Our findings suggest that there are alterations in the presynaptic release of glutamate in the ACC in this illness (Summarized in ). Furthermore, the disparate changes in VGLUT1 mRNA versus protein expression suggest that coordination of VGLUT1 gene expression is altered in this area as well. Numerous reports have implicated the ACC in schizophrenia. The ACC had the second most abnormally expressed genes in a study using microarray analyses that examined 15 discrete brain regions [28
]. Several other postmortem studies have found changes in the number of neurons and glia in this region [29
]. The ACC has also been implicated in schizophrenia by studies employing functional magnetic resonance imaging (MRI), high resolution MRI and regional cerebral blood flow [32
]. Altered glutamate transmission in this brain area is suggested by the low levels of glutamate and glutamine found in chronic schizophrenia cases [36
]. Our data add to this growing body of evidence implicating abnormalities of the ACC in the pathophysiology of schizophrenia.
Figure 4 Summary of changes and schematic of VGLUT1 (red) and VGLUT2 (green) expression in the anterior cingulate cortex in schizophrenia, based on data from the present study and earlier reports. In a previous study, increased VGLUT2 mRNA was found in the thalamus (more ...)
In contrast to our findings in the ACC, we did not detect any changes in VGLUT expression in the DLPFC. While our study was not sufficiently powered to conclude with statistical certainty that there were in fact no changes in the DLPFC, our findings are generally consistent with other studies that have found few changes in presynaptic markers in the frontal cortex [37
]. Expression of synaptosomal-associated protein-25 (SNAP-25), syntaxin, synaptophysin and growth-associated protein-43 (GAP-43) was unchanged, while synaptobrevin/vesicle-associated membrane protein (VAMP) expression was increased, in the PFC in schizophrenia [37
]. Another group found no changes in SNAP25 or syntaxin expression in subjects with schizophrenia who did not commit suicide [38
], while expression of synaptophysin, syntaxin and SNAP25 was unchanged in the PFC in two other studies [39
]. Taken together these data suggest that there is not a global change in presynaptic function in schizophrenia, and that alterations in presynaptic innervation may be found in discrete cortical regions.
Interpretation of our data is dependent upon the cellular and subcellular distribution of the VGLUTs in glutamatergic circuits, which has been well characterized. VGLUT1 and VGLUT2 protein expression is generally restricted to synaptic puncta in brain and spinal cord regions receiving glutamatergic input [41
]. VGLUT1 and VGLUT2 are colocalized with the presynaptic vesicular marker synaptophysin and are clearly segregated from the postsynaptic dendritic marker MAP2 [41
]. The complementary pattern of VGLUT1 and VGLUT2 expression suggests that there are functionally discrete subsets of glutamatergic neurons [42
]. In the DLPFC, VGLUT1 transcript and protein are expressed throughout the pyramidal neurons of layers II–VI, while expression of VGLUT2 mRNA is localized predominantly to layer IV [42
In the ACC, we found increased VGLUT1 mRNA expression across the full thickness of the cortex. Since VGLUT1 mRNA expression in this region in the primate is predominately found in the soma of pyramidal cells and in some non-pyramidal excitatory neurons (unpublished observation), our results might indicate a transcript–level compensatory response to decreased presynaptic glutamate release that is suggested by our finding of decreased VGLUT1 protein expression in this region. Increased VGLUT1 mRNA could also be secondary to decreased cortical inhibitory tone, or a reaction to abnormal subcortical input to the ACC. Several studies have demonstrated abnormalities in GABAergic interneurons in the PFC, while others have found changes in regions with dense reciprocal innervation from the PFC, such as the thalamus [24
]. For example, transcript expression for VGLUT2 and glutaminase was increased in the dorsal thalamus in schizophrenia in a study using a different sample from the same brain bank we used in this study [24
]. These data suggest increased excitatory thalamocortical innervation and/or increased release of glutamate. However, in the present study, we did not detect any alterations in VGLUT2 protein expression in the ACC or DLPFC, suggesting that changes in thalamic VGLUT2 transcript expression may not be apparent at the protein level in the regions receiving these thalamic afferent projections.
We also found decreased VGLUT1 protein expression in the ACC in schizophrenia. This result is discrepant from our finding of increased VGLUT1 mRNA in a similar sample from the same brain bank. There are several possible explanations for these potentially disparate findings. Changes in VGLUT1 protein expression could originate from intrinsic excitatory neurons of the ACC, from extrinsic presynaptic terminals expressing VGLUT1 protein, or both. If the change in protein expression is in the same population of neurons with increased VGLUT1 mRNA, this would suggest a loss of coordination of mRNA and protein expression in these cells. This could result from abnormalities of protein synthesis or from an increased rate of VGLUT1 protein degradation. Alternatively, the loss of VGLUT1 protein could be due to diminished excitatory input to the ACC from other cortical regions. We did not detect changes in VGLUT1 transcript or protein expression in the DLPFC, consistent with findings from a recent study that also found no changes in VGLUT1 protein expression in the PFC (BA 10) [48
]. However, another study has reported decreases in VGLUT1 mRNA expression in the DLPFC, a region with excitatory projections to the ACC [49
]. This study also found decreased VGLUT1 mRNA in the hippocampal formation, which is part of the efferent leg of limbic circuitry, that also includes the amygdala and the entorhinal cortex, which projects to the ACC [49
]. VGLUT1 mRNA expression has not been evaluated in the amygdala or entorhinal cortex in schizophrenia. Another source of excitatory input to the ACC is the thalamus. However, VGLUT1 mRNA expression is very low in most subcortical structures including the thalamus [24
Another possibility for our divergent VGLUT1 findings is a process involving riboswitch mRNA [51
]. These specialized RNA molecules containing structures called aptamers have been found to regulate mRNA expression by sensing the need for their protein product [51
]. If present, riboswitch mRNA in anterior cingulate cortex neurons might detect a decrease in VGLUT1 protein, and initiate increased VGLUT1 transcription.
In must also be considered that changes in VGLUT1 mRNA or protein expression could be in non-neuronal cells. While VGLUT expression was originally reported in neurons, astrocytic expression of VGLUTs has not been evaluated in postmortem tissue, but several studies have found astrocytic expression of VGLUTs and vesicular release of glutamate from astrocytes in culture and in organotypic slices [53
]. Interestingly, decreases in astrocytes have been reported in the ACC and DLPFC in schizophrenia, which could be consistent with decreased VGLUT1 protein expression, but not an increase in VGLUT1 transcripts [29
]. However, cell-level studies suggest that a majority of VGLUT1 mRNA and protein expression in the PFC is localized to neurons and presynaptic terminals forming asymmetric synapses, respectively, suggesting that the changes we have observed in the present study are likely limited to excitatory neurons [49
In contrast to the homogenously distributed VGLUT1, we detected the highest levels of VGLUT2 mRNA expression in an isodense band that encompasses layer III in the ACC and layers III and IV in the DLPFC (). Intrinsic excitatory neurons in this cortical lamina generally project within layer III (and IV in the DLPFC) and to the other superficial layers () [71
]. We did not detect changes in VGLUT2 transcript expression in the ACC or DLPFC in schizophrenia, suggesting that presynaptic function in the population of neurons expressing VGLUT2 in these regions is not affected. We also did not detect changes in VGLUT2 protein expression. In the DLPFC, VGLUT2 protein expression is highest in layer IV and is found at lower levels in layers I, II, III and VI [41
]. Thus, our results suggest that presynaptic innervation arising from intrinsic PFC neurons expressing VGLUT2 is not altered in schizophrenia. Another study has suggested that decreased spine density in the DLPFC is secondary to a reduction in excitatory thalamocortical projections [76
], which primarily express VGLUT2 and not VGLUT1 [24
]. However, we did not detect changes in VGLUT2 protein expression in the DLPFC, suggesting that thalamocortical projections from this region are unchanged, or that if there is a reduction in projections there is more VGLUT2 protein expression per terminal. Alternatively, there could be a reduction in extrinsic VGLUT2 protein expression coupled with an increase in intrinsic VGLUT2 protein expression yielding a net level of expression that is apparently unchanged.
There are several potential limitations to this study. Many of the subjects with schizophrenia were taking typical antipsychotics at the time of death, raising the concern that changes in VGLUT1 expression in the ACC could be secondary to a medication effect. However, we found no changes in VGLUT1 mRNA and protein expression in several cortical regions of the rat brain following 4 weeks of treatment with haloperidol. Consistent with our findings, another group reported no changes in VGLUT1 mRNA or protein expression following 15 day treatment with a similar dose of haloperidol (1 mg/kg/day) [77
] and another found no changes following 21 day treatment with 1.5 mg/kg/day [78
]. While these studies do not model a lifetime of antipsychotic treatment in schizophrenia, they do suggest that our findings of altered VGLUT1 may not be due to a medication effect.
In contrast to VGLUT1 expression, we did find decreased VGLUT2 protein expression in the FC in rats treated for 28 days with haloperidol. This result raises the possibility that we did not detect an increase in VGLUT2 protein expression in schizophrenia due to a masking effect of treatment with haloperidol. The effects of haloperidol treatment on VGLUT2, but not VGLUT1, protein expression also suggest that these genes are differentially regulated by antipsychotic medications.
Another limitation of our study is that we are unable to measure protein levels in specific cortical lamina. It is possible that the decrease in VGLUT1 protein expression is limited to either the superficial layers (II–III), which generally receive cortico-cortical projections, or the deep layers (V–VI), which generally receive subcortical projections. Another concern is that the cortical ribbon may be thinner in samples from patients with schizophrenia versus control subjects, possibly leading to the differential inclusion of small amounts of white matter in the dissected samples, and thus affecting our western blot results due to disease-specific differences in the grey:white matter ratio. In addition, our identification of VGLUT1–2 protein was based on molecular weight, leaving the possibility that apparent changes in expression levels could be due to a change in protein structure. Finally, our studies were performed in elderly subjects. Using linear regression we examined our data set for associations between age and gene expression and with the exception of a positive correlation between VGLUT1 transcript expression and age in the ACC, we did not find any significant associations. In this instance, we utilized analysis of covariance to test for effects of diagnosis with age as a covariate. Another study found a negative correlation between VGLUT1 mRNA expression and age in the HPC in a younger cohort, but only in schizophrenia [49
]. Significant associations between VGLUT1 protein and age have not been reported. In summary, while antipsychotic treatment and advanced age are potential limitations of this study, our data from antipsychotic treated rats, as well as our statistical analyses of the effects of age, suggest that these issues had minimal impact on our findings of altered VGLUT1 expression in the ACC.
While we did not find an effect of age on VGLUT1 protein expression, we have examined an aged cohort, and thus the question remains whether our findings are related to the end stage of the disease process or instead reflect an early pathological change. This question would best be answered by examining VGLUT expression in the ACC in a younger cohort. If our findings are only related to the latter stages of the illness, targeting changes in presynaptic function would not be a useful strategy for early pharmacological intervention. On the other hand, enhancing or reversing a loss of presynaptic function in aged patients with schizophrenia might diminish the loss of cognitive functioning associated with the end stage of this disease.
Regardless of the timing, VGLUT expression levels determine presynaptic vesicle filling and thus impact synaptic glutamate release [17
]. Thus, reduced VGLUT1 expression is consistent with a loss of excitatory neurotransmission in a region that integrates myriad cognitive functions, many of which are impaired in schizophrenia [80