Main

Abnormalities in cognitive processes involving attention and working memory are becoming accepted as hallmarks of schizophrenia (Andreasen 1997; Saykin et al. 1991). Although the classic symptoms of the disorder, including hallucinations, delusions, and emotional blunting, have traditionally led to the assumption that multiple brain regions are involved in their expression, the possibility that these may instead result from defects in a primary brain structure has also received considerable attention. To address this issue, a wide variety of approaches have been employed with the goal of identifying brain regions that deviate from normality in schizophrenics. Primary among these have been such imaging techniques as positron emission tomography (PET) and magnetic resonance imaging (MRI). However, despite the volume of data produced and extent to which these have been applied, consensus on a physiological correlate of disease symptomatology remains to be reached. An attractive theme that has emerged as a result of post-mortem, imaging, and animal studies, is that defects in the primary structures of the brain that moderate or filter incoming information may be critical to the disease process. In this regard, the concept of a defective cortico-striatal-thalamic circuit in schizophrenia has become widely recognized (Carlsson and Carlsson 1990; Carlsson et al. 1997). In support of this, all of these regions, including the frontal cortex (Buchsbaum et al. 1982; Siegel et al. 1993), striatum (Siegel et al. 1993; Shihabuddin et al. 1998), and thalamus (Andreasen et al. 1996), have been implicated as potentially defective in schizophrenics.

As a complement to imaging studies, our laboratory has applied a strategy involving the post-mortem measurement of the mitochondrial respiratory chain enzyme cytochrome-c oxidase (COX) in an attempt to localize altered brain function in schizophrenia. This approach is based upon a strong body of evidence that indicates that neuronal COX is highly regulated by the energy demands of the cell and, as such, represents an endogenous marker of cellular energy metabolism over time (Wong-Riley 1989). Interest in COX as a marker of neuronal function rests upon many of the same assumptions implicit in the use of PET in the measurement of regional glucose metabolism and blood flow. Neurons are highly dependent upon oxidative phosphorylation as the primary pathway for the generation of ATP, of which 40 to 60% is utilized in the maintenance of ion gradients by ATPases. In support of this, a strong correlation has been demonstrated between the regulation of COX and Na+,K+ ATPase in brain tissue (Hevner et al., 1992). Traditionally, however, the strongest evidence that COX is coupled to neuronal energy demands comes from studies in which changes in COX activity can be induced by experimental interventions that alter neuronal activity. Perhaps the most interesting study in this regard was performed utilizing a histochemical technique to demonstrate reduced COX activity in the brains of cats in which chronic neuronal inactivity was induced in visual cortex by monocular suture (Wong-Riley 1979). In addition, studies have shown that monocular retinal impulse inhibition with tetrodotoxin results in a decrease in COX activity in specific regions of the monkey visual cortex and thalamus (Wong-Riley and Carrol 1984). In terms of localization, evidence suggests that as much as 60% of COX activity in a particular region reflects dendritic activity; whereas, glial cell activity is responsible for less than 5% (Wong-Riley, 1989).

In this article, we have primarily explored the relative contribution of energy metabolism in various brain regions to emotional, intellectual, and motor capacity in schizophrenics. Based upon extensive research alluding to an increase in dopamine receptor binding in striatal structures in schizophrenics, primarily the putamen, an attempt was also made to address the possibility that this finding may correlate with a general increase in metabolic activity in the region.

MATERIALS AND METHODS

Materials

PD10 columns were obtained from Pharmacia (Uppsala, Sweden). [3H]raclopride was purchased from New England Nuclear (Boston, MA). Haloperidol (Haldol®) was obtained from Janssen (Beerse, Belgium). Cytochrome-c (horse heart) and all other compounds were purchased from Sigma Chemical Co. (St. Louis, MO).

Subjects and Tissue Preparation

For the primary study relating to COX, D2 binding, and cognitive measures, putamen samples were obtained from 12 schizophrenics and 10 controls with no history of psychiatric illness. The average ages for these two groups were 82 ± 11 (SD) for schizophrenics and 80 ± 7 (SD) for controls. Post-mortem intervals were 42 ± 12 (SD) for schizophrenics and 51 ± 14 (SD) for controls. Data pertaining to patient demographics are shown in Table 1 . Results in Figures 1,2, and 3 all relate specifically to these samples.

Table 1 Clinical Characteristics of Schizophrenics and Controls used for Putamen Studies
Full size table
Figure 1
figure 1

COX activity (nmol/min/mg), D2 binding (fmol/mg), and D2 Kd coefficients (nm) in the putamen of schizophrenics. Significance was determined using Student's t-test. * p < .05; ** p < .005. Significance for Bmax was p = .054

Full size image
Figure 2
figure 2

Correlations between COX (left column) and D2 binding (right column) and motor, intellectual, and emotional impairment in the putamen of schizophrenics. Graphs represent linear regression lines from simple regression analysis. Significance was determined by Student's t-test

Full size image
Figure 3
figure 3

Correlations between COX and D2 binding in the putamen of (A) controls and (B) schizophrenics. Graphs represent linear regression lines from simple regression analysis. Significance was determined using Student's t-test

Full size image

To ascertain if findings in the putamen were regionally specific, a meta-analysis was also conducted on brain samples that have previously been described biochemically but not in relation to cognitive measures (Cavelier et al. 1995; Prince et al. 1999). The number of samples employed for each brain region is listed in Table 2 . For this study, brain samples were re-analyzed, and correlation coefficients were established, with emotional, intellectual, and motor impairment scales (described below).

Table 2 Remaining Correlations between COX Activity and Emotional, Intellectual, and Motor Impairment in Various Brain Regions in Schizophrenics
Full size table

Patients were diagnosed according to the Diagnostic and Statistical Manual (DSM) III-R. All schizophrenics had suffered from a chronic course of the disease and been treated extensively with various neuroleptics. All patients had received neuroleptic treatment ranging from 50 to 400 mg/day (chlorpromazine equivalents) within the last 2 months before death (Table 1). No evidence of Alzheimer's disease or other neuropathological features of degenerative disease could be found in either schizophrenics or controls. No evidence of substance abuse was documented in any of the patients. Brain tissue specimens from the frontal cortex, caudate nucleus, putamen, nucleus accumbens, globus pallidus, thalamus, and mesencephalon were obtained at autopsy, and dissections were performed according to anatomical landmarks: the nucleus accumbens was taken from the junction between the frontal caudate nucleus and putamen. The mesencephalon was a cross section of brainstem below the superior and inferior colliculi. This rough dissection of the mesencephalon includes portions of the A8, A9, A10, and raphe nucleus. Following dissection, brain samples were frozen in liquid nitrogen and crushed into a course powder before storage at −70°C. For cytochrome-c oxidase activity measurements, brain samples were homogenized in a buffer consisting of 10 mm potassium phosphate (pH 7.6), 1 mm EDTA, 0.25 m sucrose in an Ultra-Turrax set on full speed for 30 s. Homogenates were then frozen at −70°C until assayed.

Cognitive measures in schizophrenics were assessed according to the geriatric rating scale constructed by Gottfries and Gottfries (Adolfsson et al. 1981). The rating scale consists of a series of yes/no questions relating to general patient performance and contains three subscales measuring impairment of motor performance, intellectual impairment, and emotional impairment. The system was originally designed to be applied to post-mortem brain biochemical studies primarily involving neurotransmitter/metabolite levels. Values are reported posthumously during the week following death by a trained nurse in charge of individual patient care. On the basis that they were not institutionalized nor under psychiatric care before death, no values for controls were obtained. Biochemical studies were performed blind to rating scale results.

Cytochrome-c Oxidase Assay

Cytochrome-c oxidase was assayed according to a modification of the spectrophotometric method of Yonetani and Ray (1965). Reduced cytochrome c was prepared by the addition of 30 mg Na2S2O4/100 mg cytochrome c in a 10 mm potassium phosphate buffer (pH 7.6) containing 1 mm EDTA, and separated on a PD10 column (Pharmacia). Incubations were performed in 10 mm potassium phosphate buffer (pH 7.6), 1 mm EDTA, and 25 μm reduced cytochrome c at 25°C. Upon the addition of approximately 100 μg protein, the change in absorbance at 550 nm was determined on a Jasco V-550 spectrophotometer for 5 min. Initial rates were determined differentially where -d[ferrocytochrome c]/dt is derived from polynomial plots at zero time using an extinction coefficient of 19.0 mm−1cm−1 (Yonetani and Ray 1965).

[3H]Raclopride Binding Assay

Experiments were carried out as described by Lepiku et al. (1996). Brain tissue was resuspended in ice-cold Tris-HCl (50 mm, pH 7.4) containing NaCl (120 mm), MgCl2 (5 mm) and EDTA (1 mm). After a centrifugation at 33,000 g for 20 min and homogenization using a Potter-S glass-Teflon homogenizer (1,000 rpm, six passes), the membranes were incubated at 37° for 30 min. The pellet was then re-homogenized in the same buffer. D2/D3 receptor labeling was carried out in the presence of 0.5 to 10 nm [3H]-raclopride (spec. act. 78.4 Ci/mmol, N.E.N.) at room temperature with approximately 0.2 mg protein per tube in a total incubation volume of 0.3 ml. Haloperidol (10 μm) was added to determine nonspecific binding. Incubation was terminated after 30 min by rapid filtration over Whatman GF/B filters using Brandel Cell Harvester (M-24S). The filters were washed with 9 ml cold incubation buffer, dried, and assayed for radioactivity by liquid scintillation spectrometry. All binding data were analyzed by nonlinear least-squares regression analysis using a commercial program GraphPad PRISM 2.0 (GraphPad Software, San Diego, CA)

Protein and DNA Measurements

The total amount of protein in the samples was determined according to the Markwell et al. (1978) modification of the Lowry et al. (1951) procedure using bovine albumin as a standard. Based upon extensive evidence that suggests DNA is a sensitive indicator of cell number (Downs and Wilfinger 1983; Rago et al. 1990) the total quantity of dsDNA in samples was determined fluorometrically using PicoGreen (Molecular Probes) based upon its superior selectivity for DNA over RNA (Singer et al. 1997). Samples (10 μl) were added to a 50 μl TE buffer (0.01 m Tris-HCl buffer, pH 8.0, 1 mm EDTA) containing 0.01% SDS, incubated at RT for 10 minutes and then sonicated at low power for 5 s on a Branson B15 cell disruptor. A 1 ml solution containing 0.6 μm PicoGreen was then added to the samples and incubated for 5 min at RT. Fluorescence was then determined on a Jasco FP-777 spectrofluorometer using 480 nm and 520 nm excitation and emission wavelengths. Calf thymus DNA was used as a standard, and a value of 7.23 pg DNA/cell was used to calculate cell number.

Statistics

All statistical analyses were performed using StatView v4.5.1 (Abacus Concepts) and Prism (GraphPad). Statistically significant differences in the means of biochemical data were established using Student's t-test, ANOVA, and Fisher's PLSD. Presented correlations were calculated by simple linear regression analysis, and p-values were obtained using paired t-tests. Significance was also assessed using Spearman rank correlation coefficients.

RESULTS

As an anticipated effect of long-term neuroleptic treatment (Prince et al. 1997a,b; Prince et al. 1998), COX activity was found to be increased in the putamen in this group of schizophrenics (Figure 1) Levels were essentially consistent with those of an earlier study (Prince et al. 1998). To allow for comparison between brain regions, material from other brain regions that had previously been examined was reanalyzed. No marked deviations from previously reported values were found. On the basis that clinical records typically demonstrate a varied dosing regimen for schizophrenic patients, it was also important to confirm that D2 receptor Kd levels were also elevated as compared to controls, offering indirect evidence of the presence of residual neuroleptics (Figure 1) Finally, D2 receptor levels, measured with 3H-raclopride, were also found to be elevated in schizophrenics, offering additional support for the handful of studies that have addressed this question (Burt et al. 1977; Mackay et al. 1982) and suggested that this results from neuroleptic treatment (Figure 1)

The primary finding in the present study was the significant correlation between COX activity and intellectual and emotional impairment, but not motor impairment, in the putamen of chronically medicated schizophrenics (Figure 2) Results using Spearman rank correlation coefficients were p = .0067 and p = .0121 for intellectual and emotional impairment, respectively. This effect was specific for the putamen, because none of the other regions investigated were found to correlate significantly with these parameters (Table 2). In this regard, no significant correlations could be found between these three measures and COX in the frontal cortex, caudate nucleus, nucleus accumbens, globus pallidus, thalamus, or mesencephalon. However, a tendency toward a positive correlation was evident in the globus pallidus and mesencephalon with regard to motor impairment, but significance did not fall below the p < .1 level (Table 2). In contrast with the correlation between COX and cognitive measures in the putamen, a moderate but significant positive correlation was observed between D2 receptor binding and emotional impairment in the region (Figure 2) This was not evident for motor impairment, but a tendency was apparent for intellectual impairment (Figure 2) A unique finding was also made in terms of a correlation between COX activity and D2 binding in the putamen (Figure 3) In this regard, a significant positive correlation could be demonstrated in controls, but this was apparently absent in schizophrenics. Neither of these two latter relationships, D2 binding and emotional impairment nor COX and D2 binding, were significant when Spearman rank correlation coefficients were used. Nonetheless, these results are still presented, and discussion is raised around them based on the significance evidenced by simple linear regression analysis.

Although no assessment of dendritic density was made in this study, a measure that should be reflected by both COX and D2 binding, attempts were made to correlate cell density with COX, D2 binding, and cognitive measures. The underlying assumption in this analysis was that protein measures alone would not suffice if a generalized change in synaptic density had occurred. On this basis, the relationship between protein and DNA quantity was anticipated to reveal a possible deviation. In this regard, no significant findings were made (data not shown). A high correlation was found between protein and DNA quantity (data not shown), suggesting that both measures are closely related.

A final correlation study was performed between COX activity in various brain regions and D2 binding in the putamen. Significant positive correlations were observed between the mesencephalon and thalamus and D2 binding (p < .005 in both cases; data not shown). In addition, neither COX nor D2 binding were correlated with post-mortem interval or age (data not shown).

DISCUSSION

In the present study, we provide evidence that implicates the specific involvement of the putamen in the regulation of cognitive functions that may be of relevance for schizophrenia. The concept that neuroleptics impart their effects via an enhancement of energy metabolism in specific brain structures, particularly the striatum is supported by a firm base of data (Prince et al. 1997a,b; Prince et al. 1998; DeLisi et al. 1985; Holcomb et al. 1996). On the basis that patients in this study were highly medicated before death, results strengthen the hypothesis that an elevation in energy metabolism in the putamen, possibly reflecting enhanced excitatory transmission, may underlie some of the beneficial effects of neuroleptics. However, this study is limited by several factors. The primary measure of energy metabolism (COX activity) is still subject to scrutiny based upon limited knowledge about stability of the protein in the brain over time. This is particularly relevant considering the extended post-mortem durations of patients. In addition, results reflect findings in a geriatric group and may not be indicative of what occurs in a younger population.

The search for associations between schizophrenic behavior and brain physiology has seen a wide variety of approaches over the years. The field has primarily benefited from such brain imaging techniques as PET and MRI and, from these, several cohesive themes have emerged. In this regard, the concept of “hypofrontality” has played a central role in our understanding of the disease (Siegel et al. 1993; Buchsbaum et al. 1992). However, assumptions about complex patterns of altered brain function are giving way to theories that attempt to define schizophrenia based upon alterations in a fundamental cognitive process (Sabri et al. 1997; Andreasen et al. 1997; Andreasen 1997). A convincing argument for this being the case stems from findings that a cognitive deficit in schizophrenia remains stable, even in the presence of fluctuating symptoms (Weinberger and Gallhofer 1997).

Although studies in which brain imaging has been employed to describe correlations between symptoms and brain alterations in schizophrenics are relatively common (Sabri et al. 1997; Okubo et al. 1997), findings that correlate basic cognitive measures with brain changes are rare. In this regard, correlations have been found between cognitive neuropsychological indexes and striatal size (Stratta et al. 1997) and glucose metabolic rate (Nordahl et al. 1996). Although cortical regions may also play an important role in schizophrenic pathology (Nordahl et al. 1996; Shenton et al. 1992; Humphries et al. 1996), evidence implicating the striatum is perhaps more substantial (Siegel et al. 1993). This is important considering the fact that our present results suggest a pronounced correlation with emotional and intellectual impairment and COX in the putamen, but not in the frontal cortex. The critical involvement of the putamen in information processing in the brain is well documented (Brown et al. 1997) In this regard, the concept of the putamen as a regulator of information to the thalamus is particularly pertinent to schizophrenic pathology (Carlsson et al. 1997; Smith and Bolam 1990).

Although the case for the putamen is formidable, several anomalies in the present study confound the evidence. The foremost of these is the lack of correlation between psychiatric parameters and COX activity in the caudate nucleus or nucleus accumbens, both of which contain high levels of dopamine receptors. However, we have previously reported that COX activity is decreased in the caudate, but elevated in the putamen in schizophrenics, a fact that may aid in explaining this dilemma (Prince et al. 1999). In this regard, it was suggested that the effects of neuroleptics in the caudate may be subordinate to those upon the putamen in terms of elevating functional activity (Prince et al. 1998a). This is of importance based upon recent evidence that the caudate is involved in working memory (Levy et al. 1997). Thus, an alternative explanation in this matter is that a lack of correlation in the caudate in schizophrenics may itself be “pathological” and suggestive of damaged circuitry. Indeed, the lack of ability of neuroleptics to reverse a potential deficit in the caudate may underlie some of their therapeutic shortcomings in terms of inconsistent effects on cognitive function (King 1990; Hindmarch 1994).

The second anomalous finding in this study was the positive correlation between emotional impairment and D2 receptor binding in the putamen. To our knowledge, this is the first time that elevated D2 binding has been shown to be detrimentally associated with a behavioral parameter. Although the elevation of dopamine receptors by neuroleptics is well established (Mackay et al. 1982), the behavioral consequences have been more difficult to define (Burt et al. 1977). In this regard, the main focus has been on such extrapyramidal disturbances as tardive dyskinesia, which are assumed to result from chronic neuroleptic treatment. On the basis that no correlation was found between D2 levels and motor impairment, present results suggest that extrapyramidal disturbances may not be directly coupled to D2 receptor elevation in the putamen. Instead, it seems that an elevation in D2 receptor binding may be more relevant for emotional blunting, which is also a common side effect of neuroleptic treatment (Casey 1995).

The final finding in this study that deserves attention is the correlation between energy metabolism and D2 binding in the putamen in controls and the absence of a correlation in schizophrenics. Although the biochemical basis for this effect is difficult to ascertain, it is likely to result from neuroleptic treatment. This may also have relevance for the finding of a positive association between D2 levels and emotional impairment. That this effect is dependent upon neuroleptics is supported by the finding that D2 levels in normal individuals are negatively correlated with “detachment,” a measure of social withdrawal according to the Karolinska Scales of Personality (KSP) (Farde et al. 1997). Thus, it seems that under normal circumstances, D2 binding and COX are correlated, reflecting functioning circuitry that may be disturbed by neuroleptics. The chain of events can perhaps be envisioned as follows: as D2 receptors are blocked, the initial effect is to enhance the effect of the glutamatergic system in the putamen, resulting in an increase in metabolic rate in the region and, thus, an increase in the need for metabolic machinery; that is, COX. An increase in D2 levels may then occur disproportionately with this effect. In terms of significance, the primary location of mitochondria in the brain is within dendrites, implying that increases in energy metabolism are also predominantly dendritic (Wong-Riley 1989). The concept that increased synaptic density in the putamen may result from neuroleptic treatment receives support from findings of increased synaptophysin levels after haloperidol treatment (Eastwood et al. 1994).

In summary, the present study provides evidence that an increase in energy metabolism in the putamen may underlie an augmentation of intellectual and emotional capacity in schizophrenics. Evidence that this occurs as a result of chronic neuroleptic treatment is substantial (Prince et al. 1997a,b; Prince et al. 1998; Delisi et al. 1985; Holcomb et al. 1996) In contrast, an elevation in D2 receptor binding seems to have a detrimental effect upon emotion. Together, these findings illuminate the possibility of identifying pharmacological approaches that strive to increase energy metabolism in the putamen, while at the same time, minimizing an upregulation of D2 receptors.