In contrast to our hypothesis, this study demonstrates that the lower grey matter glial cell number associated with chronic haloperidol and olanzapine exposure in macaque monkeys (9
) is due mainly to a lower astrocyte number, whereas oligodendrocyte number appears to be affected to a smaller degree. Thus, these findings provide only weak support of the interpretation that the findings of lower oligodendrocyte number in schizophrenia are attributable to antipsychotic medications. Although these findings might not be generalizable to all antipsychotics, it is important to note that both haloperidol and olanzapine, despite having very different receptor binding profiles (33
), produced similar effects on brain weight and volumes (10
), glial cell number (34
), and now astrocyte number. Therefore, it might be that treatment-associated lower astrocyte number is a common feature of both typical and atypical antipsychotic medications. Furthermore, given the role of astrocytes in glutamate homeostasis (35
), an antipsychotic-associated lower astrocyte number might be of importance in interpreting studies investigating the role of this neurotransmitter system in the disease process of schizophrenia (36
The synthesis and metabolism of glutamate in the neocortex involves trafficking of precursors and metabolites between neuronal and astrocytic compartments (35
). After the release of glutamate from the presynaptic terminal, neurotransmission is terminated by the uptake of glutamate via high affinity transporters (EAAT1−5) (38
). EAAT1 and 2 account for the bulk of glutamate transport in the brain (39
) and are localized almost exclusively on astrocytes (40
). Inside the astrocyte, glutamate is converted to glutamine via glutamine synthetase, an enzyme also localized almost exclusively to astrocytes (42
). Glutamine is shuttled to neurons via a pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons (43
). Within neurons, glutamine is converted back to glutamate via phosphate-activated glutaminase (PAG) (38
). Thus, lower astrocyte number following chronic antipsychotic exposure might affect cortical glutamate and glutamine levels.
Cortical glutamate and glutamine have been assessed in vivo
using magnetic resonance spectroscopy (MRS) in antipsychotic-naïve (44
) and chronically-ill (45
) schizophrenia subjects. In the antipsychotic-naïve subjects, glutamate levels were unchanged in the anterior cingulate cortex (ACC), whereas glutamine levels were increased (44
). In subjects with chronic schizophrenia both glutamate and glutamine levels were decreased in the ACC (45
). Although disease progression might play a role, these MRS studies raise the possibility that an antipsychotic-mediated reduction in astrocyte number might contribute to lower glutamate and glutamine levels in the frontal cortex of subjects with schizophrenia. Although astrocyte number has not been assessed in the frontal lobe, chronic exposure to antipsychotics in the same monkeys was associated with similar volume decrements in both the parietal and frontal lobes (10
) suggesting that similar effects on astrocyte number, and consequently, glutamate homeostasis might also occur in both brain regions.
The results of the current study raise the question of whether the lower number of S100B-IR astrocytes and CNP-IR oligodendrocytes accounts for the 14.2% lower total glial cell number observed previously in the antipsychotic-exposed monkeys (9
). We previously estimated the mean total glial cell number in Nissl-stained tissue sections from the sham monkeys to be 175.3×106
cells; thus, the mean glial cell number was 14.2% lower in the antipsychotic-exposed monkeys, corresponding to ~ 25.0×106
fewer glial cells. Here, in sham monkeys, we estimated the mean number of S100B-IR astrocytes to be 90.9×106
cells and the mean number of CNP-IR oligodendrocytes to be 47.8×106
cells (). In the antipsychotic-exposed monkeys, we estimated the mean numbers of S100B-IR astrocytes and CNPIR oligodendrocytes to be 20.5% and 12.9% lower relative to the sham monkeys () corresponding to 18.6×106
fewer S100B-IR cells and 6.2×106
fewer CNP-IR or a total of ~24.8×106
fewer immunoreactive cells. Interestingly, this lower number of immunoreactive cells is virtually identical to the observed lower total Nissl-stained glial cell number suggesting that the differences in S100B-IR and CNP-IR cell number across groups reflect a lower number of cells in the antipsychotic-exposed monkeys. Nevertheless, although unlikely, we cannot entirely exclude the possibility that an antipsychotic-associated loss of immunoreactivity might contribute, at least in part, to the apparent lower number of S100B-IR and CNP-IR cells.
This study also reports methods which ensured adequate visualization of immunoreactive oligodendrocytes and astrocytes throughout the entire thickness of tissue sections while minimizing final tissue shrinkage; both of these factors are critical to obtain robust and unbiased estimates of total cell number. Immunocytochemical methods using diaminobenzidene (DAB) as a chromogen can be hampered by the incomplete visualization of labeled structures in the middle of tissue sections, perhaps due to inability of DAB to polymerize to an adequate amount throughout the entire section thickness. On the other hand, immunofluorescent methods, with careful selection of antibodies and minor modifications to the tissue processing methods (e.g., inclusion or exclusion of Triton X-100), can circumvent this handicap. In addition to the improved visualization, our current methods also included a rehydration step prior to the application of a coverslip which, combined with a section drying time of <30 minutes (27
), limits the degree of final tissue shrinkage (e.g., ~30% for immunofluorescence, see also Ref. 47
). Minimizing final tissue shrinkage enhances the spatial discrimination among neighboring cells and increases the efficiency of cell counting by reducing the number of counting sites required.
In summary, the findings of this study indicate that chronic exposure to haloperidol and olanzapine is associated with significantly lower astrocyte number in the parietal cortex. Since fewer astrocytes might impact the synthesis, metabolism, and neurotransmission of cortical glutamate, this study suggests that both in vivo imaging and post-mortem studies of the glutamate system in schizophrenia should control for the possible confounding effect of chronic antipsychotic treatment. In addition, this study illustrates the use of methods which allowed robust and efficient stereological number estimates of immunofluorescently-labeled cells in the CNS and have relevance to experiments conducted in diverse areas of neuroscience.