activity was elevated in the striatum of drug-naive schizophrenia, the number of striatal D-neurons was reduced in autopsy brains of schizophrenia. It is unclear whether or not such reduction of striatal D-neurons implies downregulation. Possible pluripotentiality of D-neurons, including compensatory functions against aging and degeneration, was discussed based on recent published works.

Utilization of lectin-histochemistry in forensic neuropathology: lectin staining provides useful information for postmortem diagnosis in forensic neuropathology.

We have investigated the deposition of glycoconjugates in human brain tissue with or without brain disorders. In this review we describe the application of lectin-histochemistry techniques to forensic neuropathology. Lectin staining is able to reveal several kinds of carbohydrate-related depositions in addition to the conventional degenerative changes including senile plaques, neurofibrillary tangles and corpora amylacea. The senile plaques and neurofibrillary tangles were clearly stained by Con A, PSA and GSI lectins, the corpora amylacea which is relevant to repeated brain hypoxia and mitochondrial damage was also easily detected by these and many other kinds of lectins. Amorphous spaces were detected around blood vessels and independently from blood vessels by lectin staining in the white matter from patients with brain disorders or severe edema. The white matter lesions were not considered relevant for forensic pathology, until a large group of cerebral white matter lesions were detected in the elderly with increasing frequency by modern neuro-imaging methods. The spherical deposits were newly detected by lectin staining in the molecular layer of the dentate gyrus of the hippocampal formation chiefly from patients with schizophrenia or cognitive dysfunctions.

Effects of smoking and cytochrome P450 2D6*10 allele on the plasma haloperidol concentration/dose ratio.

OBJECTIVE: This study was carried out to evaluate the influence of CYP2D6 polymorphism and smoking on the plasma clearance of haloperidol (HAL) levels, accounting for the antipsychotic dose, body weight, and coadministration of other drugs. METHODS: Subjects were 110 Japanese patients (66 male, 44 female) diagnosed with schizophrenia, dementia, or mood disorder and treated orally with HAL. Venous blood was obtained from each patient to determine the HAL concentration/dose (C/D) ratio (plasma concentration of HAL divided by the daily dose of HAL per body weight) and for CYP2D6 genotyping. RESULTS: There was no significant difference in the HAL C/D ratio between nonsmokers and smokers. In patients with a non-2D6*10 homozygous genotype, smokers had a significantly lower HAL C/D ratio than nonsmokers, whereas smokers with a 2D6*10 homozygous genotype had a significantly higher HAL C/D ratio than those with a non-2D6*10 homozygous genotype. CONCLUSION: Our results suggest that the effect of smoking on the HAL C/D ratio depends on the CYP2D6*10 genotype.

Number of striatal D-neurons is reduced in autopsy brains of schizophrenics.

The human striatum, especially its ventral part, the nucleus accumbens, contains numerous neurons immunoreactive for aromatic L-amino acid decarboxylase (AADC, the second-step monoamine synthesizing enzyme, =DDC: dopa decarboxylase), but not for tyrosine hydroxylase (TH, the first-step catecholamine synthesizing enzyme) or tryptophan hydroxylase (TPH, the first-step serotonin synthesizing enzyme) (Neurosci Lett 232 (1997) 111-114). These AADC (+)/TH (-)/TPH (-) neurons are named as D-neurons (Jaeger CB, Ruggiero DA, Albert VR, Joh TH, Reis DJ. Immunocytochemical localization of aromatic-L-amino acid decarboxylase. In: Bjorklund A, Hokfelt T, editors. Classical transmission in the CNS, Part I, Handbook of chemical neuroanatomy, vol. 2. Amsterdam: Elsevier, 1984. pp. 387-418). The nucleus accumbens is one of the brain regions that is involved in the pathogenesis of schizophrenia. We examined the distribution of striatal D-neurons using AADC immunohistochemistry and postmortem brains obtained by legal and pathological autopsies (nine controls (27-75 years old) and nine schizophrenics (32-78 years old), postmortem interval to fixation (PMI): 2-30 h). Because the number of AADC-positive neurons per section had a tendency to reduce in the case with longer PMI, we analyzed specimens of five controls (27-64 years old) and six schizophrenics (51-78 years old) in which the PMI was less than 8 h. The number of AADC-positive neurons was reduced in the striatum of schizophrenics compared to that of controls. The reduction was significant in the nucleus accumbens (P<0.05, t-test). D-Neurons might be involved in the pathogenesis of schizophrenia. Further studies using sex-, age- and PMI-matched controls are essential.

Effects of age and the CYP2D6*10 allele on the plasma haloperidol concentration/dose ratio.

The authors studied the effect of aging and the CYP2D6*10 polymorphism on the plasma haloperidol (HAL) concentration after chronic administration of HAL. Subjects were 110 Japanese patients (66 male) treated orally with HAL. Venous blood was obtained from each patient for determination of the HAL concentration/dose (C/D) ratio (the plasma concentration of HAL divided by the daily dose of HAL per kilogram body weight) and for CYP2D6 genotyping. Overall, there was a significant linear correlation between the HAL C/D ratio and age. In subgroup analyses, the correlation was significant for patients with non-2D6*10 homozygous genotypes, but not for those with the 2D6*10 homozygous genotype. Overall, the HAL C/D ratio was significantly higher in older subjects (at least 50 years old) than younger ones (less than 50 years old). The ratio was significantly higher in older than in younger subjects for patients with non-2D6*10 homozygous genotypes, but not for those with the 2D6*10 homozygous genotype. Our results indicate that the effect of age on the HAL C/D ratio depends upon the CYP2D6*10 genotype. Because there are racial differences in the CYP2D6 genotype, further studies should investigate age effects on the HAL C/D ratio in different patient populations.

CYP2D6*10 alleles do not determine plasma fluvoxamine concentration/dose ratio in Japanese subjects.

OBJECTIVE: The purpose of the present study was to investigate whether plasma fluvoxamine (FV) concentration is associated with CYP2D6*10 allele polymorphisms. METHODS: Subjects were 46 Japanese patients (21 males) carrying neither *3, *4 nor *5 alleles and treated orally using FV. Venous blood was obtained from each patient for determination of FV concentration/dose (C/D) ratio (plasma concentration of FV divided by daily dose of FV per body weight) and CYP2D6 genotyping. RESULTS: No significant differences in FV C/D ratio were found between subjects with no (n=13), one (n=18) or two (n=15) *10 alleles. CONCLUSION: Our results indicate that CYP2D6*10 genotypes do not exert significant effects on FV C/D ratio. As CYP2D6 genotypes differ with ethnic background, further studies should be conducted in different populations.

Localization of sepiapterin reductase in the human brain.

Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR is essential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purified recombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein and SPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebral cortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPR antibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPR protein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I (GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistry revealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of the hypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPR were colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOS neuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4 biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4 biosynthesis.