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It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1(ΔPV) mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1(ΔPV) mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1(ΔPV)mice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1(ΔPV)mice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1(ΔPV)mice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease.

The GRIA1 locus, encoding the GluA1 (also known as GluRA or GluR1) AMPA glutamate receptor subunit, shows genome-wide association to schizophrenia. As well as extending the evidence that glutamatergic abnormalities have a key role in the disorder, this finding draws attention to the behavioural phenotype of Gria1 knockout mice. These mice show deficits in short-term habituation. Importantly, under some conditions the attention being paid to a recently presented neutral stimulus can actually increase rather than decrease (sensitization). We propose that this mouse phenotype represents a cause of aberrant salience and, in turn, that aberrant salience (and the resulting positive symptoms) in schizophrenia may arise, at least in part, from a glutamatergic genetic predisposition and a deficit in short-term habituation. This proposal links an established risk gene with a psychological process central to psychosis and is supported by findings of comparable deficits in short-term habituation in mice lacking the NMDAR receptor subunit Grin2a (which also shows association to schizophrenia). As aberrant salience is primarily a dopaminergic phenomenon, the model supports the view that the dopaminergic abnormalities can be downstream of a glutamatergic aetiology. Finally, we suggest that, as illustrated here, the real value of genetically modified mice is not as 'models of schizophrenia' but as experimental tools that can link genomic discoveries with psychological processes and help elucidate the underlying neural mechanisms.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes certain D-amino acids, notably the endogenous N-methyl D-aspartate receptor (NMDAR) co-agonist, D-serine. As such, it has the potential to modulate the function of NMDAR and to contribute to the widely hypothesized involvement of NMDAR signalling in schizophrenia. Three lines of evidence now provide support for this possibility: DAO shows genetic associations with the disorder in several, although not all, studies; the expression and activity of DAO are increased in schizophrenia; and DAO inactivation in rodents produces behavioural and biochemical effects, suggestive of potential therapeutic benefits. However, several key issues remain unclear. These include the regional, cellular and subcellular localization of DAO, the physiological importance of DAO and its substrates other than D-serine, as well as the causes and consequences of elevated DAO in schizophrenia. Herein, we critically review the neurobiology of DAO, its involvement in schizophrenia, and the therapeutic value of DAO inhibition. This review also highlights issues that have a broader relevance beyond DAO itself: how should we weigh up convergent and cumulatively impressive, but individually inconclusive, pieces of evidence regarding the role that a given gene may have in the aetiology, pathophysiology and pharmacotherapy of schizophrenia?

The NMDA receptor co-agonists D-serine and glycine are thought to contribute to glutamatergic dysfunction in schizophrenia. They are removed from the synapse by specific neuronal and glial transporters, the status of which is clearly relevant to theories of D-serine and glycine function in the disorder. D-serine is primarily transported by Asc-1, and glycine by GlyT1 but maybe also by SNAT2. As a first step to addressing this issue, we studied Asc-1, GlyT1 and SNAT2 expression in dorsolateral prefrontal cortex and cerebellum of 18 subjects with schizophrenia and 20 controls, using immunoblotting and in situ hybridization. Asc-1 protein and SNAT2 mRNA were decreased in schizophrenia in both regions. GlyT1 mRNA and protein, and Asc-1 mRNA, were not altered. Antipsychotic administration for 14 days did not alter expression of the genes in rat brain. Unchanged GlyT1 suggests that glycine transport is not markedly affected in schizophrenia, and therefore that increased synaptic removal is not the basis for the putative deficit in glycine modulation of NMDA receptors in the disorder. Lowered Asc-1 in schizophrenia implies that D-serine reuptake is reduced, perhaps as a response to decreased synaptic D-serine availability. However, this interpretation remains speculative. Further investigations will be valuable in the evaluation of these transporters as potential therapeutic targets in psychosis.

Group II metabotropic glutamate receptors (mGluRs) comprise mGluR2 (mGlu2; encoded by GRM2) and mGluR3 (mGlu3; encoded by GRM3) and modulate glutamate neurotransmission and synaptic plasticity. Here we review the expression and function of mGluR3 and its involvement in schizophrenia. mGluR3 is expressed by glia and neurons in many brain regions and has a predominantly presynaptic distribution, consistent with its role as an inhibitory autoreceptor and heteroceptor. mGluR3 splice variants exist in human brain but are of unknown function. Differentiation of mGluR3 from mGluR2 has been problematic because of the lack of selective ligands and antibodies; the available data suggest particular roles for mGluR3 in long-term depression, in glial function and in neuroprotection. Some but not all studies find genetic association of GRM3 polymorphisms with psychosis, with the risk alleles also being associated with schizophrenia-related endophenotypes such as impaired cognition, cortical activation and glutamate markers. The dimeric form of mGluR3 may be reduced in the brain in schizophrenia. Finally, preclinical findings have made mGluR3 a putative therapeutic target, and now direct evidence for antipsychotic efficacy of a group II mGluR agonist has emerged from a randomised clinical trial in schizophrenia. Together these data implicate mGluR3 in aetiological, pathophysiological and pharmacotherapeutic aspects of the disorder.

Alterations in the density or distribution of interstitial white matter neurons are taken as evidence in support of an early developmental component to schizophrenia. However, the existence and nature of interstitial white matter neuron changes in schizophrenia remain inconclusive. Recently, we reported that interstitial white matter neuron density is increased in the superficial white matter of the superior temporal gyrus in schizophrenia, but unchanged in deep white matter. This study extends our investigations to the dorsolateral prefrontal cortex and parahippocampal gyrus. Using the specific neuronal antibody NeuN, interstitial white matter neuron density was found to be increased in schizophrenia in the superficial white matter of the dorsolateral prefrontal cortex, with no significant changes elsewhere. As interstitial white matter neurons are presumed to be remnants of the embryonic cortical subplate, these findings provide additional evidence supportive of an early developmental abnormality in schizophrenia.

Synaptic protein gene expression is altered in schizophrenia. In the hippocampal formation there may be particular involvement of glutamatergic neurons and their synapses, but overall the profile remains unclear. In this in situ hybridization histochemistry (ISHH) study, we examined four informative synaptic protein transcripts: vesicular glutamate transporter (VGLUT) 1, VGLUT2, complexin I, and complexin II, in dorsolateral prefrontal cortex (DPFC), superior temporal cortex (STC), and hippocampal formation, in 13 subjects with schizophrenia and 18 controls. In these areas, VGLUT1 and complexin II are expressed primarily by excitatory neurons, whereas complexin I is mainly expressed by inhibitory neurons. In schizophrenia, VGLUT1 mRNA was decreased in hippocampal formation and DPFC, complexin II mRNA was reduced in DPFC and STC, and complexin I mRNA decreased in STC. Hippocampal VGLUT1 mRNA declined with age selectively in the schizophrenia group. VGLUT2 mRNA was not quantifiable due to its low level. The data provide additional evidence for a synaptic pathology in schizophrenia, in terms of a reduced expression of three synaptic protein genes. In the hippocampus, the loss of VGLUT1 mRNA supports data indicating that glutamatergic presynaptic deficits are prominent, whereas the pattern of results in temporal and frontal cortex suggests broadly similar changes may affect inhibitory and excitatory neurons. The impairment of synaptic transmission implied by the synaptic protein reductions may contribute to the dysfunction of cortical neural circuits that characterises the disorder.

This review critically summarizes the neuropathology and genetics of schizophrenia, the relationship between them, and speculates on their functional convergence. The morphological correlates of schizophrenia are subtle, and range from a slight reduction in brain size to localized alterations in the morphology and molecular composition of specific neuronal, synaptic, and glial populations in the hippocampus, dorsolateral prefrontal cortex, and dorsal thalamus. These findings have fostered the view of schizophrenia as a disorder of connectivity and of the synapse. Although attractive, such concepts are vague, and differentiating primary events from epiphenomena has been difficult. A way forward is provided by the recent identification of several putative susceptibility genes (including neuregulin, dysbindin, COMT, DISC1, RGS4, GRM3, and G72). We discuss the evidence for these and other genes, along with what is known of their expression profiles and biological roles in brain and how these may be altered in schizophrenia. The evidence for several of the genes is now strong. However, for none, with the likely exception of COMT, has a causative allele or the mechanism by which it predisposes to schizophrenia been identified. Nevertheless, we speculate that the genes may all converge functionally upon schizophrenia risk via an influence upon synaptic plasticity and the development and stabilization of cortical microcircuitry. NMDA receptor-mediated glutamate transmission may be especially implicated, though there are also direct and indirect links to dopamine and GABA signalling. Hence, there is a correspondence between the putative roles of the genes at the molecular and synaptic levels and the existing understanding of the disorder at the neural systems level. Characterization of a core molecular pathway and a 'genetic cytoarchitecture' would be a profound advance in understanding schizophrenia, and may have equally significant therapeutic implications.

Two main pieces of neurobiological evidence are adduced to support an early neurodevelopmental component to schizophrenia. Firstly, an abnormal distribution of neurons, especially interstitial white matter neurons (IWMNs). Secondly, decreased expression of reelin, a key developmental signalling molecule. Although influential, neither result is wholly established, and a possible link between them has not been examined. We addressed both issues, in superior temporal cortex, in 12 subjects with schizophrenia and 14 controls. The distribution and density of IWMNs, immunostained with the neuronal marker NeuN, was increased in the superficial white matter in schizophrenia (+16%; P=0.03). IWMN density in deep white matter was unaffected. Using in situ hybridization, reelin mRNA was found to be expressed by many IWMNs, layer I neurons, and scattered interneurons. Superficial IWMNs (P=0.008) and layer I neurons (P=0.036) both expressed less reelin mRNA per cell in schizophrenia, with a trend for deep IWMNs (P=0.055). In conclusion, we replicated findings of increased IWMN density, and of decreased reelin expression, in schizophrenia. The loss of reelin reflects, at least partly, its decreased expression by IWMNs. These findings together support neurodevelopmental theories of the disorder, and indicate a link between reelin and IWMNs in this process, consistent with evidence from the heterozygous reeler mutant mouse. The alterations may contribute to the aberrant synaptic connectivity seen in schizophrenia. However, the functional implications of the abnormalities, as well as the mechanisms involved, remain to be fully elucidated.

Four studies have reported that the mediodorsal nucleus of the thalamus (MD) is smaller and contains fewer neurons in schizophrenia. The MD is a key node in a circuit proposed to be dysfunctional in the disorder. However, one study did not find a MD volume loss in schizophrenia, and all the studies to date are relatively small. Given the importance of establishing unequivocally the presence of MD pathology, we have carried out a study of the volume and number of neurons in the left and right MD in 21 patients with schizophrenia and 27 healthy comparison subjects. We also measured the size of MD neurons, and estimated total thalamic volume. We found no difference in the volume of the MD, the number of MD neurons, or the size of MD neurons in either hemisphere in schizophrenia. Neither was total thalamic volume altered. There are no obvious methodological or clinical factors to explain our failure to replicate the finding of MD involvement in schizophrenia. Hence our negative observations, in the largest sample yet investigated, cast doubt on the robustness and/or the generalisability of MD neuropathology in schizophrenia.

The neuropathological features of schizophrenia are suggestive of a developmentally induced impairment of synaptic connectivity. Semaphorin 3A (sema3A) might contribute to this process because it is a secreted chemorepellant which regulates axonal guidance. We have investigated sema3A in the cerebellum (an area in which expression persists in adulthood), and measured its abundance in 16 patients with schizophrenia and 16 controls. In adults, sema3A was predominantly localized to the inner part of the molecular layer neuropil, whereas infants and rats showed greater labelling of Purkinje cell bodies. Sema3A was increased in schizophrenia, as shown by enzyme-linked immunosorbent assay (+28%; P<0.05) and immunohistochemistry (+45%; P<0.01). We also measured reelin mRNA, since reelin is involved in related developmental processes and is decreased in other brain regions in schizophrenia. Reelin mRNA showed a trend reduction in the subjects with schizophrenia (-26%; P=0.07) and, notably, was negatively correlated with sema3A. Sema3A also correlated negatively with synaptophysin and complexin II mRNAs. The results show that sema3A is elevated in schizophrenia, and is associated with downregulation of genes involved in synaptic formation and maintenance. In this respect, sema3A appears to contribute to the synaptic pathology of schizophrenia, perhaps via ongoing effects of persistent sema3A elevation on synaptic plasticity. The findings are consistent with an early neurodevelopmental origin for the disorder, and the reciprocal changes in sema3A and reelin may be indicative of a pathogenic mechanism that affects the balance between trophic and inhibitory factors regulating synaptogenesis.

5-Hydroxytryptamine (5-HT; serotonin) 5-HT(6) receptors (5-HT(6)R) and 5-HT(7) receptors (5-HT(7)R) have been implicated in schizophrenia and as targets of atypical antipsychotic drugs. We have studied the expression of these receptors in the hippocampal formation and dorsolateral prefrontal cortex (DLPFC) of 17 subjects with schizophrenia and 17 controls using reverse transcription-polymerase chain reaction (RT-PCR) with cyclophilin co-amplification. In schizophrenia, 5-HT(6)R mRNA was decreased in the hippocampal formation, and 5-HT(7)R mRNA was decreased in the dorsolateral prefrontal cortex. The mRNAs were unchanged in rats treated for 2 weeks with haloperidol, chlorpromazine, risperidone, olanzapine or clozapine. Regional decreases in 5-HT(6)R and 5-HT(7)R expression in schizophrenia may contribute to the overall serotonergic alterations which occur in the disorder, in part through their interactions with other neurotransmitter systems including glutamate and acetylcholine., (Copyright 2002 Elsevier Science B.V.)

The occurrence of human cerebellar serotonin 5-HT(2A) receptors (5-HT(2A)R) is equivocal and their status in schizophrenia unknown. Using a range of techniques, we investigated cerebellar 5-HT(2A)R expression in 16 healthy subjects and 16 subjects with schizophrenia. Immunocytochemistry with a monoclonal antibody showed labelling of Purkinje cell bodies and dendrites, as well as putative astrocytes. Western blots showed a major band at approximately 45 kDa. Receptor autoradiography and homogenate binding with [(3)H]ketanserin revealed cerebellar 5-HT(2A)R binding sites present at levels approximately a third of that in prefrontal cortex. 5-HT(2A)R mRNA was detected by reverse transcriptase-polymerase chain reaction, with higher relative levels in men than women. Several aspects of 5-HT(2A)R expression were altered in schizophrenia. 5-HT(2A)R immunoreactivity in Purkinje cells was partially redistributed from soma to dendrites and was increased in white matter. 5-HT(2A)R mRNA was decreased in the male patients. 5-HT(2A)R measured by dot blots and [(3)H]ketanserin binding (B(max) and K(d)) were not significantly altered in schizophrenia. These data show that 5-HT(2A)R gene products (mRNA, protein, binding sites) are expressed in the human cerebellum at nonnegligible levels; this bears upon 5-HT(2A)R imaging studies which use the cerebellum as a reference region. 5-HT(2A)R expression is altered in schizophrenia; the shift of 5-HT(2A)R from soma to dendrites is noteworthy since atypical antipsychotics have the opposite effect. Finally, the results emphasise that expression of a receptor gene is a mutifaceted process. Measurement of multiple parameters is necessary to give a clear picture of the normal situation and to show the profile of alterations in a disease., (Copyright 2001 Wiley-Liss, Inc.)

There are several reports of ultrastructural and protein changes affecting synapses in the anterior cingulate cortex in schizophrenia. Altered cytoarchitecture has also been described in this region in schizophrenia as well as in mood disorders. In this paper we review the literature and present a new study investigating synaptic abnormalities in the anterior cingulate cortex (area 24) in the Stanley Foundation brain series. We used Western blotting to assess four synaptic proteins: synaptophysin, growth-associated protein-43 (GAP-43), complexin I and complexin II, which inform about somewhat different aspects of the synaptic circuitry. Synaptophysin, complexin II and GAP-43 were reduced in bipolar disorder. The decreases correlated with the duration of illness and tended to be greater in subjects without a family history. Complexin II was also reduced in major depression. Complexin I and the housekeeping protein beta-actin did not differ between groups. None of the proteins changed significantly in schizophrenia. The results indicate the presence of a synaptic pathology in the anterior cingulate cortex in mood disorders, especially bipolar disorder. The abnormalities may contribute to the dysfunction of cingulate neural circuits. The loss of synaptophysin is suggestive of decreased synaptic density whilst the decrease in GAP-43 may denote impaired synaptic plasticity and the reduction of complexin II but not complexin I implies that the alterations particularly affect excitatory connections. The reductions may be progressive.

5-HT(2C) receptor (5HT(2C)R, serotonin-2C) RNA undergoes editing to produce several receptor variants, some with pharmacological differences. This investigation comprised two parts: the characterisation of 5-HT(2C)R RNA editing in a larger human control sample than previously examined, and a comparative study in subjects with schizophrenia. Secondary structure analysis of the putative edited region of the human 5-HT(2C)R gene predicted the existence of a double stranded (ds) RNA loop, essential for RNA editing in this receptor. RNA was then extracted from frontal cortex of five controls and five subjects with schizophrenia. RT-PCR products of the edited region were cloned and sequenced (n = 100). Reduced RNA editing, increased expression of the unedited 5-HT(2C-INI) isoform in schizophrenia (P = 0.001) and decreased expression of the 5-HT(2C-VSV) and 5-HT(2C-VNV) isoforms were detected in the schizophrenia group. In addition, two novel mRNA edited variants were identified: 5-HT(2C-MNI) and 5-HT(2C-VDI). Screening of the 5-HT(2C)R gene did not reveal any mutations likely to disrupt the dsRNA loop, suggesting that the reduced RNA editing in schizophrenia may instead be caused by altered activity of the editing enzyme(s). Since the unedited 5-HT(2C-INI) is more efficiently coupled to G proteins than the other isoforms, its increased expression in schizophrenia may lead to enhanced 5-HT(2C)R-mediated effects. The results also illustrate that potentially important receptor alterations may occur in schizophrenia which are not detectable merely in terms of receptor abundance.

Background: It has been suggested that there is frontal lobe involvement in schizophrenia, and that it may be lateralised and gender-specific., Aims: To clarify the structure of the frontal lobes in schizophrenia in a post-mortem series., Method: The volume of white matter and cortical components of the frontal lobes was measured in brains of controls and patients with schizophrenia using planimetry and the Cavalieri principle. The components measured were: superior frontal gyrus, middle frontal gyrus, a composite of inferior frontal gyrus and orbito-frontal cortex, as well as total frontal lobe cortex and white matter. In addition, the anterior cingulate gyrus was measured., Results: No diagnosis, gender, diagnosis x side, diagnosis x gender or diagnosis x gender x side interactions were observed in the volume of any of the components, the grey matter as a whole or the white matter. No evidence for volumetric inter-group differences was found for the anterior cingulate gyrus., Conclusions: Such structural abnormalities as are present in the frontal lobes are more subtle than straightforward alterations in tissue volume; they may include changes in shape and the pattern of gyral folding.

A cortico-subcortico-cerebellar neural circuit has been postulated to be important in the pathophysiology of schizophrenia. This study investigated whether there are synaptic changes in the cerebellum to accompany its putative involvement in the disorder. We measured the expression of three synaptic proteins (synaptophysin, complexin I and complexin II) in the cerebellar cortex of 16 subjects with schizophrenia and 16 controls using in situ hybridisation histochemistry and immunoautoradiography. Complexin I and II are expressed predominantly by inhibitory and excitatory neurones respectively. In schizophrenia, synaptophysin mRNA was decreased, as was complexin II and its mRNA. Complexin I mRNA and protein levels were unaltered. Expression of the mRNAs in the rat cerebellum was unaffected by 2 weeks administration of antipsychotic drugs (haloperidol, chlorpromazine, risperidone, olanzapine or clozapine). We conclude that there is synaptic pathology in the cerebellum in schizophrenia. By disrupting neural circuits, the alterations may contribute to the cerebellar dysfunction thought to occur in the disorder.

Cytoarchitectural changes in the hippocampal formation have been prominent among the various neuropathological abnormalities reported in schizophrenia. Replicated positive findings include decreased neuronal size and alterations in presynaptic and dendritic markers. These findings, in the absence of neurodegenerative changes, suggest that there are alterations in the neural circuitry in schizophrenia. These may represent the anatomical correlate of the aberrant functional connectivity described in neuroimaging studies, which in turn contributes to the psychotic and cognitive symptomatology of the disorder. The identity of the affected hippocampal circuits remains unclear; there is evidence for both glutamatergic and GABAergic involvement, and perhaps for a gradient of pathology in which changes are most apparent in CA4 and the subiculum, and least in CA1. The data, their interpretation, and their limitations are discussed, with particular emphasis upon molecular and immunological studies of synaptic protein gene expression.

Complexin (cx) I and cx II are synaptic proteins preferentially expressed by inhibitory and excitatory hippocampal neurons respectively. We previously reported decreased hippocampal formation cx mRNA and protein expression in schizophrenia, with a greater loss of cx II than cx I. The present in situ hybridization study was both an attempt at replication, and an extension to include bipolar and unipolar mood disorders, using sections from the Stanley Foundation brain series. In schizophrenia, both mRNAs were decreased in some hippocampal subfields, especially CA4, but were preserved in subiculum. The cx II/cx I mRNA ratio was unchanged. In bipolar disorder, the mRNAs were reduced in CA4, subiculum and parahippocampal gyrus, with the deficit in subiculum being diagnostically specific. No alterations in cx mRNAs were found in major depression. Treatment of rats with antipsychotics (haloperidol or chlorpromazine) for 2 weeks had no effect on hippocampal cx mRNAs. These data replicate the finding of decreased cx I and cx II expression in the hippocampus in schizophrenia and show a similar or greater abnormality in bipolar disorder. Non-replication of the cx II > cx I mRNA loss in schizophrenia means that the hypothesis of a preferential involvement of excitatory connections was not supported. The results extend the emerging evidence that altered circuitry may be a component of the neuroanatomy of both schizophrenia and bipolar mood disorder.

Background: Decreased expression of proteins such as synaptophysin in the hippocampus and prefrontal cortex in schizophrenia is suggestive of synaptic pathology. However, the overall profile of changes is unclear., Aims: To investigate synaptophysin gene expression in the cerebral cortex in schizophrenia., Method: The dorsolateral prefrontal (Brodmann area [BA] 9/46), anterior cingulate (BA 24), superior temporal (BA 22) and occipital (BA 17) cortex were studied in two series of brains, totalling 19 cases and 19 controls. Synaptophysin was measured by immunoautoradiography and immunoblotting. Synaptophysin messenger RNA (mRNA) was measured using in situ hybridisation., Results: Synaptophysin was unchanged in schizophrenia, except for a reduction in BA 17 of one brain series. Synaptophysin mRNA was decreased in BA 17, and in BA 22 in the women with schizophrenia. No alterations were seen in BA 9/46., Conclusions: Synaptophysin expression is decreased in some cortical areas in schizophrenia. The alterations affect the mRNA more than the protein, and have an unexpected regional distribution. The characteristics of the implied synaptic pathology remain to be determined.

Background: The substance P receptor (neurokinin-1 receptor) has been implicated in stress responses and anxiety traits in the rodent, and neurokinin-1 receptor antagonism may have antidepressant and anxiolytic effects. This suggests that the function and/or expression of neurokinin-1 receptor might be affected in subjects with mood disorders., Methods: We measured neurokinin-1 receptor densities in the anterior cingulate cortex in subjects with unipolar (major) depression (n = 13), bipolar disorder (n = 13), schizophrenia (n = 14), and controls (n = 14) using quantitative autoradiography with [125I]BH-substance P. The anterior cingulate cortex was chosen for initial analysis since recent positron emission tomography, magnetic resonance imaging, and neuropathological data suggest its involvement in mood disorders., Results: Neurokinin-1 receptor densities were higher in superficial than in deep laminae. Neurokinin-1 receptor densities increased with age and declined with prolonged autopsy interval. No differences were seen between the four groups. However, the ratio of superficial to deep laminar binding was lower in the subjects with unipolar depression compared with all other groups (p < .01) Neurokinin-1 receptor binding and the laminar ratio were unaffected by sex, medication history, pH, suicide, comorbid substance abuse, or a family psychiatric history., Conclusions: No overall change in neurokinin-1 receptor densities occurs in the cingulate cortex in subjects with mood disorders or schizophrenia. However, the changed laminar ratio in unipolar depression may reflect alterations in specific neural circuits expressing neurokinin-1 receptor.

In addition to their neurochemical effects, antipsychotic (neuroleptic) drugs produce structural brain changes. This property is relevant not only for understanding the drugs' mode of action, but because it complicates morphological studies of schizophrenia. Here the histological neuropathological effects of antipsychotics are reviewed, together with brief mention of those produced by other treatments sometimes used in schizophrenia (electroconvulsive shock, lithium and antidepressants). Most data come from drug-treated rats, though there are also some human post-mortem studies with broadly congruent findings. The main alteration associated with antipsychotic medication concerns the ultrastructure and proportion of synaptic subpopulations in the caudate nucleus. In rats, synapses and dendrites in lamina VI of the prefrontal cortex are also affected. The changes are indicative of a drug-induced synaptic plasticity, although the underlying mechanisms are poorly understood. Similarly, it is unclear whether the neuropathological features relate primarily to the therapeutic action of antipsychotics or, more likely, to their predisposition to cause tardive dyskinesia and other motor side-effects. Clozapine seems to cause lesser and somewhat different alterations than do typical antipsychotics, albeit based on few data. There is no good evidence that antipsychotics cause neuronal loss or gliosis, nor that they promote neurofibrillary tangle formation or other features of Alzheimer's disease.

Despite a hundred years' research, the neuropathology of schizophrenia remains obscure. However, neither can the null hypothesis be sustained--that it is a 'functional' psychosis, a disorder with no structural basis. A number of abnormalities have been identified and confirmed by meta-analysis, including ventricular enlargement and decreased cerebral (cortical and hippocampal) volume. These are characteristic of schizophrenia as a whole, rather than being restricted to a subtype, and are present in first-episode, unmedicated patients. There is considerable evidence for preferential involvement of the temporal lobe and moderate evidence for an alteration in normal cerebral asymmetries. There are several candidates for the histological and molecular correlates of the macroscopic features. The probable proximal explanation for decreased cortical volume is reduced neuropil and neuronal size, rather than a loss of neurons. These morphometric changes are in turn suggestive of alterations in synaptic, dendritic and axonal organization, a view supported by immunocytochemical and ultrastructural findings. Pathology in subcortical structures is not well established, apart from dorsal thalamic nuclei, which are smaller and contain fewer neurons. Other cytoarchitectural features of schizophrenia which are often discussed, notably entorhinal cortex heterotopias and hippocampal neuronal disarray, remain to be confirmed. The phenotype of the affected neuronal and synaptic populations is uncertain. A case can be made for impairment of hippocampal and corticocortical excitatory pathways, but in general the relationship between neurochemical findings (which centre upon dopamine, 5-hydroxytryptamine, glutamate and GABA systems) and the neuropathology of schizophrenia is unclear. Gliosis is not an intrinsic feature; its absence supports, but does not prove, the prevailing hypothesis that schizophrenia is a disorder of prenatal neurodevelopment. The cognitive impairment which frequently accompanies schizophrenia is not due to Alzheimer's disease or any other recognized neurodegenerative disorder. Its basis is unknown. Functional imaging data indicate that the pathophysiology of schizophrenia reflects aberrant activity in, and integration of, the components of distributed circuits involving the prefrontal cortex, hippocampus and certain subcortical structures. It is hypothesized that the neuropathological features represent the anatomical substrate of these functional abnormalities in neural connectivity. Investigation of this proposal is a goal of current neuropathological studies, which must also seek (i) to establish which of the recent histological findings are robust and cardinal, and (ii) to define the relationship of the pathological phenotype with the clinical syndrome, its neurochemistry and its pathogenesis.

Most in situ hybridization histochemistry studies of messenger RNA in human brain have been carried out on frozen tissue. Recently, autoclaving has been reported to enable routinely processsed material to be used for in situ localization of messenger RNA. We have investigated whether autoclaving also permits in situ hybridization histochemistry to be used quantitatively. To do this, we targeted synaptophysin messenger RNA with a 35S-labelled oligonucleotide probe in autoclaved, formalin-fixed, paraffin wax-embedded sections of the hippocampal formation of 11 schizophrenics and 11 controls. We compared the results with those seen on frozen sections from adjacent blocks, which had been used previously to demonstrate a loss of the messenger RNA in schizophrenia. Synaptophysin messenger RNA was readily detected in the autoclaved sections. The hybridization signal correlated strongly with that seen in the frozen sections. We found a similar pattern and magnitude of decreased synaptophysin messenger RNA in schizophrenia in the autoclaved sections as we had in the frozen sections, including the selective preservation of synaptophysin messenger RNA in CA1. The reduction of synaptophysin messenger RNA was replicated when six subjects with schizophrenia not included in the earlier study were considered separately. We conclude that autoclaving renders formalin-fixed, paraffin wax-embedded sections of human brain suitable for quantitative in situ hybridization histochemistry. This has considerable implications, given the wider availability, better morphology and easier handling of fixed than frozen human brain tissue. Using this material, we confirmed the finding of decreased synaptophysin messenger RNA in the hippocampal formation in schizophrenia, furthering the evidence for synaptic pathology in this region in the disorder.

Background: The anatomical basis of schizophrenia involves the cytoarchitecture of the cerebral cortex, but the phenotype of the affected neurons and synapses remains unclear. In mice, the presynaptic protein complexin I is a marker of axosomatic (inhibitory) synapses, whereas complexin II is a marker of axodendritic (mainly excitatory) synapses. These findings suggest that the complexins might be useful in the investigation of the synaptic pathology of schizophrenia., Methods: We characterised the expression of the complexins in tissue taken at necropsy from human medial temporal lobe (hippocampus, parahippocampal gyrus) and cerebellum using in-situ hybridisation and immunoautoradiography. We then measured the concentrations of the complexins and their messenger RNAs (mRNAs) in the medial temporal lobe of 11 patients with schizophrenia and 11 non-schizophrenic controls., Findings: The distribution of complexin I and II was consistent with the data on mice, with predominant expression of complexin I by inhibitory neurons, and complexin II by excitatory neurons. The amounts of both complexin mRNAs were lower in schizophrenic than in control patients (p<0.001), but the difference of complexin II mRNA was greater. The amount of complexin I protein was unchanged in schizophrenia, but complexin II protein was decreased (p<0.001). For both mRNA and protein, the complexin II/complexin I ratio was lower in schizophrenia, confirming the relatively greater loss of the excitatory marker. The findings did not seem attributable to medication., Interpretation: The synaptic pathology of schizophrenia, at least in medial temporal lobe, primarily affects excitatory (glutamatergic) neurons. The inferred imbalance between excitatory and inhibitory circuitry may contribute to the involvement of this region in the pathophysiology of the disorder.

Growth-associated protein-43 is involved in maturational and plasticity-associated processes, and changes in growth-associated protein-43 expression are a marker of altered plasticity following experimental and neuropathological lesions. Using in situ hybridization, we have investigated growth-associated protein-43 mRNA in the medial temporal lobe and cerebral cortex in 11 normal subjects and 11 matched subjects with schizophrenia, a disorder in which perturbed neurodevelopment and aberrant plasticity are implicated. In the schizophrenia group, growth-associated protein-43 messenger RNA was decreased in the medial temporal lobe, primary visual cortex and anterior cingulate gyrus, but was unaltered in the superior temporal and dorsolateral prefrontal cortices. Correlations of growth-associated protein-43 messenger RNA signal between areas were stronger and more numerous in the schizophrenics than in the controls, suggesting a more global regulation of growth-associated protein-43 expression. Finally, the ratio of growth-associated protein-43 messenger RNA to synaptophysin messenger RNA--a putative index of the production of new synapses--was decreased in the medial temporal lobe in the schizophrenics. Our findings imply that neuronal plasticity as indexed by growth-associated protein-43 expression is impaired, and perhaps aberrantly regulated, in schizophrenia. The data support the emerging view that the disease pathophysiology is one which affects the hippocampal and cortical circuitry and that the abnormalities are reflected in the altered expression of specific neuronal genes.

The abundance of polyadenylated messenger RNA (poly(A)+mRNA) reflects overall gene expression and provides an index on neuronal activity. Poly(A)+mRNA was measured in the hippocampal formation and in occipital, temporal, cingulate and frontal cortices (Brodmann areas 17, 22, 24 and 46, respectively) of 11 schizophrenic and 17 control subjects post mortem by in situ hybridization histochemistry with a 35S-oligodeoxythymidine probe. There were no differences in poly(A)+mRNA between cases and controls, except for a modest decrease in the parahippocampal gyrus of the schizophrenics which may be attributable to cytoarchitectural differences in this area in the disease. The unchanged level of poly(A)+mRNA in all other regions argues against the existence of a widespread or sustained alteration in the metabolic activity of cortical neurons in schizophrenia. It also provides a further indication that the differential expression of individual transcripts reported in the disease is not merely reflecting changes in overall gene expression.

Objective: Schizophrenia may involve the two cerebral hemispheres differentially. This study was conducted to determine whether left and right hippocampal neuronal size, shape, and orientation are normally asymmetrical or asymmetrically affected in schizophrenia., Method: The authors examined postmortem tissue from the left and right hippocampus of 17 normal individuals and 14 individuals with schizophrenia. They measured the size, shape, and variability in orientation of pyramidal neurons in hippocampal subfields CA1-CA4 and the subiculum in computer images of 10-micron coronal sections stained with cresyl violet., Results: Both neuronal size and shape showed significant effects of diagnosis and a three-way interaction between diagnosis, hemisphere, and subfield. Neurons of the schizophrenic subjects were smaller than those of the normal subjects in the left CA1, left CA2, and right CA3 subfields; their shape differed from that of the normal subjects in the left CA1, left subiculum, and right CA3 subfields. There were no group differences in variability of neuronal orientation, but neurons in the CA3 genu in the schizophrenic subjects were less variable on the right than on the left. In the normal subjects, except for larger neurons in the left than in the right CA2 subfield and some left-right differences in variability of neuronal orientation, no statistically significant asymmetries were observed., Conclusions: The data confirm that hippocampal neuronal size is decreased in schizophrenia and reveal that the shape of neurons is altered, supporting the view that hippocampal cytoarchitectural abnormalities may be part of the cerebral substrate of schizophrenia. They also provide further evidence that the abnormalities are localized and lateralized.

WAY-100635 is the first selective, silent 5-HT1A (5-hydroxytryptamine1A, serotonin-1A) receptor antagonist. We have investigated the use of [3H]WAY-100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5-HT1A receptor agonist, [3H]8-OH-DPAT. Saturation studies showed an average Kd for [3H]WAY-100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [3H]WAY-100635 binding and [3H]8-OH-DPAT binding were similar. The density of [3H]WAY-100635 binding sites was 60-70% more than that of [3H]8-OH-DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [3H]WAY-100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [3H]WAY-100635, we confirmed an increase of 5-HT1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [3H]8-OH-DPAT. Compared to [3H]8-OH-DPAT, [3H]WAY-100635 has two advantages: it has a higher selectivity and affinity for the 5-HT1A receptor, and it recognizes 5-HT1A receptors whether or not they are coupled to a G-protein, whereas [3H]8-OH-DPAT primarily detects coupled receptors. Given these considerations, the [3H]WAY-100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [3H]8-OH-DPAT binding seen in the same cases is attributable to an increase of 5-HT1A receptors rather than any other binding site. Second, the enhanced [3H]8-OH-DPAT binding in schizophrenia reflects an increased density of 5-HT1A receptors, not an increased percentage of 5-HT1A receptors which are G-protein-coupled. We conclude that [3H]WAY-100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5-HT1A receptors in the human brain.

Background: The histological basis of schizophrenia is unknown, but it appears to affect the hippocampal and neocortical cytoarchitecture. Some cytoarchitectural parameters normally differ between the two cerebral hemispheres. Moreover, schizophrenia is associated with altered structural cerebral asymmetry. However, few cytoarchitectural studies of schizophrenia have taken the question of asymmetry fully into account., Methods: We performed a morphometric post mortem study of neuronal density in sections from the left and right hippocampus (dentate gyrus, CA4, CA3, CA1 and subiculum) of 22 schizophrenics and 18 normal subjects. We also determined the correlations of neuronal density between pairs of subfields as an index of their inter-relationship; a previous study had found correlations in the left but not the right hippocampus of normal subjects., Results: There were three differences in the schizophrenics compared to the controls. (1) neuronal density was increased in right CA3 (by 25%) and right CA1 (by 22%); (2) neuronal density correlated strongly between homologous left and right subfields (i.e. inter-hippocampally) for CA4, CA3, CA1 and subiculum, in normals this occurs only for dentate gyrus and CA4; and (3) intrahippocampal correlations of neuronal density between pairs of subfields were similar in both hippocampi of the schizophrenia cases, unlike their asymmetrical distribution in controls., Conclusions: The alterations may be part of the histological substrate of schizophrenia. The nature of the findings is consistent with a neurodevelopmental origin, and with a disease process that affects cerebral asymmetry and leaves its imprint upon the hippocampal cytoarchitecture.

For the first time, there is a pathogenic hypothesis of schizophrenia based upon reasonable empirical data. The hypothesis is that schizophrenia is a disorder arising from aberrant brain development. The neurodevelopmental view of schizophrenia is supported by neuropathological, epidemiological and clinical findings. Here, the evidence in favour of the model is summarized, together with a consideration of its weaknesses.

The mRNAs encoding kainic acid (KA) preferring glutamate receptor subunits (GluR5-7, KA1 and KA2) are differentially expressed in rat brain. We have used regional and cellular in situ hybridization histochemistry with subunit-specific 35S-labelled oligodeoxyribonucleotides to examine these mRNAs in adult human hippocampus, neocortex and cerebellum. GluR5 mRNA was detected only in Purkinje cells and a few scattered hippocampal neurons. GluR6 mRNA was relatively abundant in all areas, notably in dentate gyrus, pyramidal neurons of CA3, and cerebellar granule cells, as well as being present in superficial and deep laminae of the neocortex. Moderate signal for GluR7 mRNA was seen in deep laminae of the neocortex with a weak signal in the dentate gyrus; in dipped sections GluR7 mRNA was also apparent over some pyramidal and non-pyramidal cells in hippocampus and over putative cerebellar stellate/basket cells. KA1 mRNA was detected in the dentate gyrus but not reliably elsewhere. The expression profile and abundance of KA2 mRNA was similar to that of GluR6 mRNA. For all five transcripts, concurrent hybridization of rat brain sections produced the anticipated distribution of signal. The data indicate that the regional and cellular distribution of KA receptor subunit mRNAs in human hippocampus, neocortex and cerebellum largely parallels that in the corresponding areas of rat brain, albeit at lower levels, especially with regard to GluR5 and KA1 transcripts. In schizophrenia there is a partial loss of hippocampal non-NMDA receptors, but there are no data concerning KA receptor subunit expression. KA2 and GluR6 mRNAs were sufficiently abundant for a comparison in the left and right hippocampus between 11 schizophrenics and 13 controls. Using film autoradiography, both mRNAs were significantly reduced in the schizophrenics, having controlled for the effects of brain pH, post mortem interval and age. GluR6 mRNA was also quantitated in cerebellum, wherein no differences were found between cases and controls. In conjunction with earlier findings of reduced hippocampal GluR1 and GluR2 expression and a loss of [3H]KA binding sites, these data show that schizophrenia is associated with impaired expression of both AMPA- and KA-preferring ionotropic glutamate receptors. These deficits are likely to contribute to the glutamatergic component of the disease pathophysiology.

Decreased expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-preferring non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) occurs in the medial temporal lobe of schizophrenics in terms of reduced abundance of GluR1 and GluR2 subunit mRNAs. To investigate further these receptors in schizophrenia, we have performed a quantitative immunoautoradiographic study in medial temporal lobe sections of 11 schizophrenics and 10 well-matched controls. GluR1 and GluR2/3 were detected with polyclonal antisera coupled to 35S-labeled secondary antibodies. Both subunits were vulnerable to a prolonged postmortem interval and poor agonal state as indicated by brain pH. GluR1 also tended to decline with increasing age. These factors were therefore used as covariates. GluR1 abundance was reduced in schizophrenics in parahippocampal gyrus (p < .025), while GluR2/3 was lower in most subfields in the schizophrenics, significantly so in CA4 (p < .02). The present data extend the evidence for decreased expression of the AMPA subtype of non-NMDA receptors in the medial temporal lobe in schizophrenia, although the magnitude and spatial extent of the loss is smaller than that affecting the encoding mRNAs. Impaired AMPA receptor expression is consistent with a neurodevelopmental origin and with hypotheses of glutamatergic hypofunction in the disease; however, its true pathophysiological significance and relationship to the other neuropathological and pathochemical abnormalities in the medial temporal lobe in schizophrenia remain to be determined.

GluR2 is the key subunit of heteromeric AMPA-preferring glutamate receptors. GluR2 mRNA has been shown by in situ hybridization histochemistry to be decreased in the hippocampal formation in schizophrenics. Here, a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to investigate GluR2 expression further and to examine the relative abundance of its alternatively spliced mRNA isoforms ('flip' and 'flop') in 11 schizophrenics and 11 matched controls. Compared to the controls, schizophrenics showed reduced expression of both isoforms relative to cyclophilin mRNA, but a greater loss of the flop isoform led to a higher flip:flop ratio. These differences were observed having controlled for the confounding effects of brain pH and age upon the mRNAs. We also found that the abundance of GluR2 mRNA correlates with that of the encoded subunit. This study has confirmed that, in schizophrenia, hippocampal GluR2 mRNA is reduced, and indicates that GluR2 subunits are composed of a higher proportion of the flip variant. These data extend the evidence for glutamatergic dysfunction in the disease. They suggest that signal transduction through hippocampal AMPA receptors is impaired in schizophrenia both by an overall loss of GluR2 expression, and by the change in flip:flop ratio which is predicted to alter the desensitization kinetics of the remaining GluR2 subunits.

We have investigated 5-HT1A (serotonin1A) and 5-HT2A (serotonin2A) receptor mRNA abundance and binding site densities in various neocortical and hippocampal regions of schizophrenics and control subjects. Age, agonal state (brain pH), and post mortem interval were included where necessary as covariates in our analyses. In schizophrenics, 5-HT1A binding site densities, determined autoradiographically by [3H]8-hydroxy-2,3-(dipropylamino)-tetralin ([3H]8-OH-DPAT), were significantly increased (+23%) in the dorsolateral prefrontal cortex, with a similar trend in anterior cingulate gyrus. These increases were not accompanied by any change in 5-HT1A receptor mRNA. No differences between the groups in [3H]8-OH-DPAT binding or 5-HT1A receptor mRNA were seen in superior temporal gyrus, striate cortex, or hippocampus. 5-HT2A binding sites, determined by [3H]ketanserin, were decreased in the dorsolateral prefrontal cortex (-27%) and parahippocampal gyrus (-38%) of schizophrenics, with a similar trend in cingulate gyrus, but not in superior temporal gyrus or striate cortex. 5-HT2A receptor mRNA abundance was reduced in schizophrenics in the dorsolateral prefrontal (-49%), superior temporal (-48%), anterior cingulate (-63%) and striate (-63%) cortices, but not in parahippocampal gyrus. Parallel analyses of rat brain tissue showed no changes in 5-HT1A or 5-HT2A receptor mRNAs or binding site densities after chronic administration of haloperidol. These data show that schizophrenia is associated with alterations in the expression of central 5-HT1A and 5-HT2A receptors. They confirm reports of increased 5-HT1A and decreased 5-HT2A binding site densities in prefrontal cortex, and reveal more extensive decreases in 5-HT2A receptor gene expression at the mRNA level. The resulting imbalance in the 5-HT1A to 5-HT2A receptor ratio, when considered in terms of the chemoarchitectural distribution of these receptors, may contribute to an impairment of corticocortical association pathways. The apparent dissociation of the normal relationships between the abundance of each 5-HT receptor and its mRNA in schizophrenia introduces a separate complexity to the data, which may give clues to the underlying molecular mechanisms.

Synaptic alterations have been suggested, largely on theoretical grounds, to occur in the brain in schizophrenia. The messenger RNA encoding synaptophysin, a presynaptic terminal protein, is reduced in the medial temporal lobe in the disease, but immunocytochemical and immunoblotting data have not produced clear evidence for a loss of the encoded protein. Here we have used immunoautoradiography with an antisynaptophysin monoclonal antibody and a 35S-labelled secondary antibody in medial temporal lobe sections from 11 schizophrenics and 14 matched controls. In the schizophrenic cases there was an overall loss of synaptophysin (P < 0.02). Analysis by subfield showed significant reductions in the right dentate gyrus molecular layer, subiculum and parahippocampal gyrus, with similar trends in most other subfields. These data confirm that synaptophysin expression is decreased within the medial temporal lobe in schizophrenia. In the respect that synaptophysin is a marker of synaptic density, our findings suggest that reduced synaptic density may be a feature of the molecular neuropathology of the disease.