We have previously found that, in layer 2 of the ACCx, the expression of NR2A mRNA in approximately 53% and 35% of the GABA cells in schizophrenia and bipolar disorder, respectively, appears to be decreased to a level that is no longer experimentally detectable (9
). Because CB-containing GABA cells tend to be more heavily localized to layer 2 of the cortex (16
), we hypothesized that they would be among the GABA neurons that are affected. Contrary to this hypothesis, our data suggest that the density of the CB-containing cells that express NR2A mRNA seems to be unchanged in bipolar disorder. Furthermore, it appears that the density of these cells may actually be increased in schizophrenia.
There are at least two scenarios that could explain the observation of increased density of CB+/NR2A+ neurons in schizophrenia. First, this finding may indicate that some of the CB-containing cells that normally do not express NR2A become NR2A-expressing in schizophrenia. In this case, the number of CB+/NR2A- cells would be expected to decrease correspondingly, which our analysis failed to detect. However, it should be noted that because only about 10% of all CB mRNA-containing cells express NR2A mRNA, a ~40% increase in the density of these neurons would amount to only about a 4% decrease in the CB+/NR2A- neurons. Such a small magnitude of change is below the detection sensitivity of in situ hybridization. A second possible explanation of our finding is that the number of CB mRNA-expressing cells may actually be increased in schizophrenia and, additionally, these cells express NR2A. In this scenario, according to the discussion above, the density of the entire population of CB mRNA-expressing cells would increase by only ~4%, which, again, is unlikely to be detectable by in situ hybridization. An alternative methodology with superior sensitivity, such as single cell real-time polymerase chain reaction, will be required before we can distinguish between these two scenarios, which also need not be mutually exclusive. Although to our knowledge there have been no previous studies examining the expression of CB mRNA in schizophrenia, several studies have found changes in the density of CB-immunoreactive neurons. For example, Daviss and Lewis have shown that the density CB-immunoreactive neurons may be increased in the dorsolateral prefrontal cortex in subjects with schizophrenia (30
); this observation may therefore seem to lend support to one of the scenarios discussed above about a possible increase in the density of CB mRNA-expressing cells in the ACCx. However, in another study, the density of CB-immunoreactive cells in layer 2 of the ACCx has actually been found to be decreased in subjects with schizophrenia (19
). Similarly, a decrease in the density of these cells has also been observed in the temporal cortex (31
). Nevertheless, transcript and protein expression do not necessarily always correlate (32
); it is conceivable that, in schizophrenia, one mechanism to compensate for decreased CB protein expression is by increasing transcription.
Although the majority of the CB-expressing cells are GABA neurons; a small number of pyramidal cells (mostly in the lower layers) in the normal cortex also contain CB (16
). Therefore, it is possible that at least some of the NR2A-expressing CB-containing cells that are affected may be pyramidal cells. Although this possibility cannot be ruled out, it is not supported by our observation that the average area (mean±S.D.) of grain clusters on CB+ cells, which can be considered a reflection of somal size, in layer 2 of the ACCx was found to be significantly (t=1.98; p=0.048) smaller in subjects with schizophrenia (158.3±62.1 µm2
) compared to the normal control subjects (164.9±65.5 µm2
). Importantly, this difference was not observed in any of the other cortical layers (data not shown). This finding would seem to support the argument that the majority of the CB+/NR2A+ cells that are affected in schizophrenia may in fact be GABA interneurons, which have smaller somal areas than pyramidal cells.
Specific subsets of GABA interneurons subserve distinct functions to ensure the integrity of information processing in the cerebral cortex (33
). Many of the CB-containing GABA cells, for instance, are thought to be double-bouquet cells, which selectively target the distal dendrites of pyramidal neurons (20
). The firing of the distal dendrite-targeting inhibitory neurons may play a very important role in phasing the activity of the pyramidal cells they innervate to temporally match the spike pattern of the incoming excitatory stimuli in order to facilitate activity-dependent synaptic plasticity (34
). Furthermore, there is also evidence suggesting that activation of these neurons may be very important in regulating the stability of network activity by filtering out distracting stimuli (11
). Our finding of increased density of CB+/NR2A+ cells implies that glutamatergic inputs to the CB-containing neuronal network in layer 2 of the ACCx may be altered in schizophrenia, although the exact nature of this alteration cannot at present be determined. A possible pathophysiologic manifestation of altered glutamatergic modulation of CB-containing neurons is that the inhibition provided by these neurons to time excitatory inputs to pyramidal cells may be compromised and hence the process of activity-dependent synaptic plasticity on pyramidal cells may be disturbed. In addition, in functional terms, these pyramidal cells may also become more susceptible to the distracting effects of stimuli that are context-irrelevant, rendering the pyramidal cell network more easily destabilized. Together these mechanisms may contribute to aberrant information processing in the ACCx in schizophrenia. Finally, it would be of significant interest to identify the source of the glutamatergic inputs that may be altered in schizophrenia; one of the possibilities is that they may represent, at least in part, inputs from the amygdala, as layer 2 is one of the main layers where the axonal projections of this structure terminate (35
). Interestingly, amygdalocortical projections to the ACCx appear to undergo progressive sprouting throughout the adolescent period, achieving the adult pattern of innervation only during early adulthood (35
), which coincides with the period of time when the symptomatology of schizophrenia typically begins to emerge. Together these observations raise an interesting possibility that disturbances of the periadolescent maturation of the amygdalocortical innervation of CB-containing neurons in the ACCx, which may, at least in part, be mediated by stress, may play a role in the pathophysiology of the onset of schizophrenia.