Abstract

Schizophrenia has been associated with anatomical and functional abnormalities of the dorsolateral prefrontal cortex (DLPFC), which may reflect abnormal connections of DLPFC neurons. We measured mRNA levels of growth-associated protein (GAP-43), a peptide linked to the modifiability of neuronal connections, in post-mortem brain tissue from two cohorts of patients with schizophrenia and controls. Using the RNase protection assay (RPA), we found a significant reduction in GAP-43 mRNA in the DLPFC, but not in the hippocampus, of patients with schizophrenia. With in situ hybridization histo- chemistry (ISHH), performed on a separate cohort, we confirmed the reduction of GAP-43 mRNA in the DLPFC of patients with schizophrenia. We detected reduced GAP-43 mRNA per neuron in layers III, V and VI of patients with schizophrenia compared with normal controls and patients with bipolar disorder. Thus, glutamate neurons in DLPFC of schizophrenic patients may synthesize less GAP-43, which could reflect fewer and/or less modifiable connections than those in normal human brain, and which may be consistent with the deficits of prefrontal cortical function that characterize schizophrenia.

Introduction

There is considerable evidence that schizophrenia is associated with anatomical and functional abnormalities of the dorsolateral prefrontal cortex (DLPFC) (Goldman-Rakic, 1991; Weinberger, 1993; Lewis, 1997; Weickert and Kleinman, 1998). Clinically, the cognitive problems of patients with schizophrenia resemble those of patients with prefrontal brain lesions and include reduced mental flexibility and working-memory deficits [reviewed by Goldberg and Gold (Goldberg and Gold, 1995)] (Goldman-Rakic, 1991, 1994). Compared with healthy individuals, patients with schizophrenia have reduced blood flow in DLPFC when performing executive cognitive tasks that normally increase blood flow to DLPFC (Weinberger et al., 1986; Andreasen et al., 1992; Ganguli et al., 1997). While the precise anatomical correlates of these behavioral and functional abnormalities are unknown, recent brain-imaging studies suggest that the integrity of cortical neurons and/or their connections may be altered (Weinberger et al., 1992; Frith et al., 1995; Bertolino et al., 1996, 1998). Increases in neuronal cell density in Brodmann's areas 9 and 46 (Selemon et al., 1995, 1998), along with decreases in DLPFC synaptic associated proteins (Glantz and Lewis, 1997; Thompson et al., 1998; Honer et al., 1999; Karson et al., 1999), in patients with schizophrenia suggest that reduced neuronal connections or reduced neuropil may underlie functional abnormalities in the DLPFC of patients with schizophrenia.

The formation and maintenance of neuronal connections are complex processes involving the regulation of many genes and proteins. One such protein, growth-associated protein 43 (GAP-43), is a neuron-specific phosphoprotein localized to the presynaptic membrane and is a substrate for protein kinase C [reviewed by Benowitz and Routtenberg (Benowitz and Routtenberg, 1997)]. Manipulations that abolish GAP-43 expression result in disruption of axon outgrowth and can lead to premature death of the organism (Shea et al., 1991; Aigner and Caroni, 1993, 1995; Shea and Benowitz, 1995; Strittmatter et al., 1995), whereas transgenic overexpression of GAP-43 results in the spontaneous formation of new synapses (Zuber et al., 1989; Aigner and Caroni, 1995). In developing rodent brain, GAP-43 expression is high during times of axon elongation (Jacobson et al., 1986), but declines as the animals mature. In humans, GAP-43 messenger ribonucleic acid (mRNA) and protein are found in embryonic telencephalon during periods of differentiation and axon extension (Honig et al., 1996; Kanazir et al., 1996). Thus, GAP-43 may be integral to neurite outgrowth in both rodents and humans.

The function of GAP-43 in the adult brain is less well understood, but studies in rodents demonstrate that GAP-43 may mediate experience-dependent ‘plasticity’ and long-term potentiation (Nelson and Routtenberg, 1985; Lovinger et al., 1986). Levels of GAP-43 vary according to the region of primate brain examined and roughly correlate with the level of functional complexity associated with a particular cortical region (Nelson et al., 1987). In the adult human brain, GAP-43 mRNA and protein are abundant in neocortical areas associated with high degrees of behavioral ‘plasticity’ and learning, including the prefrontal cortex, and less abundant in neocortical primary sensory areas (Neve et al., 1987; Neve et al., 1988; Benowitz et al., 1989). In adults, GAP-43 protein may function to enhance both growth and retraction of presynaptic terminals in cortical brain areas.

Although the anatomical and molecular substrates of behavioral change in humans are unknown, it has been proposed that the functional ‘plasticity’ of DLPFC may depend on maintenance of proper connections between prefrontal regions and other cortical and subcortical structures (Weinberger, 1993). In primates, the granular prefrontal cortex is interconnected with multi-modal association areas in the inferior parietal, superior temporal and orbital prefrontal regions (Nauta, 1971; Fuster et al., 1985), the head of the caudate nucleus and the mediodorsal nucleus of the thalamus (Arikuni et al., 1983; Yeterian and Pandya, 1994). Proteins that are found in the presynaptic terminals of these projections, such as GAP-43, are synthesized in the cell bodies in the DLPFC and are anterogradely transported to terminals at target sites within the prefrontal cortex and distant from the prefrontal cortex. As a correlate of the proposed abnormal DLPFC neuronal connectivity and possible synaptic pathology in the DLPFC of patients with schizophrenia, we hypothesized that GAP-43 mRNA levels would be reduced.

Materials and Methods

Brain Collection

Cohort 1

Cohort 1, used in the RNase protection assay (RPA), included 21 patients with schizophrenia and 17 normal controls (Table 1) matched for age, sex, race, brain pH, post-mortem interval (PMI, defined as time between death and brain freezing) and time in the freezer (defined as the number of months between brain freezing and RNA extraction). No statistical differences among the groups for the demographic variables were detected. Post-mortem brains were collected at the Clinical Brain Disorders Branch (NIMH, St Elizabeths) as previously described (Kleinman et al., 1995). Briefly, 1.5 cm coronal slabs through the entire cerebrum of each human brain were rapidly frozen in a pre-chilled dry-ice isopentane slurry bath and stored at –80°C. The time the tissue was stored at –80°C, before RNA was extracted (freezer time), was not significantly different between the brains of normal individuals (91.3 ± 28.6 months, mean ± SD) and those with schizophrenia (88.8 ± 24.5 months). The number of psychiatric charts available for review varied according to duration of illness (26.5 ± 15.2 years) and typically amounted to >1000 pages of clinical information. Diagnosis was determined by independent reviews of clinical records by two board certified psychiatrists who used the Diagnostic Evaluation After Death (Zalcman et al., 1983; Keks et al., 1999) as a guide to review the material available on each case. Cases that met DSM-IV criteria for schizophrenia were used in our study (American Psychiatric Association, 1994). Disagreements between the two independent reviews were resolved by requesting a third psychiatrist's review of the case. Out of the 21 patients in Cohort 1 who were diagnosed with schizophrenia, 15 patients were of the chronic undifferentiated subtype, 2 patients of the chronic disorganized subtype and 4 patients of the chronic paranoid subtype. The average age of disease onset was 22.4 ± 5.2 years of age (Table 2). All patients diagnosed with schizophrenia in Cohort 1 had documented auditory hallucinations. All but one patient had documented paranoid delusions; 18 patients showed evidence of thought disorder, and another 18 exhibited negative symptoms. IQ data were available for 14/21 patients (full-scale IQ for these 14 patients = 81 ± 20, mean ± SD). The total dose of neuroleptic medication given to the patients was calculated by adding the various daily medication levels as determined from available medical records and converting these levels to chlorpromazine (CPZ) equivalents as previously formulated (Torrey, 1983). A median value of drug dosage was then calculated from the CPZ equivalents to give the estimated average daily dose; this value was multiplied by the duration of illness (estimated from the earliest age of definable symptoms or age at first hospitalization) to give the estimated lifetime CPZ equivalents (Table 2).

Characterization of Normal Controls

Cases were screened by police and/or by telephone interviews of family members for a history of medical and/or psychiatric problems, including alcohol abuse and illicit drug use. Any positive history of a psychiatric problem or excessive alcohol or drug use led to the exclusion of that case from the normal control group. In addition, 80% of all cases had toxicological screens of blood. If the medical examiner believed that the index of suspicion for illicit drugs or alcohol abuse was low, a toxicological screen was not performed.

All brains (both normal controls and patients with schizophrenia) were screened for signs of macroscopic pathology at the time of autopsy; brain sections were examined microscopically with the use of Bielschowsky's silver stain on multiple cerebral areas to exclude the presence of neuritic pathology as seen in Alzheimer's disease (AD). Those cases with an unclear psychiatric diagnosis, evidence of cocaine or PCP abuse (history and/or toxicology), cerebrovascular disease, autolysis, subdural hematoma, neuritic pathology or other pathological features were excluded from the analysis.

Cohort 2

The brains composing Cohort 2, used in the in situ hybridization histo- chemistry (ISHH), were obtained from the Stanley Foundation Brain Bank and included six normal controls, six patients with schizophrenia and five patients with bipolar disease who received neuroleptics. The brains in Cohort 2 were collected, evaluated and screened in the same manner as those in Cohort 1 (Table 3). For Cohort 2, five out of the six individuals diagnosed with schizophrenia were of the chronic undifferentiated type and one individual was of the chronic paranoid type. The average age of disease onset was 21.8 ± 9.3 years of age for the patients with schizophrenia and 24.2 ± 8.6 years of age for the patients with bipolar disorder. The three groups in Cohort 2 were matched as to PMI, brain pH and postnatal age. No statistical differences among the groups for the demographic variables were detected (Table 3).

pH Determinations

The brain tissue pH was determined from homogenized cerebellar tissue; in <5% of the cases, frontal cortex was used. Tissue (0.4–0.8 g) was thawed and homogenized in 10 times the tissue volume in double-distilled (dd) H2O (pH 7.0) and the homogenate pH was measured with a Sentron pH meter. The pH electrode was washed thoroughly in ddH2O (pH 7.0) after each sample and recalibrated after every 10 samples.

RNA Extractions

Tissue was excised from either the right or left side of the middle third of the superior or middle frontal gyrus immediately anterior to the genu of the corpus callosum. Hippocampal tissue was dissected from along the temporal horns of the lateral ventricle from either the right or left temporal lobe. Approximately 400 mg of frozen pulverized human brain tissue from each case was assigned an RNA isolation number, weighed frozen and transferred into a pre-chilled, DEPC-treated, glass homogenizer. Each sample was homogenized in lysis buffer, layered over a cushion buffer with a higher sucrose concentration and centrifuged at 5000 g for 20 min at 4°C as described previously (Jakubowski and Roberts, 1992). The cytoplasmic RNA was further purified by proteinase K digestions and phenol:chloroform extractions (Jakubowski and Roberts, 1992). A spectrophotometer was used to quantitate the total RNA (μg) in each sample, and the integrity of ribosomal RNA bands was verified by agarose gel electrophoresis for each sample. Reisolation of RNA was performed as necessary. Total RNA was aliquoted, dried down and stored in 30 μl of hybridization buffer (80% deionized formamide, 40 mM PIPES, pH 6.7, 400 mM NaCl, 1 mM EDTA) at –80°C.

Northern Blot and RPA

The human GAP-43 DNA template used for the preparation of riboprobes corresponded to base pairs 54–832 of the cDNA (Kosik et al., 1988) (gene bank XM25667), and was constructed by J.E. Cheetham, of the University of Rochester (Callahan et al., 1994). Northern blot analysis was used to confirm the specificity of the GAP-43 probe for a 1.6 kB transcript in human cortical RNA (Kosik et al., 1988). A 32P-labeled riboprobe with a sp. act. of 1 × 109 c.p.m./μg was synthesized from the GAP-43 cDNA template. An RNA blot containing cortical regions from adult human brain (cat. no. 7755-1, Clonetech, Palo Alto, CA) was pre-hybridized in buffer containing 12.5 M formamide, 50 μg/ml salmon sperm DNA, 0.025 M KPO4, 5× Denhardt's solution and 5× saline sodium citrate (SSC) in RNase-free water for 2 h at 42°C. Radioactive probe at 5 ng probe/ml was added to the hybridization buffer (prepared as above, with the addition of 10% Dextran sulfate) and the mixture was pre-equilibrated to 42°C, added to the blot and allowed to hybridize overnight at 42°C. The blot was rinsed three times in 1× SSC/0.1%SDS at room temperature (15 min), three times in 0.25× SSC/0.1%SDS at 55°C (15 min) and three times in 0.2× SSC/0.1%SDS at 68°C (15 min). The blot was air-dried and exposed to BioMax film (Kodak, Rochester, NY) overnight.

On the day of the RPA, sample tubes containing the RNA were removed from the freezer, thawed and reassigned random sample numbers using a random number table, by an individual not performing the assay. The experimenters were blind to diagnosis during sample handling and the samples were processed in a random order. RPAs were preformed as previously described (Jakubowski and Roberts, 1992; Lazar and Blum, 1992; Weickert and Blum, 1995). Riboprobes were labeled to a sp. act. of 1 × 109 c.p.m./μg RNA with [32P]UTP (Amersham, Arlington Heights, IL), purified by ethanol precipitation and added (1 μl of 200 pg/μl) to the samples of 2 or 4 μg of total RNA. Standard curves for the GAP-43 RPA were generated by adding increasing amounts of in vitro transcribed sense strand GAP-43 RNA ranging from 0.1 pg (0.36 amol) to 100 pg (363.63 amol), and by normalizing the total RNA levels with yeast total RNA. A tube containing no sense strand (0 pg) but with total yeast RNA was included as a control to ensure that the digestion of radiolabeled antisense probe was complete. Two or four micrograms of total yeast RNA were added to the tubes containing the standards so the total amount of RNA was equivalent to the samples. Hybridization of the riboprobe to standards and samples was allowed to proceed in a 45°C water bath overnight. For RNase digestion of non-protected fragments of RNA, RNase A (10 μg/ml) and RNase T1 (2 μg/ml) were used. The protected hybrids were purified by phenol:chloroform extractions and ethanol precipitations, and were size separated on a 5% acrylamide gel. The gels were dried and exposed to autoradiographic film. The most prominent band corresponding to the appropriate size protected fragment for the standard curves and the samples were cut from the dried gels and placed in a scintillation counter. A regression analysis using known amounts of plus strand GAP-43 mRNA was used to determine the amount of GAP-43 mRNA in the samples. Aliquots from the same RNA samples were also assayed for three constitutively expressed genes, cyclophilin, β-actin and glyceraldehyde-3-phosphate-dehydrogenase (GADPH), to address potential systematic differences in RNA integrity between patients and controls that might be related to agonal state, post-mortem handling, tissue processing or RNA isolation procedures. Human cDNA templates for cyclophilin, β-actin and GADPH were purchased from Ambion (Austin, TX) as linearized triple-script plasmids. The RPAs were performed twice for both cyclophilin and GAP-43 mRNA with similar results each time; therefore the average value of mRNA from the two assays was used in the final statistical analysis.

In situ Hybridization and Quantitation

Fixation, acetylation, delipidation and dehydration of the slides containing 14-μm-thick sections of frontal cortex were performed as previously described (Whitfield et al., 1990). Two hundred microliters of hybridization buffer containing the [35S]UTP-labeled GAP-43 riboprobe (5 ng/ml) was added to each section and hybridization was allowed to occur at 55°C overnight in humidified chambers. After the in situ hybridization procedure, slides, along with 14C standards (American Radiolabeled Chemicals, Inc., St Louis, MO), were exposed to Kodak autoradiographic film (X-OMAT) for 3 days. With the aid of a microscope, the boundary of Brodmann's area 46 (BA 46) was delineated on Nissl-stained sections, applying the criteria described previously (Rajkowska and Goldman-Rakic, 1995). The criteria used can be outlined as follows: (i) the presence of a well defined granular layer IV; (ii) the columnar arrangement of pyramidal neurons in layer III; (iii) the increase in size of pyramidal neurons in layer III with an increase in cortical depth; and (iv) the presence of a clear transition from layer VI into the white matter. Sampling was done in BA 46 where the laminae were running parallel to the pial surface. Quantitation of optical density from the film was done blind to diagnosis (with numbers assigned by the Stanley Foundation) with the aid of NIH Image (Rasband, NIH) and Excel (Microsoft) software programs. For each slide, three lines of 170 μm width, traversing the entire cortical gray matter, were drawn perpendicular to the pial surface. Optical density, interpolated along the 14C standard curve, was sampled at 85 μm pixel intervals along these lines. These data were used to construct profile plots of μCi/g of GAP-43 mRNA as it varied with cortical gray matter depth. The profiles along each line were then linearly interpolated to a common anatomical scale in units of percent cortical depth. Two sections per case were analyzed. For each case, the μCi/g of GAP-43 averaged from the six sampling lines was used in the statistical analysis, so that each individual had only one representative measurement at each cortical depth. Measurements for the representative laminae were taken from within the borders of each layer, based on percent cortical depth for each layer. These measurements were derived from average values for laminar boundaries described elsewhere (Rajkowska and Goldman-Rakic, 1995). Layer III was considered to be at 20–46% of the total cortical depth, layer V at 54–70%, and layer VI at 74–84%. Each case was analyzed independently by two neuroscientists with significantly correlated results (intraclass correlation coefficient, ICC = 0.69, P < 0.001).

Silver Grain Analysis

In order to determine whether GAP-43 mRNA reductions in the DLPFC of patients with schizophrenia were due to a decrease in the amount of GAP-43 mRNA per neuron, we counted the number of silver grains overlying pyramidal neurons. We chose to focus on pyramidal neurons because GAP-43 mRNA is distributed preferentially over large pyramid-shaped cells. While smaller non-pyramid-shaped neurons of the human DLPFC occasionally appeared to have positive hybridization signal and thus may have contained GAP-43 mRNA, the many smaller, more round-shaped cells appeared unlabeled and were not analyzed. Additionally, cells with a small, darkly stained nucleus with little cytoplasmic stain (possible astrocytes) did not appear to express GAP-43 mRNA in any of the groups.

Slides were dipped in NT-B2 emulsion (Kodak), stored in light-tight boxes in the dark for 9 days and then developed in D-19 developer, dehydrated and lightly stained with a Nissl counterstain. Silver grain analysis was conducted blind to diagnosis on a Zeiss Axiophot microscope equipped with a video camera and Bioquant image analysis system. We counted grains in layers III, V and VI, because it was in these layers that we had the most robust signal on the autoradiographs. Neurons in layers III, V and VI from each case were identified under bright field by their large cell size, light Nissl stain and triangular (layers III, V and VI) or fusiform shape (layer VI). The shape of the cells could be outlined by combining demarcation of cells in the light Nissl stain and GAP-43 mRNA silver grain distribution, which often extended into apical dendrites. Circles of 35 μm diameter were drawn, centered over every large triangular or fusiform-shaped neuron in a field including ~5–6 neurons. The illumination was then switched to dark field and the silver grains overlying a neuron, within the boundaries of this circle, were counted with the aid of a grain-counting macro-program written with Bioquant software. The threshold for determining positive grains was set using the numerical value of 118–255 in the green measurement window, and was held constant for every case and for all sampling fields within each case. On average, seven microscopic fields per layer (at a final magnification of 40×) were examined and only those neurons containing numbers of silver grains fourfold above background (criteria as set in Akbarian et al., 1995) were entered into the quantitative analysis. Background was determined by averaging the number of grains in three 35-μm-diameter circles over areas of gray matter neuropil from within fields in which neurons were being sampled. The background level of grains was determined separately for each case. Overall, >95% of neurons counted contained enough silver grains to reach inclusion criteria, and this percentage was similar in all three diagnostic groups. At least 30 neurons within each layer for each case (a total of 1620 neurons) were sampled within layers III, V and VI from contiguous fields using an arbitrary start point within BA 46. The average number of grains per cell for each layer for each case was used in the statistical analysis.

Statistical Analysis

The Spearman rank order correlations were run between measurements of RNA and brain cohort characteristics (Spearman R, Statistica, Statsoft, Tulsa, OK). For the RPAs, the data were analyzed by ANCOVA, where diagnosis or diagnosis and hemisphere were the independent factors, GAP-43 mRNA and cyclophlin mRNA were the dependent factors, and factors that correlated with GAP-43 mRNA levels were co-varied for. For the data derived from the ISHH, there were three GAP-43 measurements per case, for layers III, V and VI respectively. The average μCi/g for each layer was used in a MANOVA (Statistica), where diagnosis was the independent variable and the GAP-43 mRNA in layers III, V and VI were the dependent variables. When a significant overall main effect of diagnosis was found in the MANOVA, the groups were compared by post-hoc t-tests run separately for layers III, V and VI.

Results

RNA

The total yield of RNA extracted from the DLPFC did not significantly differ between the normal controls (0.15 ± 0.08 μg RNA/mg tissue, mean ± SD) and the patients with schizophrenia (0.19 ± 0.10 μg RNA/mg tissue, mean ± SD) in Cohort 1 (t = –1.30, df = 36, P = 0.20). No qualitative relationship between integrity of ribosomal RNA bands and PMI was noted and no significant correlation between PMI or freezer storage time and total yield of RNA was detected (R = –0.01 and 0.02, respectively). This is in agreement with other reports of general RNA stability in human brain tissue over a wide range of post-mortem intervals (Johnson et al., 1986; Barton et al., 1993). Total RNA and pH measurements were positively correlated (R = 0.37, P < 0.05, pH range 5.70–6.76), suggesting that brain pH measurements may be predictive of overall RNA stability as reported previously (Harrison et al., 1995). We were unable to detect any significant correlation between yield of total RNA from prefrontal cortical tissue and age of subject (R = –0.14, P = 0.42, age range 20–80 years). PMI and brain tissue pH were not correlated (R = 0.05, P = 0.75).

Northern Blot

The GAP-43 riboprobe used in our experiments recognized one major band at the expected 1.6 kB transcript size in RNA extracted from human brain (Fig. 1). Additionally, GAP-43 mRNA levels were lowest in medulla and spinal cord (lanes 3 and 4), intermediate in cerebellum and putamen (lanes 1 and 8) and highest in cortical regions, including the occipital cortex (lane 5), frontal cortex (lane 6) and temporal cortex (lane 7).

GAP-43 mRNA RPA

The autogradiographic films generated from the RNase protection assays showed a protected fragment at the predicted 778 bp in all cases (Fig. 2). The radioactive signal found below the protected band in each sample lane is also found in the standard curve as is commonly seen when using RNase protection assay techniques (Lazar and Blum, 1992), and does not reflect degradation of extracted RNA. The lack of a signal in the 0 lane (which contained no input RNA from brain) shows that there was complete digestion of the radiolabeled probe. We found a 38% reduction in GAP-43 mRNA levels/μg total RNA in the DLPFC of patients with schizophrenia (mean ± SD = 14.43 ± 10.68 amol), as compared with normal controls (mean ± SD = 23.38 ± 15.13 amol) (Fig. 3A). GAP-43 mRNA levels correlated significantly with both PMI (R = –0.32, P = 0.05) and pH (R = 0.35, P = 0.03) (Fig. 4A,C). GAP-43 mRNA and postnatal age were not significantly correlated (R = –0.02, P = 0.92, Fig. 4E). An analysis of DLPFC GAP-43 mRNA levels using an ANCOVA with pH and PMI entered as covariates revealed a significant effect of diagnostic group (F = 5.70, df = 1,34, P = 0.023), where patients with schizophrenia had reduced GAP-43 mRNA compared with controls (a significant decrease of GAP-43 mRNA was also found by using a t-test, t = 2.14, df = 36, P = 0.04).

Next, we considered if brain hemisphere had a significant effect on GAP-43 mRNA levels. Using a two-way ANCOVA, there was again a significant main effect of diagnosis on GAP-43 mRNA levels (F = 8.31, df = 1,32, P = 0.007), where patients with schizophrenia had significantly less GAP-43 mRNA. GAP-43 mRNA levels were increased by 45% on the right side of the brain compared with the left for both normal individuals and patients with schizophrenia; however, this effect approached, but did not reach, statistical significance (F = 2.80, df = 1,32, P = 0.10). No interaction effect between hemisphere and diagnosis was detected (F = 1.93, df = 1,32, P = 0.17).

As a test of the regional specificity of the GAP-43 mRNA reduction, we used RPA to determine the GAP-43 mRNA level in total RNA extracted from hippocampus (Cohort 1) but did not detect a significant difference in GAP-43 mRNA levels between schizophrenic patients and controls (using ANCOVA with PMI and pH as covariates, F = 0.20, df = 1,28, P = 0.65). In fact, hippocampal GAP-43 mRNA was slightly (17%) higher in patients with schizophrenia compared with normal controls. This suggests that the reduction in GAP-43 mRNA in the DLPFC of patients with schizophrenia is not due to uncontrolled differences in the agonal state of patients or post-mortem brain handling, which would not be expected to have regionally selective effects.

Lastly, we tested to see whether levels of GAP-43 mRNA correlated with neuroleptic medication histories. There was a significant negative correlation between GAP-43 mRNA levels and average daily doses of medication converted to chlorpromazine equivalents, in both the DLPFC (R = –0.60, P = 0.008) and the hippocampus (R = –0.67, P = 0.05), among schizophrenic patients. GAP-43 mRNA levels were also correlated with the estimated lifetime neuroleptic dose in the DLPFC (R = –0.47, P = 0.05) and the hippocampus (R = –0.58, P = 0.04). GAP-43 mRNA levels did not correlate significantly with daily dose of medication at death (in DLPFC R = –0.27, P = 0.28; in hippocampus R = –0.30, P = 0.33) nor with the duration of illness in the DLPFC (R = –0.16, P = 0.50) or in the hippocampus (R = –0.08, P = 0.76).

Cyclophilin RNA RPA

In preliminary studies, we found that the expression of cyclophilin, β-actin and GADPH did not differ between patients with schizophrenia and normal controls in the DLPFC. Cyclophilin mRNA proved to have the least amount of subject-to-subject variability and was chosen for further analysis. Cyclophilin mRNA was not altered in patients with schizophrenia relative to controls (ANCOVA with PMI and pH as covariates, F = 0.16, df = 1,34, P = 0.69, Fig. 2B). Unlike GAP-43 mRNA, the level of cyclophilin mRNA did not correlate with PMI (R = 0.06, P = 0.73), suggesting that the effect of PMI on mRNA levels may be message selective (Fig. 4B). Cyclophilin mRNA levels did correlate significantly with brain pH (R = 0.39, P = 0.02, Fig. 4D), but not with age (R = –0.04, P = 0.80, Fig. 4F). We found that the ratio of GAP-43 to cyclophilin was significantly reduced in the DLPFC of patients with schizophrenia (ANCOVA with pH and PMI as covariates, F = 5.65, df = 1,34, P = 0.023).

GAP-43 mRNA In Situ Hybridization

GAP-43 mRNA hybridization signal was primarily distributed over pyramidal neurons in both the supragranular and infragranular layers in all three diagnostic groups (Figs 5 and 7). There were very few scattered silver grains present in sections hybridized with radiolabeled GAP-43 sense strand control (Fig. 5D). The GAP-43 mRNA hybridization signal from the autoradiographic films was significantly reduced in the schizophrenic brains as compared with normal controls [Wilks' Lambda = 0.34 (df = 3,8), P < 0.05] in layers III (mean 29% reduction), V (mean 28% reduction) and VIa (mean 25% reduction, post-hoc tests, t = 2.56, P = 0.01, t = 3.29, P < 0.01 and t = 2.16, P < 0.05, respectively). In contrast, the GAP-43 mRNA levels in the individuals with bipolar disorder did not differ significantly from the normal controls [Wilks' Lambda = 0.834 (df = 3,7), P = 0.72]. In the bipolar group, the level of GAP-43 mRNA did not correlate with medication dosages in any layer (all P > 0.10). Quantitative analysis at the cellular level revealed that the GAP-43 silver grains per cell varied significantly in the pyramidal neurons according to diagnostic group (MANOVA, Wilks' Lambda = 0.34, df = 6, 22, P = 0.04, Fig. 6). Neurons residing in layer III, V and VI of the schizophrenics had a 40, 35 and 36% reduction, respectively, in number of GAP-43 mRNA silver grains per neuron compared with controls. The reduction in GAP-43 mRNA as detected by silver grain analysis in Cohort 2 was similar in magnitude to that detected by RPA in Cohort 1 and was statistically significant in all layers examined (post-hoc t-tests all P < 0.01). When patients with schizophrenia were compared with individuals diagnosed with bipolar disorder, layers III, V and VI showed a 29, 24 and 34% reduction, respectively, in the number of silver grains per neuron in patients with schizophrenia (Fig. 6). This reduction in GAP-43 mRNA/pyramidal neuron in the patients with schizophrenia compared with the neuroleptic control (bipolar disorder) group was significant in layers III and VI (P < 0.05) and approached significance in layer V (P = 0.09). The number of silver grains/neuron in patients with bipolar disorder was not significantly reduced compared with normal non-neuroleptic-medicated controls (all layers, P > 0.05). The amount of GAP-43 silver grains/neuron did not correlate with the lifetime dose of neuroleptic medication in the patients with bipolar disorder (all layers, P > 0.10).

Discussion

We have found a reduction in GAP-43 mRNA levels in the DLPFC of patients with schizophrenia using two complementary techniques in two separate cohorts. Additionally, we found several factors that may contribute to post-mortem human brain DLPFC GAP-43 mRNA levels, i.e. brain pH, PMI, brain hemisphere and medication history. This may explain the high degree of subject-to-subject variability found in our study and may explain some of the inconsistencies across studies of GAP-43 in post-mortem human brain tissue. While the GAP-43 mRNA reduction may be a component of the primary disease process in schizophrenia, it is important to consider an alternative possibility; that this represents an epiphenomenon related to the experience of having schizophrenia, or exposure to neuroleptic drugs or to other events associated with a lifetime of unremitting mental illness. We have detected a relationship between average daily dose and lifetime dose of neuroleptic medication and GAP-43 mRNA levels, and this suggests that neuroleptics may affect GAP-43 mRNA levels. Alternatively, this correlation may reflect another common variable that determines both GAP-43 mRNA levels and neuroleptic dose, such as illness severity, rather than suggesting a direct causal relationship between neuroleptics and GAP-43 mRNA levels.

Several lines of data argue against neuroleptic exposure and nonspecific illness factors as the cause of the reduction of GAP-43 mRNA in the DLPFC. First, we did not observe a decrease in GAP-43 mRNA levels in the hippocampus from the same group of patients in which we found the reduction in DLPFC. Second, we did not detect a significant decrease in GAP-43 mRNA levels in the neuroleptic-treated and chronically ill bipolar disorder group compared with normal controls, nor did GAP-43 mRNA levels correlate with neuroleptic exposure in the group of bipolar patients examined. Also, the amount of GAP-43 mRNA per neuron was significantly reduced in layers III and VI in individuals with schizophrenia compared with individuals with bipolar disorder. Third, chronic treatment with haloperidol does not significantly alter GAP-43 mRNA levels in the rodent neocortex (Eastwood et al., 1997).

Our findings may appear inconsistent with two earlier studies of GAP-43 mRNA and protein in the DLPFC of patients with schizophrenia. A similar reduction in GAP-43 mRNA levels was found in some cortical areas (primary visual and anterior cingulate) of patients with schizophrenia, but no significant changes were found in the DLPFC (Eastwood and Harrison, 1998). Although there are no obvious reasons for the discrepancy between the Eastwood and Harrison study and our own, the differences could relate to subtle differences in cohort characteristics or brain storage conditions. Another study has reported that GAP-43 protein in brain homogenates from DLPFC (BA9) is increased; they used a fairly small number of cases (five patients with schizophrenia and four normal controls) and a quantitative study of GAP-43 mRNA levels was not conducted (Perrone-Bizzozero et al., 1996). Since many axon terminals in the DLPFC arise from intrinsic sources, one may expect that changes in GAP-43 protein may be mirrored by similar changes in GAP-43 mRNA levels. However, this is not what we found. The putative increase in GAP-43 protein in DLPFC may arise from a compensatory response of afferent terminals originating in distant sites. Alternatively, the increase in GAP-43 protein in the DLPFC could result from a deficit in fast axonal transport of the GAP-43 protein (Liu et al., 1991), and thus GAP-43 protein may inappropriately accumulate in DLPFC cell bodies. Further work evaluating the anatomical and subcellular localization of altered GAP-43 protein in the brains of patients with schizophrenia is necessary to explore these possibilities.

The reduction of GAP-43 mRNA reported here suggests that there may be reduced GAP-43 protein in presynaptic terminals, less phosphoprotein activity in presynaptic terminals and a diminished ‘plasticity’ of DLPFC pyramidal neurons in patients with schizophrenia. Layers III, V and VI in the schizophrenic DLFPC had less GAP-43 mRNA hybridization signal, which could represent fewer GAP-43 producing neurons or could reflect less GAP-43 mRNA per neuron. In support of the latter interpretation, silver grain analysis has confirmed that there were reduced levels of GAP-43 mRNA overlying pyramidal neurons in the DLPFC of patients with schizophrenia. This suggests that glutamate pyramidal neurons in the DLPC may synthesize less GAP-43 protein in patients with schizophrenia. Our data do not rule out the possibility that there may also be fewer GAP-43 producing neurons in the DLPFC of patients diagnosed with schizophrenia. Prior studies have reported normal numbers of pyramidal neurons in the DLPFC of patients with schizophrenia (Pakkenberg, 1993; Akbarian et al., 1995), an increase in pyramidal cell density (Selemon et al., 1995, 1998) and a smaller size of pyramidal neurons (Rajkowska et al., 1998). In our study, GAP-43 mRNA was expressed in almost every pyramidal neuron in each case examined. These observations support the conclusion that there is less GAP-43 mRNA per pyramidal neuron in the DLPFC of patients with schizophrenia.

We did not find decreased GAP-43 mRNA in the hippocampus of patients with schizophrenia. This lack of a reduction was somewhat surprising, because evidence for hippocampal pathology is common in the brain of patients with schizophrenia [reviewed by Weickert and Kleinman (Weickert and Kleinman, 1998)]. Also, damage to the hippocampus in the neonatal rodent can model some aspects of schizophrenia [reviewed by Weinberger and Lipska (Weinberger and Lipska, 1995)]; thus, one might expect to see altered levels of growth-associated markers, such as GAP-43, in the hippocampus of individuals with schizophrenia. Our measurements of GAP-43 mRNA were made in adult humans and may not adequately reflect hippocampal GAP-43 mRNA levels found earlier in development. Additionally, there are, in fact, anatomically specific decreases in GAP-43 mRNA in the hippocampus (Eastwood and Harrison, 1998; Webster et al., 2001) that may not be detected in assays using hippocampal homogenates as we utilized in the RPA analysis in our study.

The decrease of GAP-43 mRNA in the DLPFC of patients with schizophrenia involves pyramidal neurons in at least three different cortical layers. Pyramidal neurons located in cortical layer III can terminate broadly throughout the cortex, in the adjacent cortex and in areas of axon origin through recurrent collaterals (Barbas and Pandya, 1989; Levitt et al., 1993). The reduction of GAP-43 mRNA in pyramidal neurons of layer III in patients with schizophrenia may reflect reduced numbers of cortico-cortical connections. There are reports of a decreased number of postsynaptic spines on layer III pyramidal neurons (Garey et al., 1998; Glantz and Lewis, 2000), reduced levels of the presynaptic vesicular-associated proteins (synaptophysin and SNAP-25) (Glantz and Lewis, 1997; Thompson et al., 1998; Honer et al., 1999; Karson et al., 1999) and increased packing density of neurons (Selemon et al., 1995, 1998) in the DLPFC of patients with schizophrenia. Pyramidal neurons in deep layer III have been reported to be smaller in size in patients with schizophrenia compared with controls (Rajkowska et al., 1998). These results, taken together with our own, support the notion that reductions in the vitality and connectivity of the supragranular pyramidal neuron may be a prominent pathological feature of the DLPFC in patients with schizophrenia. In our study, we also found that subcortically projecting infragranular glutamate pyramidal neurons (layers V and VIa) of patients with schizophrenia had significantly reduced GAP-43 mRNA levels in DLPFC. In primates, neurons in the infragranular layers can project to subcortical sites; DLPFC axons terminating in the caudate nucleus preferentially arise from neurons in layer V and those terminating in the thalamus preferentially arise from neurons in layer VI (Yeterian and Pandya, 1994). Our results suggest that terminals of excitatory corticocaudate and corticothalamic neurons in the brain of patients with schizophrenia may contain less GAP-43 protein and, thus, may be functionally altered. Recent evidence from in vivo brain imaging suggests that the frontostriatal circuitry is dysfunctional in the brains of patients with schizophrenia (Manoach et al., 2000).

DLPFC efferents arising in layers III, V and VI can extend to distant cortical regions, including the inferior parietal cortex; the superior, middle and inferior temporal cortices; and the anterior cingulate cortex (Pearlson et al., 1996). A measure of neuronal pathology in the prefrontal cortex of patients with schizophrenia correlates with the activation of the temporal cortex and inferior parietal cortex during working memory tasks (Bertolino et al., 2000). Therefore, it is possible that a decrease in GAP-43 mRNA in cortically projecting neurons of the schizophrenic DLPFC may relate to a decrease in the ability of DLPFC neurons to recruit other cortical areas necessary to perform integrative cognitive tasks (Weinberger, 1993; Goldman-Rakic and Selemon, 1997; Lewis, 1997).

Notes

We would like to thank Dr D. Jones, Anne K. Brouha, Yeva Snitskovsky, Dr Juraj Cervenak, Tara Romanczyk and Judith O'Grady for help with this project. Also, we would like to acknowledge Drs P.D. Coleman and J.E. Cheetham for supplying the GAP-43 cDNA, the Stanley Foundation Brain Collection for supplying brain tissue for Cohort 2 and Dr B. Lipska for her helpful review of the manuscript.

Address correspondence to Cynthia Shannon Weickert, NIH, Building 10, Room 4N 308, MSC 1385, Bethesda, MD 20892-1385, USA. Email: shannowc@intra.nimh.nih.gov.

Table 1

Demographic information on Cohort 1

Case no. Diagnosis Age/sex Race Side pH PMI (h) Months in freezer COD MOD Toxicology 
Means and standard deviations are printed below the last individual in each group. Abbreviations are as follows: PMI = post-mortem interval, COD = cause of death, MOD = manner of death, CON = normal control, CUS = chronic undifferentiated schizophrenia, CDS = chronic disorganized schizophrenia, CPS = chronic paranoid schizophrenia, TD = tardive dyskinesa, m = male, f = female. B = Black, W = White, H = Hispanic, R = right, L = left, ASCVD = arteriosclerotic cardiovascular disease, GSW = gun shot wound, MI = myocardial infarction, COPD = chronic obstructive pulmonary disease, n.a. = not available. 
CON 53/f 5.76 22  96 burns pending n.a. 
CON 34/m 6.60 37 106 ASCVD natural blood EtOH 0.06%, 
CON 68/m 6.57 20 113 ASCVD natural n.a. 
CON 47/m 6.62 57 118 MI natural n.a. 
CON 47/m 6.54 24.5 119 GSW to chest homicide blood EtOH 0.10%, 
CON 58/f 6.54 26.5 147 ASCVD natural negative 
CON 39/f 6.34 41.5 147 ASCVD natural negative 
CON 40/m 6.44 48.5  83 ASCVD natural negative 
CON 46/f 5.93 21.5  68 cardiomyopathy natural negative 
10 CON 74/m 6.65 33.5  80 stab wounds to chest homicide blood EtOH 0.04%, 
11 CON 45/m 6.61 17  65 crushing injury to chest accident n.a. 
12 CON 47/m 6.03 59.5  78 acute bronchial asthma natural blood morphine 0.015 mg/dl 
13 CON 46/m 6.71 29  70 ASCVD natural negative 
14 CON 55/m 6.00 10.5  67 MI (ASCVD) natural lidocaine ‘detected’ 
15 CON 48/f 6.08 18.5  66 pulmonary artery thrombosis natural phensuximide ‘detected’ 
16 CON 60/f 6.40 10  66 ASCVD natural lidocaine ‘detected’ 
17 CON 61/f 6.15 63.5  63 multiple blunt force injuries accident n.a. 
Mean (SD)  51 (11)   6.35 (0.30) 31.8 (16.9)  91.3 (28.6)    
18 CUS 53/f 6.35 27.5 107 ASCVD natural lidocaine & thioridazine ‘detected’ 
19 CUS/TD 71/f 6.41 47.5 103 ASCVD natural phenothiazine metabolites ‘detected’ 
20 CDS 36/f 6.33 60 109 acute peritonitis (ruptured appendix) natural negative 
21 CUS 48/m 6.42 48.5 103 ASCVD natural carbamazepine ‘detected’ 
22 CDS 48/m 5.70 40.5 107 internal obstruction (volvulus of colon) natural blood acetone 20 mg 
23 CUS 36/m 6.56 13  97 blunt force injuries (fall) suicide phenothiazine metabolites ‘detected’ 
24 CUS 46/m 6.35 24.5  42 ASCVD natural lidocaine & benztopine ‘detected’ 
25 CPS/TD 44/f 6.51 36.5  95 cardiomegaly (hypertension) natural haloperidol & clonidine ‘detected’ 
26 CPS 26/m 6.76 14.5  81 asphyxia (hanging) suicide n.a. 
27 CUS 71/f 6.63 32.5 113 drowning accident thioridazine ‘detected’ 
28 CUS 46/m 6.73 25 116 blunt force injuries (fall) suicide haloperidol & diphenhydramine ‘detected’ 
29 CPS 45/m 6.15 30 112 ASCVD natural negative 
30 CUS 34/m 6.50 23  80 acute benztropine intoxication undetermined blood EtOH 0.10%, 
31 CUS 54/m 6.31 24  67 subarachnoid hemorrhage natural blood EtOH 0.32%, benztropine & doxepin ‘detected’ 
32 CUS 80/f 6.52 59.5  63 ASCVD (aspiration of food) natural negative 
33 CUS 48/m 6.29 15  64 dilutional hyponatremia (hypo-osmolar coma) natural lidocaine ‘detected’ 
34 CUS 59/f 6.54 7.5 118 pulmonary embolism natural n.a. 
35 CUS 49/f 6.23 15  39 COPD natural negative 
36 CUS 34/m 6.23 36.5  68 acute benztropine intoxication undetermined benztropine ‘detected’ 
37 CUS/TD 64/f 6.48 20.5  71 asphyxia (aspiration) accident negative 
38 CPS 34/f 6.28 84 109 overdose (tricyclics) suicide amitriptyline & nortriptyline ‘detected’ 
Mean (SD)  49 (14)   6.39 (0.23) 32.6 (18.9)  88.8 (24.5)    
Case no. Diagnosis Age/sex Race Side pH PMI (h) Months in freezer COD MOD Toxicology 
Means and standard deviations are printed below the last individual in each group. Abbreviations are as follows: PMI = post-mortem interval, COD = cause of death, MOD = manner of death, CON = normal control, CUS = chronic undifferentiated schizophrenia, CDS = chronic disorganized schizophrenia, CPS = chronic paranoid schizophrenia, TD = tardive dyskinesa, m = male, f = female. B = Black, W = White, H = Hispanic, R = right, L = left, ASCVD = arteriosclerotic cardiovascular disease, GSW = gun shot wound, MI = myocardial infarction, COPD = chronic obstructive pulmonary disease, n.a. = not available. 
CON 53/f 5.76 22  96 burns pending n.a. 
CON 34/m 6.60 37 106 ASCVD natural blood EtOH 0.06%, 
CON 68/m 6.57 20 113 ASCVD natural n.a. 
CON 47/m 6.62 57 118 MI natural n.a. 
CON 47/m 6.54 24.5 119 GSW to chest homicide blood EtOH 0.10%, 
CON 58/f 6.54 26.5 147 ASCVD natural negative 
CON 39/f 6.34 41.5 147 ASCVD natural negative 
CON 40/m 6.44 48.5  83 ASCVD natural negative 
CON 46/f 5.93 21.5  68 cardiomyopathy natural negative 
10 CON 74/m 6.65 33.5  80 stab wounds to chest homicide blood EtOH 0.04%, 
11 CON 45/m 6.61 17  65 crushing injury to chest accident n.a. 
12 CON 47/m 6.03 59.5  78 acute bronchial asthma natural blood morphine 0.015 mg/dl 
13 CON 46/m 6.71 29  70 ASCVD natural negative 
14 CON 55/m 6.00 10.5  67 MI (ASCVD) natural lidocaine ‘detected’ 
15 CON 48/f 6.08 18.5  66 pulmonary artery thrombosis natural phensuximide ‘detected’ 
16 CON 60/f 6.40 10  66 ASCVD natural lidocaine ‘detected’ 
17 CON 61/f 6.15 63.5  63 multiple blunt force injuries accident n.a. 
Mean (SD)  51 (11)   6.35 (0.30) 31.8 (16.9)  91.3 (28.6)    
18 CUS 53/f 6.35 27.5 107 ASCVD natural lidocaine & thioridazine ‘detected’ 
19 CUS/TD 71/f 6.41 47.5 103 ASCVD natural phenothiazine metabolites ‘detected’ 
20 CDS 36/f 6.33 60 109 acute peritonitis (ruptured appendix) natural negative 
21 CUS 48/m 6.42 48.5 103 ASCVD natural carbamazepine ‘detected’ 
22 CDS 48/m 5.70 40.5 107 internal obstruction (volvulus of colon) natural blood acetone 20 mg 
23 CUS 36/m 6.56 13  97 blunt force injuries (fall) suicide phenothiazine metabolites ‘detected’ 
24 CUS 46/m 6.35 24.5  42 ASCVD natural lidocaine & benztopine ‘detected’ 
25 CPS/TD 44/f 6.51 36.5  95 cardiomegaly (hypertension) natural haloperidol & clonidine ‘detected’ 
26 CPS 26/m 6.76 14.5  81 asphyxia (hanging) suicide n.a. 
27 CUS 71/f 6.63 32.5 113 drowning accident thioridazine ‘detected’ 
28 CUS 46/m 6.73 25 116 blunt force injuries (fall) suicide haloperidol & diphenhydramine ‘detected’ 
29 CPS 45/m 6.15 30 112 ASCVD natural negative 
30 CUS 34/m 6.50 23  80 acute benztropine intoxication undetermined blood EtOH 0.10%, 
31 CUS 54/m 6.31 24  67 subarachnoid hemorrhage natural blood EtOH 0.32%, benztropine & doxepin ‘detected’ 
32 CUS 80/f 6.52 59.5  63 ASCVD (aspiration of food) natural negative 
33 CUS 48/m 6.29 15  64 dilutional hyponatremia (hypo-osmolar coma) natural lidocaine ‘detected’ 
34 CUS 59/f 6.54 7.5 118 pulmonary embolism natural n.a. 
35 CUS 49/f 6.23 15  39 COPD natural negative 
36 CUS 34/m 6.23 36.5  68 acute benztropine intoxication undetermined benztropine ‘detected’ 
37 CUS/TD 64/f 6.48 20.5  71 asphyxia (aspiration) accident negative 
38 CPS 34/f 6.28 84 109 overdose (tricyclics) suicide amitriptyline & nortriptyline ‘detected’ 
Mean (SD)  49 (14)   6.39 (0.23) 32.6 (18.9)  88.8 (24.5)    
Table 2

Clincal information on patients with schizophrenia and bipolar disorder in Cohort 1 and Cohort 2

Case Age of onset Illness duration Psychiatric medications at death (mg PO) Last CPZ equivalent Daily CPZ equivalent Lifetime CPZ (mg/eq) 
Ages of onset and illness durations are given in years. Abbreviations are as follows: CPZ = chlorpromazine, mg/eq = milligram equivalent. 
18 24 29 thioridazine 200 TID, lithium 600 hs  600  250 2.6 ×106 
19 15 56 mesoridazine 25–50 BID  100  500 0.6 × 106 
20 23 13 thiothixene, lithium, benztropine mesylate  n.a.  n.a. n.a. 
21 22 26 haloperidol 25 QID, carbamazepine 400 BID, benztropine mesylate 2 BID 2000  275 2.6 × 106 
22 19 29 haloperidol 45 mg po qd  900  533 5.6 × 106 
23 21 16 fluphenazine 20 qd-wk  400  850 5.0 × 106 
24 23 23 fluphenazine decanoate i.m., lithium 300 qd, benztropine mesylate 2 qd  n.a.  n.a. n.a. 
25 29 15 haloperidol 5 BID  200  200 1.1 × 106 
26 17 chlorpromazine 50 qhs  50  450 1.5 × 106 
27 22 49 mesoridazine 300 qd  400  650 7.1 × 106 
28 36 10 haloperidol 5 TID  300  300 1.1 × 106 
29 20 25 fluphenazine 10 qd, benztropine mesylate 2 BID  200  900 8.2 × 106 
30 19 15 fluphenazine 40 qd, lithium 1200 qd, benztropine mesylate 8 qd, doxepine 50 qhs  800  700 3.8 × 106 
31 23 31 fluphenazine decanoate 50 mq wk 2400  800 9.1 × 106 
32 24 56 haloperidol 10 TID, benztropine mesylate 2 BID  600  600 7.0 × 106 
33 33 15 chlorpromazine 300 qd, carbamazepine 400 qd, diphenhydramine 50 qd  300  300 1.6 × 106 
34 19 40 chlorpromazine 100 BID  200  200 2.2 × 106 
35 17 32 fluphenazine 30 qd, lithium 900 qd  600  600 7.0 × 106 
36 26 thiothixene, amitryptiline, benztropine mesylate  n.a.  n.a. n.a. 
37 19 45 haloperidol decanoate 50 i.m. q 4 wks, haloperidol 5 TID, clonazepam 2 TID  900  400 5.3 × 106 
38 19 15 fluphenazine decanoate 37.5 i.m. wk, benztropine mesylate (dose n.a.) 1800 1000 5.5 × 106 
Mean (SD) 22.4 (5.2) 26.5 (15.2)   708 (683)  528 (252) 4.3×106 
45 20 17 none (untreated for 20 years)   0.18 × 106 
46 27 33 thioridazine, amitryptiline   1.6 × 106 
47 38 24 none (untreated for several months)   1.0 × 106 
48 13 17 risperidone   1.0 × 106 
49 18 13 clozapine   0.08 × 106 
50 15 45 none (never given)   
Mean (SD) 21.8 (9.3) 24.8 (12.1)    0.64 × 106 
51 22 risperidone, lithium   0.18 × 106 
52 39 14 lithium, bupropion, clonazepam, lorazepam   0.05 × 106 
53 30 27 haloperidol, diphenhydramine   1.2 × 106 
54 19 15 risperidone, valproate, venlafaxine   0.14 × 106 
55 19 thiothixene, carbamazepine, lithium, trazadone   0.15 × 106 
Mean (SD) 25.8 (8.7) 13.8 (8.4)    0.34 × 106 
Case Age of onset Illness duration Psychiatric medications at death (mg PO) Last CPZ equivalent Daily CPZ equivalent Lifetime CPZ (mg/eq) 
Ages of onset and illness durations are given in years. Abbreviations are as follows: CPZ = chlorpromazine, mg/eq = milligram equivalent. 
18 24 29 thioridazine 200 TID, lithium 600 hs  600  250 2.6 ×106 
19 15 56 mesoridazine 25–50 BID  100  500 0.6 × 106 
20 23 13 thiothixene, lithium, benztropine mesylate  n.a.  n.a. n.a. 
21 22 26 haloperidol 25 QID, carbamazepine 400 BID, benztropine mesylate 2 BID 2000  275 2.6 × 106 
22 19 29 haloperidol 45 mg po qd  900  533 5.6 × 106 
23 21 16 fluphenazine 20 qd-wk  400  850 5.0 × 106 
24 23 23 fluphenazine decanoate i.m., lithium 300 qd, benztropine mesylate 2 qd  n.a.  n.a. n.a. 
25 29 15 haloperidol 5 BID  200  200 1.1 × 106 
26 17 chlorpromazine 50 qhs  50  450 1.5 × 106 
27 22 49 mesoridazine 300 qd  400  650 7.1 × 106 
28 36 10 haloperidol 5 TID  300  300 1.1 × 106 
29 20 25 fluphenazine 10 qd, benztropine mesylate 2 BID  200  900 8.2 × 106 
30 19 15 fluphenazine 40 qd, lithium 1200 qd, benztropine mesylate 8 qd, doxepine 50 qhs  800  700 3.8 × 106 
31 23 31 fluphenazine decanoate 50 mq wk 2400  800 9.1 × 106 
32 24 56 haloperidol 10 TID, benztropine mesylate 2 BID  600  600 7.0 × 106 
33 33 15 chlorpromazine 300 qd, carbamazepine 400 qd, diphenhydramine 50 qd  300  300 1.6 × 106 
34 19 40 chlorpromazine 100 BID  200  200 2.2 × 106 
35 17 32 fluphenazine 30 qd, lithium 900 qd  600  600 7.0 × 106 
36 26 thiothixene, amitryptiline, benztropine mesylate  n.a.  n.a. n.a. 
37 19 45 haloperidol decanoate 50 i.m. q 4 wks, haloperidol 5 TID, clonazepam 2 TID  900  400 5.3 × 106 
38 19 15 fluphenazine decanoate 37.5 i.m. wk, benztropine mesylate (dose n.a.) 1800 1000 5.5 × 106 
Mean (SD) 22.4 (5.2) 26.5 (15.2)   708 (683)  528 (252) 4.3×106 
45 20 17 none (untreated for 20 years)   0.18 × 106 
46 27 33 thioridazine, amitryptiline   1.6 × 106 
47 38 24 none (untreated for several months)   1.0 × 106 
48 13 17 risperidone   1.0 × 106 
49 18 13 clozapine   0.08 × 106 
50 15 45 none (never given)   
Mean (SD) 21.8 (9.3) 24.8 (12.1)    0.64 × 106 
51 22 risperidone, lithium   0.18 × 106 
52 39 14 lithium, bupropion, clonazepam, lorazepam   0.05 × 106 
53 30 27 haloperidol, diphenhydramine   1.2 × 106 
54 19 15 risperidone, valproate, venlafaxine   0.14 × 106 
55 19 thiothixene, carbamazepine, lithium, trazadone   0.15 × 106 
Mean (SD) 25.8 (8.7) 13.8 (8.4)    0.34 × 106 
Table 3

Demographic information on Cohort 2

Case Diagnosis Age/sex Race Side pH PMI (h) Months in freezer COD MOD 
Means and standard deviations are printed below the last individual in each group. Abbreviations not given for Table 1: Bw/P = bipolar disorder with psychotic features, Bw/o P = bipolar disorder without psychotic features, A=Asian. 
39 CON 59/m 6.4 26 ASCVD natural 
40 CON 34/m 6.3 23 ASCVD natural 
41 CON 48/m 6.2 17 10 ASCVD natural 
42 CON 18/m 6.3 53 14 multiple injuries, transected spinal cord (T6) accident 
43 CON 48/m 6.1 12 ASCVD natural 
44 CON 52/m 6.5 28 12 ASCVD natural 
Mean (SD)  43 (15)   6.3 (0.1) 26.5 (14.2) 10.2 (2.4)   
45 CUS 52/m 6.0 61 17 ASCVD natural 
46 CUS 60/m 6.2 31 asphyxiation (drowning) accident 
47 CPS 62/f 6.1 26 13 multiple blunt force injuries accident 
48 CUS 30m 5.8 32 14 bronchopneumonia natural 
49 CUS 31/m 5.8 14 blunt force injuries (fall) suicide 
50 CUS 60/f 6.2 40 13 ASCVD natural 
Mean (SD)  49 (15)   6.0 (0.2) 34 (15.8) 12.2 (3.6)   
51 B w/P 29/m 6.0 48 blunt force injuries (fall) suicide 
52 B w/o P 54/m 5.8 39 12 blunt force injuries to head (fall), subdural hematoma accident 
53 B w/ P 57/m 6.2 19 ASCVD natural 
54 B w/ P 34/m 6.3 23 blunt force injuries (jump) suicide 
55 B w/ P 25/f 6.1 24 13 hanging suicide 
Mean (SD)  40(15)   6.1 (0.2) 30.6 (12.3) 10 (2.3)   
Case Diagnosis Age/sex Race Side pH PMI (h) Months in freezer COD MOD 
Means and standard deviations are printed below the last individual in each group. Abbreviations not given for Table 1: Bw/P = bipolar disorder with psychotic features, Bw/o P = bipolar disorder without psychotic features, A=Asian. 
39 CON 59/m 6.4 26 ASCVD natural 
40 CON 34/m 6.3 23 ASCVD natural 
41 CON 48/m 6.2 17 10 ASCVD natural 
42 CON 18/m 6.3 53 14 multiple injuries, transected spinal cord (T6) accident 
43 CON 48/m 6.1 12 ASCVD natural 
44 CON 52/m 6.5 28 12 ASCVD natural 
Mean (SD)  43 (15)   6.3 (0.1) 26.5 (14.2) 10.2 (2.4)   
45 CUS 52/m 6.0 61 17 ASCVD natural 
46 CUS 60/m 6.2 31 asphyxiation (drowning) accident 
47 CPS 62/f 6.1 26 13 multiple blunt force injuries accident 
48 CUS 30m 5.8 32 14 bronchopneumonia natural 
49 CUS 31/m 5.8 14 blunt force injuries (fall) suicide 
50 CUS 60/f 6.2 40 13 ASCVD natural 
Mean (SD)  49 (15)   6.0 (0.2) 34 (15.8) 12.2 (3.6)   
51 B w/P 29/m 6.0 48 blunt force injuries (fall) suicide 
52 B w/o P 54/m 5.8 39 12 blunt force injuries to head (fall), subdural hematoma accident 
53 B w/ P 57/m 6.2 19 ASCVD natural 
54 B w/ P 34/m 6.3 23 blunt force injuries (jump) suicide 
55 B w/ P 25/f 6.1 24 13 hanging suicide 
Mean (SD)  40(15)   6.1 (0.2) 30.6 (12.3) 10 (2.3)   
Figure 1.

Northern hybridization of GAP-43 32P-labeled probe to cortical poly-A RNA extracted from human brain showing one major 1.6 kB band (arrow) in lanes 1–8. Lanes contain poly(A) mRNA extracted from the pooled right and left hemispheres from multiple normal adult individuals from the following regions (1) whole cerebellum; (2) whole cerebral cortex; (3) medulla; (4) spinal cord; (5) occipital pole; (6) frontal lobe; (7) temporal lobe; (8) putamen.

Figure 1.

Northern hybridization of GAP-43 32P-labeled probe to cortical poly-A RNA extracted from human brain showing one major 1.6 kB band (arrow) in lanes 1–8. Lanes contain poly(A) mRNA extracted from the pooled right and left hemispheres from multiple normal adult individuals from the following regions (1) whole cerebellum; (2) whole cerebral cortex; (3) medulla; (4) spinal cord; (5) occipital pole; (6) frontal lobe; (7) temporal lobe; (8) putamen.

Figure 2.

Autoradiographic film produced from the RNase protection assay. The black bar on the left denotes the migration of a 622 bp and a 527 bp piece of the Msp1-digested pBR322 marker DNA (M). The standard curve (top) generated with known amounts of in vitro transcribed sense strand GAP-43 mRNA ranges from 0 to 100 pg (left to right) and is followed by the 38 samples which showed a 778 bp protected GAP-43 mRNA fragment (N = normal control, S= schizophrenic).

Figure 2.

Autoradiographic film produced from the RNase protection assay. The black bar on the left denotes the migration of a 622 bp and a 527 bp piece of the Msp1-digested pBR322 marker DNA (M). The standard curve (top) generated with known amounts of in vitro transcribed sense strand GAP-43 mRNA ranges from 0 to 100 pg (left to right) and is followed by the 38 samples which showed a 778 bp protected GAP-43 mRNA fragment (N = normal control, S= schizophrenic).

Figure 3.

Mean RNA levels in the prefrontal cortex of normal controls and schizophrenics from the RNase protection assays: attomoles of GAP-43/μg total cytoplasmic RNA (A) and of cyclophilin/μg total cytoplasmic RNA (B). Error bars represent the standard error. The mean GAP-43 mRNA level was 14.43 ± 10.68 amol in patients with schizophrenia (S) and 23.38 ± 15.13 amol in normal controls (N) (*P < 0.05).

Figure 3.

Mean RNA levels in the prefrontal cortex of normal controls and schizophrenics from the RNase protection assays: attomoles of GAP-43/μg total cytoplasmic RNA (A) and of cyclophilin/μg total cytoplasmic RNA (B). Error bars represent the standard error. The mean GAP-43 mRNA level was 14.43 ± 10.68 amol in patients with schizophrenia (S) and 23.38 ± 15.13 amol in normal controls (N) (*P < 0.05).

Figure 4.

Scattergrams of DLPFC GAP-43 mRNA in amol (y-axis) and PMI in hours (x-axis, A), brain pH (x-axis, C) and postnatal age in years (x-axis, E) for all 38 samples are displayed. Scattergrams of DLPFC cyclophilin mRNA in amol (y-axis) and PMI in hours (x axis, B), brain pH (x axis, D) and postnatal age in years (x-axis, F) for all 38 samples are shown (filled circles = normal controls, filled triangles = schizophrenics).

Figure 4.

Scattergrams of DLPFC GAP-43 mRNA in amol (y-axis) and PMI in hours (x-axis, A), brain pH (x-axis, C) and postnatal age in years (x-axis, E) for all 38 samples are displayed. Scattergrams of DLPFC cyclophilin mRNA in amol (y-axis) and PMI in hours (x axis, B), brain pH (x axis, D) and postnatal age in years (x-axis, F) for all 38 samples are shown (filled circles = normal controls, filled triangles = schizophrenics).

Figure 5.

Darkfield photomicrograph showing hybridization signal for GAP-43 mRNA in layer III and in layers V/VI overlying pyramidal neurons (arrows) in the normal (A), schizophrenic (B) and bipolar disorder (C) brains. (D) The absence of labeling when the GAP-43 mRNA sense strand was hybridized to the sections. A relative decrease in GAP-43 mRNA hybridization signal is seen over cortical pyramidal neurons in layers III and V/VI of the schizophrenic prefrontal cortex as compared with the bipolar disorder and control subjects. (Scale bar = 200 μm).

Figure 5.

Darkfield photomicrograph showing hybridization signal for GAP-43 mRNA in layer III and in layers V/VI overlying pyramidal neurons (arrows) in the normal (A), schizophrenic (B) and bipolar disorder (C) brains. (D) The absence of labeling when the GAP-43 mRNA sense strand was hybridized to the sections. A relative decrease in GAP-43 mRNA hybridization signal is seen over cortical pyramidal neurons in layers III and V/VI of the schizophrenic prefrontal cortex as compared with the bipolar disorder and control subjects. (Scale bar = 200 μm).

Figure 6.

The average number of silver grains per neuron per lamina III, V and VI are plotted for each normal control (circles), and schizophrenic (triangles) and bipolar disorder patients (squares). The number of GAP-43 mRNA silver grains per neuron is reduced in layers III, V and VI in the patients with schizophrenia compared with both control groups.

Figure 6.

The average number of silver grains per neuron per lamina III, V and VI are plotted for each normal control (circles), and schizophrenic (triangles) and bipolar disorder patients (squares). The number of GAP-43 mRNA silver grains per neuron is reduced in layers III, V and VI in the patients with schizophrenia compared with both control groups.

Figure 7.

High power bright field (A,B) of cells in layer III are shown. Note that pyramidal neurons in the brain of normal individuals (A) and of patients with schizophrenia (B) contain GAP-43 silver grains (solid arrows in A and B). Many non-pyramidal, round cells that contain lightly Nissl-stained nuclei contain a low level of silver grains (open arrow in A and B). Small darkly Nissl-stained nuclei have no apparent silver grain clusters localized over them. (Scale bar = 20 μm).

Figure 7.

High power bright field (A,B) of cells in layer III are shown. Note that pyramidal neurons in the brain of normal individuals (A) and of patients with schizophrenia (B) contain GAP-43 silver grains (solid arrows in A and B). Many non-pyramidal, round cells that contain lightly Nissl-stained nuclei contain a low level of silver grains (open arrow in A and B). Small darkly Nissl-stained nuclei have no apparent silver grain clusters localized over them. (Scale bar = 20 μm).

References

Aigner L, Caroni P (
1993
) Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones.
J Cell Biol
 
123
:
417
–429.
Aigner L, Caroni P (
1995
) Absence of persistent spreading, branching, and adhesion in GAP-43-depleted growth cones.
J Cell Biol
 
128
:
647
–660.
Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney WE Jr, Jones EG (
1995
) Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics.
Arch Gen Psychiat
 
52
:
258
–266.
American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders, 4th edn. Washington, DC: American Psychiatric Association.
Andreasen NC, Rezai K, Alliger R, Swayze VW II, Flaum M, Kirchner P, Cohen G, O'Leary DS (
1992
) Hypofrontality in neuroleptic-naive patients and in patients with chronic schizophrenia. Assessment with xenon 133 single-photon emission computed tomography and the Tower of London.
Arch Gen Psychiat
 
49
:
943
–958.
Arikuni T, Sakai M, Kubota K (
1983
) Columnar aggregation of prefrontal and anterior cingulate cortical cells projecting to the thalamic mediodorsal nucleus in the monkey.
J Comp Neurol
 
220
:
116
–125.
Barbas H, Pandya DN (
1989
) Architecture and intrinsic connections of the prefrontal cortex in the rhesus monkey.
J Comp Neurol
 
286
:
353
–375.
Barton AJL, Pearson RCA, Najlerahim A, Harrison PJ (
1993
) Pre- and postmortem influences on brain RNA.
J Neurochem
 
61
:
1
–11.
Benowitz LI, Routtenberg A (
1997
) GAP-43: an intrinsic determinant of neuronal development and plasticity.
Trends Neurosci
 
20
:
84
–91.
Benowitz LI, Perrone-Bizzozero NI, Finklestein SP, Bird ED (
1989
) Localization of the growth-associated phosphoprotein GAP-43 (B-50, F1) in the human cerebral cortex.
J Neurosci
 
9
:
990
–995.
Bertolino A, Nawroz S, Mattay VS, Barnett AS, Duyn JH, Moonen CTW, Frank JA, Tedeschi G, Weinberger DR (
1996
) Regionally specific pattern of neurochemical pathology in schizophrenia as assessed by multislice proton magnetic resonance spectroscopic imaging.
Am J Psychiat
 
153
:
1554
–1563.
Bertolino A, Callicott JH, Elman I, Mattay VS, Tedeschi G, Frank JA, Breier A, Weinberger DR (
1998
) Regionally specific neuronal pathology in untreated patients with schizophrenia: a proton magnetic resonance spectroscopic imaging study.
Biol Psychiat
 
43
:
641
–648.
Bertolino A, Esposito G, Callicott JH, Mattay VS, Van Horn JD, Frank JA, Berman KF, Weinberger DR (
2000
) Specific relationship between prefrontal neuronal N-acetylaspartate and activation of the working memory cortical network in schizophrenia.
Am J Psychiat
 
157
:
26
–33.
Callahan LM, Selski DJ, Martzen MR, Cheetham JE, Coleman PD (
1994
) Preliminary evidence: decreased GAP-43 message in tangle-bearing neurons relative to adjacent tangle-free neurons in Alzheimer's disease parahippocampal gyrus.
Neurobiol Aging
 
15
:
381
–386.
Eastwood SL, Harrison PJ (
1998
) Hippocampal and cortical growth-associated protein-43 messenger RNA in schizophrenia.
Neuroscience
 
86
:
437
–448.
Eastwood SL, Heffernan J, Harrison PJ (
1997
) Chronic haloperidol treatment differentially affects the expression of synaptic and neuronal plasticity-associated genes.
Mol Psychiat
 
2
:
322
–329.
Frith CD, Friston KJ, Herold S, Silbersweig D, Fletcher P, Cahill C, Dolan RJ, Frackowiak RSJ, Liddle PF (
1995
) Regional brain activity in chronic schizophrenic patients during the performance of a verbal fluency task.
Br J Psychiat
 
167
:
343
–349.
Fuster JM, Bauer RH, Jervey JP (
1985
) Functional interactions between inferotemporal and prefrontal cortex in a cognitive task.
Brain Res
 
330
:
299
–307.
Ganguli R, Carter C, Mintun M, Brar J, Becker J, Sarma R, Nichols T, Bennington E (
1997
) PET brain mapping study of auditory verbal supraspan memory versus visual fixation in schizophrenia.
Biol Psychiat
 
41
:
33
–42.
Garey LJ, Ong WY, Patel TS, Kanani M, Davis A, Mortimer AM, Barnes TRE, Hirsch SR (
1998
) Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia.
J Neurol Neurosurg Psychiat
 
65
:
446
–453.
Glantz LA, Lewis DA (
1997
) Reduction of synaptophysin immuno-reactivity in the prefrontal cortex of subjects with schizophrenia: regional and diagnostic specificity.
Arch Gen Psychiat
 
54
:
943
–952.
Glantz LA, Lewis DA (
2000
) Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia.
Arch Gen Psychiat
 
57
:
65
–73.
Goldberg TE, Gold JM (1995) Neurocognitive deficits in schizophrenia. In: Schizophrenia (Hirsch SR, Weinberger DR, eds), pp. 146–162. Oxford: Blackwell Science.
Goldman-Rakic PS (1991) Prefrontal cortical dysfunction in schizophrenia: the relevance of working memory. In: Psychopathology and the brain (Carroll BJ, Barrett JE, eds), pp. 1–23. New York: Raven Press.
Goldman-Rakic PS (
1994
) Working memory dysfunction in schizophrenia.
J Neuropsychiat Clin Neurosci
 
6
:
348
–357.
Goldman-Rakic PS, Selemon LD (
1997
) Functional and anatomical aspects of prefrontal pathology in schizophrenia.
Schizophr Bull
 
23
:
437
–458.
Harrison PJ, Heath PR, Eastwood SL, Burnet PWJ, McDonald B, Pearson RCA (
1995
) The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins.
Neurosci Lett
 
200
:
151
–154.
Honer WG, Falkai P, Chen C, Arango V, Mann JJ, Dwork AJ (
1999
) Synaptic and plasticity-associated proteins in anterior frontal cortex in severe mental illness.
Neuroscience
 
91
:
1247
–1255.
Honig LS, Herrmann K, Shatz CJ (
1996
) Developmental changes revealed by immunohistochemical markers in human cerebral cortex.
Cereb Cortex
 
6
:
794
–806.
Jacobson RD, Virag I, Skene JHP (
1986
) A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS.
J Neurosci
 
6
:
1843
–1855.
Jakubowski M, Roberts JL (
1992
) Multiplex solution hybridization– ribonuclease protection assay for quantification of different ribonucleic acid transcripts from snap-frozen neuroendocrine tissues of individual animals.
J Neuroendocrinol
 
4
:
79
–89.
Johnson SA, Morgan DG, Finch CE (
1986
) Extensive postmortem stability of RNA from rat and human brain.
J Neurosci Res
 
16
:
267
–280.
Kanazir S, Ruzdijic S, Vukosavic S, Ivkovic S, Milosevic A, Zecevic N, Rakic L (
1996
) GAP-43 mRNA expression in early development of human nervous system.
Brain Res Mol Brain Res
 
38
:
145
–155.
Karson CN, Mrak RE, Schluterman KO, Sturner WQ, Sheng JG, Griffin WST (
1999
) Alterations in synaptic proteins and their encoding mRNAs in prefrontal cortex in schizophrenia: a possible neurochemical basis for ‘hypofrontality’.
Mol Psychiat
 
4
:
39
–45.
Keks NA, Hill C, Opeskin K, Copolov D, Dean B (1999) Psychiatric diagnosis after death: the problems of accurate diagnosis from case history review and relative interviews. In: Using CNS tissue in psychiatric research: a practical guide (Dean B, Kleinman JE, eds), pp. 19–37. Amsterdam: Overseas Publishers Association.
Kleinman JE, Hyde TM, Herman MM (1995) Methodological issues in the neuropathology of mental illness. In: Psychopharmacology: the fourth generation of progress (Bloom FE, Kupfer DJ, eds), pp. 859–864. New York: Raven Press.
Kosik KS, Orecchio LD, Bruns GAP, Benowitz LI, MacDonald GP, Cox DR, Neve RL (
1988
) Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human.
Neuron
 
1
:
127
–132.
Lazar LM, Blum M (
1992
) Regional distribution and developmental expression of epidermal growth factor and transforming growth factor-α mRNA in mouse brain by a quantitative nuclease protection assay.
J Neurosci
 
12
:
1688
–1697.
Levitt JB, Lewis DA, Yoshioka T, Lund JS (
1993
) Topography of pyramidal neuron intrinsic connections in macaque monkey prefrontal cortex (areas 9 and 46).
J Comp Neurol
 
338
:
360
–376.
Lewis DA (
1997
) Development of the prefrontal cortex during adolescence: insights into vulnerable neural circuits in schizophrenia.
Neuropsychopharmacology
 
16
:
385
–398.
Liu Y, Chapman ER, Storm DR (
1991
) Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.
Neuron
 
6
:
411
–420.
Lovinger DM, Colley PA, Akers RF, Nelson RB, Routtenberg A (
1986
) Direct relation of long-term synaptic potentiation to phosphorylation of membrane protein F1, a substrate for membrane protein kinase C.
Brain Res
 
399
:
205
–211.
Manoach, DS, Gollub, RL, Benson, ES, Searl, MM, Goff, DC, Halpern, E, Saper, CB, Rauch, SL. (
2000
) Schizophrenic subjects show aberrant fMRI activation of dorsolateral prefrontal cortex and basal ganglia during working memory performance.
Biol Psychiat
 
48
:
99
–109.
Nauta WJH (
1971
) The problem of the frontal lobe: a reinterpretation.
J Psychiat Res
 
8
:
167
–187.
Nelson RB, Routtenberg A (
1985
) Characterization of protein F1 (47 kDa, 4.5 pI): a kinase C substrate directly related to neural plasticity.
Exp Neurol
 
89
:
213
–224.
Nelson RB, Friedman DP, O'Neill JB, Mishkin M, Routtenberg A (
1987
) Gradients of protein kinase C substrate phosphorylation in primate visual system peak in visual memory storage areas.
Brain Res
 
416
:
387
–392.
Neve RL, Finch EA, Bird ED, Benowitz LI (
1988
) Growth-associated protein GAP-43 is expressed selectively in associative regions of the adult human brain.
Proc Natl Acad Sci USA
 
85
:
3638
–3642.
Neve RL, Perrone-Bizzozero NI, Finklestein S, Zwiers H, Bird E, Kurnit DM, Benowitz LI (
1987
) The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of human and rat mRNAs.
Brain Res Mol Brain Res
 
2
:
177
–183.
Pakkenberg B (
1993
) Total nerve cell number in neocortex in chronic schizophrenics and controls estimated using optical disectors.
Biol Psychiat
 
34
:
768
–772.
Pearlson GD, Petty RG, Ross CA, Tien AY (
1996
) Schizophrenia: a disease of heteromodal association cortex?
Neuropsychopharmacology
 
14
:
1
–17.
Perrone-Bizzozero NI, Sower AC, Bird ED, Benowitz LI, Ivins KJ, Neve RL (
1996
) Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia.
Proc Natl Acad Sci USA
 
93
:
14182
–14187.
Rajkowska G, Goldman-Rakic PS (
1995
) Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria.
Cereb Cortex
 
5
:
307
–322.
Rajkowska G, Selemon LD, Goldman-Rakic PS (
1998
) Neuronal and glial somal size in the prefrontal cortex: a postmortem morphometric study of schizophrenia and Huntington disease.
Arch Gen Psychiat
 
55
:
215
–224.
Selemon LD, Rajkowska G, Goldman-Rakic PS (
1995
) Abnormally high neuronal density in the schizophrenic cortex. A morphometric analysis of prefrontal area 9 and occipital area 17.
Arch Gen Psychiat
 
52
:
805
–818.
Selemon LD, Rajkowska G, Goldman-Rakic PS (
1998
) Elevated neuronal density in prefrontal area 46 in brains from schizophrenic patients: application of a three-dimensional, stereologic counting method.
J Comp Neurol
 
392
:
402
–412.
Shea TB, Benowitz LI (
1995
) Inhibition of neurite outgrowth following intracellular delivery of anti-GAP-43 antibodies depends upon culture conditions and method of neurite induction.
J Neurosci Res
 
41
:
347
–354.
Shea TB, Perrone-Bizzozero NI, Beermann ML, Benowitz LI (
1991
) Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis.
J Neurosci
 
11
:
1685
–1690.
Strittmatter SM, Fankhauser C, Huang PL, Mashimo H, Fishman MC (
1995
) Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43.
Cell
 
80
:
445
–452.
Thompson PM, Sower AC, Perrone-Bizzozero NI (
1998
) Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia.
Biol Psychiat
 
43
:
239
–43.
Torrey EF (1983) Surviving schizophrenia. New York: Harper & Row.
Webster MJ, Weickert CS, Herman MM, Hyde TM, Kleinman JE (2001) Synaptophysin and GAP-43 mRNA levels in the hippocampus of subjects with schizophrenia. Schizophr Res (in press).
Weickert CS, Blum M (
1995
) Striatal TGF-alpha: postnatal developmental expression and evidence for a role in the proliferation of subependymal cells.
Brain Res Devl Brain Res
 
86
:
203
–216.
Weickert CS, Kleinman JE (
1998
) The neuroanatomy and neurochemistry of schizophrenia.
Psychiat Clin North Am
 
21
:
57
–75.
Weinberger DR (
1993
) A connectionist approach to the prefrontal cortex.
J Neuropsychiat Clin Neurosci
 
5
:
241
–253.
Weinberger DR, Lipska BK (
1995
) Cortical maldevelopment, antipsychotic drugs, and schizophrenia: a search for common ground.
Schizophr Res
 
16
:
87
–110.
Weinberger DR, Berman KF, Zec RF (
1986
) Physiologic dysfunction of dorsolateral prefrontal cortex in schizophrenia. I. Regional cerebral blood flow evidence.
Arch Gen Psychiat
 
43
:
114
–124.
Weinberger DR, Berman KF, Suddath R, Torrey EF (
1992
) Evidence of dysfunction of a prefrontal-limbic network in schizophrenia: a magnetic resonance imaging and regional cerebral blood flow study of discordant monozygotic twins.
Am J Psychiat
 
149
:
890
–897.
Whitfield HJ Jr, Brady LS, Smith MA, Mamalaki E, Fox RJ, Herkenham M (
1990
) Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol.
Cell Mol Neurobiol
 
10
:
145
–157.
Yeterian EH, Pandya DN (
1994
) Laminar origin of striatal and thalamic projections of the prefrontal cortex in rhesus monkeys.
Exp Brain Res
 
99
:
383
–398.
Zalcman S, Endicott J, Clayton P, Winokur G (1983) Diagnostic Evaluation After Death (DEAD). Rockville, MD: National Institute of Mental Health.
Zuber MX, Goodman DW, Karns LR, Fishman MC (
1989
) The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells.
Science
 
244
:
1193
–1195.