Phospholipid abnormalities in postmortem schizophrenic brains detected by 31P nuclear magnetic resonance spectroscopy: a preliminary study.

It has been hypothesized that schizophrenia arises from cell membrane abnormalities due to changes in phospholipid (PL) composition and metabolism. We have used high resolution, in vitro 31P nuclear magnetic resonance (NMR) to characterize the PLs in left frontal cortex (gray matter) of postmortem brain from four schizophrenics and five controls. High resolution 31P NMR spectra were obtained in an organic-solvent system to resolve PL classes (headgroups) and in a sodium-cholate, aqueous dispersion system to resolve phosphatidylcholine (PC) molecular species. Multivariate analysis which included the major PC molecular species and phosphatidylinositol (PI) showed a significant difference between schizophrenics and controls. Analysis of specific interactions showed that the PI was significantly higher in the schizophrenic group than in the control group. There were no differences between the two groups for other individual PL classes, or for individual PL subclasses determined by the linkage type at the sn-1 position on glycerol. There was a trend for total PL content to be higher in schizophrenics than in controls. There was no evidence for elevated lysophosphatidylcholine or lysophosphatidylethanolamine in schizophrenia. The intensity of the PC peak representing molecular species with one saturated and one unsaturated (one or two double bonds) acyl chain was higher for the schizophrenic group than for the control group. Although these results are not in complete agreement with previous studies, they support the idea that PL abnormalities occur in the brain in schizophrenia and that fatty acid metabolism may be abnormal.

Differential and region-specific activation of mitogen-activated protein kinases following chronic administration of phencyclidine in rat brain.

We have previously demonstrated elevation of the extracellular signal-regulated kinase (ERK) pathway in the cerebellum from patients with schizophrenia, an illness that may involve dysfunction of the N-methyl-D-aspartate (NMDA) receptor. Since the NMDA antagonist, phencyclidine (PCP), produces schizophrenic-like symptoms in humans, and abnormal behavior in animals, we examined the effects of chronic PCP administration in time- and dose-dependent manner on ERK and two other members of mitogen-activated protein kinase family, c-Jun N-terminal protein kinase (JNK) and p38, in rat brain. Osmotic pumps for PCP (18 mg/kg/day) and saline (controls) were implanted subcutaneously in rats for three, 10, and 20 days. Using Western blot analysis, we found no change at three days, but a significant increase in the phosphorylation of ERK1, ERK2 and MEK in the cerebellum at 10- and 20-days of continuous PCP infusion. For the experiments involving various doses of PCP, rats were infused with PCP at concentrations of 2.5, 10, 18, or 25 mg/kg/day, or saline for 10 days. We observed a dose-dependent elevation in the phosphorylation of ERK1 and ERK2 only in the cerebellum but not in brainstem, frontal cortex or hippocampus. The activities of JNK and p38 were unchanged in all investigated brain regions including cerebellum. These results demonstrate that chronic infusion of PCP in rats produces a differential and brain region-specific activation of MAP kinases, suggesting a role for the ERK signaling pathway in PCP abuse and perhaps in schizophrenia.

Increased levels of transcription factors Elk-1, cyclic adenosine monophosphate response element-binding protein, and activating transcription factor 2 in the cerebellar vermis of schizophrenic patients.

BACKGROUND: We investigated the levels of transcription factors associated with activation of the mitogen-activated protein (MAP) kinase pathway in schizophrenics using postmortem brain samples. These studies were done to determine whether our previous findings of abnormal levels of the MAP kinases in the cerebellar vermis were linked to additional downstream targets of this signal transduction pathway. METHOD: We measured the protein levels of 3 transcription factors in nuclear fractions of postmortem samples from cerebellar vermis of 10 patients with schizophrenia and 13 control subjects: Elk-1, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and activating transcription factor 2 (ATF-2). Studies in rats examined the postmortem stability and effect of haloperidol and risperidone on levels of Elk-1, cAMP, and ATF-2 proteins. RESULTS: We found a significant increase in the protein levels of Elk-1 (mean+SD, 4489+/-659 vs 2915+/-583 arbitrary densitometric units [P<.001]), CREB (mean +/- SD, 2149 1061 vs 904+/-711 arbitrary densitometric units [P=.003]) and ATF-2 (mean+/-SD, 1421 854 vs 512+/-394 arbitrary densitometric units [P=.003]) in the cerebellar vermis of schizophrenic subjects. Complementary studies in rats indicate that these findings can not be attributed to subacute treatment with antipsychotic medications. CONCLUSION: Taken together with the alterations of MAP kinases previously reported, and the findings of elevations of downstream transcription targets, we suggest that the MAP kinase signal transduction pathway contributes to the cerebellar abnormalities in schizophrenia.

Contribution of trifluoperazine metabolites to the in vivo (19)F NMR spectrum of rat brain.

Fluorine-19 NMR spectra were acquired from extracts of tissues from heads of rats given the antipsychotic drug trifluoperazine (TFP). Contributions to the in vivo (19)F spectra from tissues other than brain were negligible. The in vivo (19)F resonance at -62.3 ppm from CCl(3)F consisted of 6-8 resolved resonances in vitro. Some in vitro resonances were assigned to previously identified TFP metabolites. Multiple resonances in vitro partially explain the relatively large line width seen in vivo for TFP. Unidentified metabolites were observed at about -74 to -75 ppm in a number of spectra of extracts of brain and muscle.

Effects of antipsychotic drugs on metabolite ratios in rat brain in vivo.

Localized in vivo proton magnetic resonance spectroscopy at 4.7 T was used to examine the brains of rats that were given the antipsychotic drugs haloperidol, clozapine, or olanzapine for 1 week. Spectra were collected before and during treatment. The ratios of N-acetylaspartate (NAA) to creatine (Cr) and choline to Cr were determined from the spectra. No significant differences in these ratios were seen among the rats given the various antipsychotic medications or between the control rats and the treated rats. No significant time-dependent changes were seen in most cases, except for a small reduction of NAA/Cr after 7 days of olanzapine administration. These results suggest that differences in brain metabolite ratios in vivo in schizophrenics relative to controls, at least for short-term treatment, arise from the disease, and not as a metabolic effect of the medication.

Mitogen-activated protein kinases in schizophrenia.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) are important mediators of signal transduction from the cell surface to the nucleus and have been implicated in the integration of a variety of physiologic processes in most cells, including neurons. To investigate the possible involvement of MAPKs in schizophrenia, we compared the levels of the MAPK intermediates in postmortem brain tissue obtained from schizophrenic and control subjects. Our focus was on the cerebellar vermis because of evidence suggesting that schizophrenia is associated with abnormalities of structure, function, and signal transduction in this brain region. METHODS: Cytosolic proteins were fractionated by gel electrophoresis and subjected to Western blot analysis using polyclonal MAPK antibody, which detects total extracellular signal-regulated kinases (ERKs) 1 and 2 levels, and monoclonal MAP kinase phosphatase (MKP) 2 antibody. RESULTS: Schizophrenic subjects had increased levels of ERK2 [2763 +/- (SD) 203 vs. 2286 +/- 607 arbitrary units, U = 17, p < .05] in cerebellar vermis. The levels of a dual specificity tyrosine phosphatase, MKP2, were significantly decreased in cerebellar vermis (1716 +/- 465 versus 2372 +/- 429 arbitrary units, U = 12, p < .02) from schizophrenic patients. ERK1/MKP2 and ERK2/MKP2 ratios in cerebellar vermis, but not in other brain regions, were significantly different in schizophrenic subjects as compared to control subjects (U = 15, p < or = .027; U = 3, p < .001, respectively). CONCLUSIONS: MAPK levels are elevated in the cerebellar vermis of schizophrenic subjects. This could result from a protein dephosphorylation defect in vivo and might be involved in the pathology of the disease.

Reduced sensory gating of the P1 potential in rape victims and combat veterans with posttraumatic stress disorder.

The P1 midlatency auditory evoked potential was studied in female rape victims with Posttraumatic Stress Disorder (PTSD) and compared to an age-matched female control group; and in male combat veterans with PTSD and compared to three groups of age-matched male control subjects. Sensory gating of the P1 potential was determined using a paired click stimulus paradigm in which the stimuli were presented at 250, 500 and 1000 msec interstimulus intervals (ISI). Results showed that sensory gating of the P1 potential was significantly decreased at the 250 msec ISI, and that there was a numerical, but not a statistically significant, decrease in sensory gating at the other intervals tested in both male and female PTSD subjects compared to all control groups. Since the P1 potential may be generated, at least in part, by the reticular activating system, dysregulation of sensory processing by elements of this system may be present in PTSD.

Effects of gender and region on proton MRS of normal human brain.

Localized, in vivo 1H magnetic resonance spectroscopy has been performed in a number of brain regions of neuropsychiatric interest in male and female control subjects to determine if gender and region affect the measured metabolite ratios. In contrast to some previous reports, no significant differences were seen in any region for any metabolite ratio between males and females. As expected, significant variations with brain region were seen for metabolite ratios for the total group of subjects.

Alterations in synaptic proteins and their encoding mRNAs in prefrontal cortex in schizophrenia: a possible neurochemical basis for 'hypofrontality'.

An impairment of prefrontal cortical functioning in schizophrenia ('hypofrontality') has been suggested by clinical, neuroimaging, and postmortem brain tissue studies. We used Western immunoblot and Northern hybridization analyses of postmortem brain tissue obtained from 14 schizophrenic patients and 12 control patients of similar ages to measure tissue levels of synaptophysin (a structural synaptic vesicle protein) and of SNAP-25 (a 25-kDa presynaptic protein), and their encoding mRNAs, in Brodmann's area 10 of prefrontal cortex. There were significant decreases in tissue levels of both of these proteins in prefrontal cortex of schizophrenic patients relative to controls. In contrast, tissue levels for the mRNAs encoding these proteins were not decreased in schizophrenic patients. Subsequent labeling of the same Western immunoblots showed no difference in tissue levels of glial fibrillary acidic protein (GFAP) in schizophrenic and control patients. Similarly, subsequent hybridization of the same Northern hybridization membranes showed no difference in tissue levels of GFAP mRNA or of 28S rRNA in schizophrenic and control patients. These alterations in tissue levels of synaptophysin and SNAP-25 are consistent with the idea that the clinically observed 'hypofrontality' of schizophrenia arises from abnormalities of synaptic number or structural integrity in prefrontal cortex.

Regional proton magnetic resonance spectroscopy in schizophrenia and exploration of drug effect.

Schizophrenia is a disorder with an unclear pathophysiology, despite numerous attempts to elucidate its etiology. We have employed proton magnetic resonance spectroscopy in vivo to explore the neurochemistry of several brain regions (left frontal and temporal cortices, left basal ganglia, and left and right thalamus) in patients with schizophrenia and in normal control subjects. We have also examined patients in different medication states. A trend toward a decreased level of inositol/creatine was found in the left temporal lobe of patients with schizophrenia, as was a trend toward a reduced level of N-acetylaspartate/creatine in the left thalamus of patients. In schizophrenic patients treated with atypical antipsychotics, decreased levels of choline were found in the left basal ganglia, while increased levels of N-acetylaspartate were found in the left frontal cortex. These results suggest altered metabolism in patients with schizophrenia, and imply that further study is needed to clarify the effects of the more recently available antipsychotics.

In vivo proton magnetic resonance spectroscopy of the medial temporal lobes of subjects with combat-related posttraumatic stress disorder.

Recent findings using volumetric MRI techniques have revealed that patients with combat-related and noncombat-related posttraumatic stress disorder (PTSD) have reductions in right hippocampal volume. Twenty-one veterans with PTSD and eight age-matched control veterans were studied using proton magnetic resonance spectroscopy to test the hypothesis that the N-acetyl-L-aspartic acid/creatine (NAA/Cr) ratio would be decreased in the right medial temporal lobe structures of patients with PTSD compared to controls. Patients with PTSD displayed significantly lower NAA/Cr ratio for the right medial temporal lobe relative to the left (P < or = 0.011). Patients with PTSD also had lower NAA/Cr in right medial temporal lobe (P < or = 0.013) and lower choline/Cr in left medial temporal lobe (P < or = 0.030) compared to control subjects. Because NAA is regarded as an indicator of neuronal density, this finding suggests that the neuronal density of right-sided medial temporal structures in patients with combat-related PTSD may be decreased.

In vitro 1H-magnetic resonance spectroscopy of postmortem brains with schizophrenia.

Some evidence suggests that thalamic dysfunction could explain some of the signs and symptoms of schizophrenia. We measured the absolute concentrations of amino acid metabolites in thalamus, frontal pole, and cerebellar vermis in extracts of postmortem brains from 8 schizophrenics and 10 controls using high-resolution 1H-magnetic resonance spectroscopy. The concentrations of N-acetyl aspartate, glutamate, and valine tended to be reduced in the thalamus of the schizophrenic group. Although it is difficult to ascribe significance to the "tendencies," these data may tend to support other data suggesting decreased thalamic volume or neuronal number in schizophrenia.

Combat veterans with posttraumatic stress disorder exhibit decreased habituation of the P1 midlatency auditory evoked potential.

The current study used a paired stimulus paradigm to investigate the P1 midlatency auditory evoked potential in Vietnam combat veterans with posttraumatic stress disorder (PTSD) and three comparison groups: alcohol dependents, combat-exposed normals, and combat-unexposed normals. Compared to each comparison group, PTSD subjects exhibited significantly diminished habituation of the P1 potential. P1 potential habituation within the PTSD group, correlated significantly with intensity of PTSD reexperiencing symptoms, such as trauma-related nightmares and flashbacks. These findings are discussed as consistent with a sensory gating defect at the brainstem level in PTSD, and are further discussed in the context of other psychophysiological measures in PTSD and of P1 potential findings in psychiatric disorders other than PTSD.

Decreased mesopontine choline acetyltransferase levels in schizophrenia. Correlations with cognitive functions.

The objective was to replicate a reported decrease of choline acetyltransferase (ChAT) in the mesopontine tegmentum of deceased schizophrenics and to see if such a decrease is related to their cognitive status as measured during life. Rigorous antemortem psychiatric evaluations were performed on our large population of schizophrenic patients. Mesopontine tissue was collected promptly following death from eight of these patients, from an additional five schizophrenics without systematic premortem psychiatric evaluation, and from control subjects. ChAT content of this brain tissue was determined using Western immunoblot analysis. There were 13 schizophrenic patients and 8 control subjects. The mean age of subjects in the two groups was similar (64 +/- 9 yr vs 63 +/- 10 yr). Even in the face of reduced post mortem intervals in the patients with schizophrenia, mesopontine tegmental ChAT concentrations were depressed by 70% in schizophrenic patients (1.28 +/- 1.74 vs 4.39 +/- 3.20 ng ChAT/micrograms tissue protein, P < 0.01), and correlated with orientation and reasoning (rs = 0.90 and 0.98, respectively) in those subjects assessed antemortem. Mesopontine ChAT concentrations are depressed in schizophrenia and correlate significantly with measures of cognitive performance in patients with this disorder.

Nitric oxide synthase (NOS) in schizophrenia: increases in cerebellar vermis.

A high proportion of neurons in the cerebellum and in cholinergic brainstem nuclei stain positive for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd), which is a nitric oxide synthase (NOS). Recent evidence suggests that schizophrenia may involve increased numbers of NADPHd-stained neurons in different areas of the subcortex. This led us to examine the actual concentration of NOS in postmortem brain specimens of cerebellum, and the relevant regions of brainstem tegmentum, to see if NOS concentrations were also increased in schizophrenia. Postmortem brain tissue was obtained at autopsy from schizophrenics and controls who did not have other brain disease. In patients with schizophrenia, NOS concentration was higher.

Mesopontine neurons in schizophrenia.

Findings reported here show that there is a significant increase in the number of neurons in the pedunculopontine nucleus in most schizophrenic patients compared to age-matched controls. Nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to label putative cholinergic neurons in the pedunculopontine nucleus and laterodorsal tegmental nucleus, while noradrenergic locus coeruleus neurons were labeled immunocytochemically using an antibody to tryosine hydroxylase. Cell counts of these neuronal groups were carried out using a Biographics image analysis system. We found significantly increased cell numbers in the pedunculopontine nucleus of schizophrenic patients compared to controls. The number of laterodorsal tegmental nucleus neurons was increased but this was not statistically significant. However, the total cell counts for pedunculopontine and laterodorsal tegmental nuclei were significantly higher in schizophrenic subjects. The number of locus coeruleus noradrenergic neurons was similar in both groups. These results implicate the brainstem reticular formation as a pathophysiological site in at least some patients with schizophrenia. In addition, these findings suggest a developmental etiology for the disease and account for some, but not all, of the symptoms of schizophrenia, including sensory gating abnormalities, sleep-wake disturbances and, perhaps, hallucinations. Overdriving of thalamic and substantia nigra function by cholinergic afferents from the midbrain may account for some of the symptoms seen in schizophrenia. These findings suggest that, at least in some schizophrenic patients, there is an increased number of neurons in the cholinergic arm of the reticular activating system. This may explain some of the symptoms of schizophrenia and points to a prenatal disturbance as one of the possible causes of the disease.

In vivo 19F spin relaxation and localized spectroscopy of fluoxetine in human brain.

Fluorine-19 NMR spectroscopy was used to monitor the anti-depressant drug fluoxetine (and its metabolite norfluoxetine) in vivo in human brain. A quadrature birdcage head coil, developed for operation at 60.1 MHz, yielded a signal from the head 2 to 4 times stronger than for surface coils. It was used to measure the in vivo 19F spin-lattice relaxation time (T1) of fluoxetine for five patients by the inversion-recovery technique. The individual T1s varied from 149 to 386 ms, which was attributed in part to interindividual differences based on the reproducibility of a phantom T1. The individual T1 correlated weakly with approximate brain concentration. A lower limit of 3 to 4 ms was found for the spin-spin relaxation time from line width measurements. Low resolution 4-dimensional spectroscopic imaging confirmed that the single in vivo 19F resonance for fluoxetine arose primarily from brain. The spectrum of a cerebral hemisphere (in formalin) obtained at autopsy from a patient on 40 mg/day of fluoxetine for 19 weeks was comparable with that seen for patients in vivo. The in vivo signal arose about equally from fluoxetine and the active metabolite norfluoxetine, as demonstrated by the in vitro 19F NMR spectrum of the lipophilic extract of a small section of brain. In vitro quantitation of frozen samples from three brain regions yielded combined fluoxetine/norfluoxetine concentrations of 12.3 to 18.6 micrograms/ml, which is higher than typically determined in vivo, and suggests that the fluorinated drugs may not be 100% visible in vivo.

Clinical correlates of body weight changes in schizophrenia.

Body weight was examined in chronic schizophrenic patients. Underweight medicated patients had normal ventricular brain ratios (VBRs) on CT. Overweight patients had both normal and abnormal VBRs. Weight decreased during neuroleptic withdrawal; caloric intake and weight increased when neuroleptics were reinstituted. Weight gain on neuroleptics correlated with symptom improvement independent of VBR or gender. Weight changes and psychosis in schizophrenia may be mediated by similar neurochemical systems.

The neuropathology of schizophrenia. A focus on the subcortex.

Investigations into the neuropathology of schizophrenia have increasingly altered the perception of the illness. Early studies focused on finding consistent and discrete areas of cortical pathology in the brain material of schizophrenic patients. After nearly a half century of study, little evidence emerged from a great body of data suggesting any consistent, discrete neuropathologic finding associated with this illness. This lack of evidence led to obvious frustration on the part of researchers and movement within the psychiatric community towards significantly less brain-based theories of the genesis of schizophrenia. With the advent of new technologies such as enhanced structural imaging (CT, MRI), functional imaging (PET, SPECT), and better neuropathologic methods, the focus of schizophrenia research has again turned towards the brain. Ultimately, hypotheses regarding the cause of schizophrenia will be proved or disproved on neuropathologic evidence. Few, if any, modern schizophrenia researchers would make the argument that there is a single, consistent neuropathologic lesion that is responsible for the entire illness of schizophrenia. Current theories tend to interpret the wide variety of neuropathologic changes in this illness as evidence of disturbed nervous system maturation--either acquired or inherent--or perhaps as a response to damage with aberrant neuronal regeneration. Evidence for a neurodegenerative disorder has not proved to be compelling. Furthermore, these theories emphasize dysfunction of elements of distributed neuronal systems, including subcortical systems, not discrete "lesions." The danger with the theoretic approach of distributed systems is, as Steriade and McCarley noted so well, that the flexibility of this idea can "mean a retreat into vagueness so that hypotheses concerning the role of multiple interrelated structures in the genesis of a given behavioral state cannot be proved wrong." (p 23). Despite the risks, this approach to investigation promises to offer more than just information pertinent to schizophrenia research; it offers insights into the mechanisms of behavior as a product of integrated brain function.

In vivo 7Li nuclear magnetic resonance study of lithium pharmacokinetics and chemical shift imaging in psychiatric patients.

New data are presented on the application of 7Li in vivo nuclear magnetic resonance (NMR) spectroscopy to human studies. The technique was used to monitor the between-dose pharmacokinetics of lithium (Li) in brain for three patients on Li therapy. Brain Li concentrations were at their highest from 0 to 2 hours after the peak occurred in serum concentration. Elimination from brain tissue took longer than elimination from muscle, and no signal could be detected from brain at 10 days after termination of therapy. A birdcage radiofrequency coil for 7Li was constructed and used to measure the 7Li spin-lattice relaxation time of 4.6 seconds in vivo in human head, and to acquire preliminary spectroscopic images of a phantom and human brain.