GSK3β plays a diverse role in normal brain function, and its dysregulation is believed to underlie some psychiatric disorders and neurodegenerative diseases 
. In light of the critical role of GSK3β in the CNS, several groups have studied the expression pattern and activity of endogenous GSK3β in the brain by immunohistochemistry and light microscopy during brain development, in brain disease processes, and in response to various stimuli 
. In this regard the light microscopy data reported in the present study is supportive of many of the earlier reports. However, to further elucidate the distribution of GSK3β at the subcellular level, the expression of this kinase was also evaluated by electron microscopy. Although Hoshi et al. 
originally described expression of GSK3β in brain mitochondria by electron microscopy, a detailed analysis of GSK3β expression at the ultrastructural level has not been reported previously. Thus, the main goals of this study were to corroborate the distribution of GSK3β in the brain, and to examine in detail the subcellular distribution of this protein.
Initial examination by light microscopy revealed that GSK3β is expressed in brain neurons throughout all the rostrocaudal extent of the adult mouse brain. Overall, the distribution observed in our light microscopy study mostly concurs with findings in previous studies. Takahashi et al. 
had originally conducted an extensive immunohistochemical survey of GSK3β expression in the developing and adult rat cerebellum. In their study, in which GSK3β is referred by its other name, τ protein kinase I, they reported GSK3β expression in axonal fibers of the axonal tract and in the granular layer. Expression of which they reported decreased during later stages of development 
. They also found that GSK3β immunostaining in the molecular layer increased in later stages of development, and the Purkinje cells in the Purkinje cell layer had staining mainly in their cytoplasm 
. These findings were subsequently confirmed by Leroy and Brion 
. In our study we have found that in the mature mouse brain, the Purkinje cell layer and the molecular cell layer (where the dendritic processes of Purkinje cells end) present strong GSK3β labeling, while the granular cell layer was devoid of immunostaining and non-axonal staining was observed. These data are generally in accordance with previously reported studies, however Takahashi et al 
and Leroy and Brion 
reported some immunostaining in the granular cell layer of the adult rat brain. This discrepancy could be due to several reasons including a difference in species (mice in our study versus rats), gender (i.e. the studies in rat do not state if they have used male or female animals), or age of the animals used. In addition, it was previously reported that in the adult rat brain there was strong staining in the hippocampus in the CA and dentate gyrus regions, the deeper cortical layers, thalamic nuclei, and the substantia nigra pars compacta 
. However, our findings indicated a lack of GSK3β staining in most hypothalamic areas of the adult mouse brain, whereas previously strong labeling of GSK3β was noted in this region of the brain in the adult rat 
. This difference could also potentially reflect some variance between the two species or perhaps a gender difference. Overall, the present light microcopy data mostly concurred with previous findings on GSK3β localization in the brain, and was meant to establish a frame of reference for examination of GSK3β at the subcellular level.
GSK3β immunolocalization was also examined by electron microscopy to scrutinize its subcellular distribution. The most salient feature is the abundance of GSK3β immunostaining in the cytosol of the neuronal soma, dendritic shaft, dendrites, and dendritic spines, but no GSK3β labeling was observed in axons. Within neurons GSK3β labeling was clearly present in the rough endoplasmic reticulum (RER), free ribosomes and in the outer membrane of mitochondria. Furthermore, we also found clear presence of GSK3β within astrocytes, especially on their RER, free ribosomes, mitochondria, and in astrocytic processes. In contrast, other glial types, such as the oligodendrocytes and microglia showed little evidence of GSK3β labeling. Although several studies have already noted GSK3β signaling activity in isolated astrocytes 
, and elevated staining of phospho-serine-9 GSK3β in astrocytes by light microscopy has been reported in cases of human tauopathies 
, other immunohistochemical studies 
, could not detect, or did not report, staining of GSK3β in astrocytes by light microscopy. However, our data shows that GSK3β labeling is clearly present in astrocytes at the electron microscope level and GSK3β appears in several subcellular structures such as the RER, ribosomes and mitochondria, albeit at much lower levels than in neurons.
Electron microscopy data of the intracellular distribution GSK3β in neurons revealed that GSK3β is expressed in the mitochondrial membranes, and robust GSK3β labeling was found on ribosomes and the rough endoplasmic reticulum. In the mitochondria, GSK3β was previously reported to be resident in the mitochondrial membranes of cultured SH-SY5Y cells 
, and activated GSK3β is believed to regulate mitochondrial metabolic output 
, mitochondrial motility 
, and mitochondria-linked apoptosis signaling 
. In addition, GSK3β signaling is known to impact protein translation through its phosphorylation of eukaryotic initiation factor 2B 
possibly at the vicinity of the ribosomes and the RER. Increased GSK3β activity is also known to accentuate ER stress 
. Thus, the strong labeling of GSK3β at the RER and ribosomes provides further support of its known signaling activities at these sites. In contrast to the robust staining of GSK3β in the endoplasmic reticulum and the mitochondria was the lack of GSK3β staining in brain cell nuclei under both light and electron microscopy. Previously it has been shown by immunoblot analysis that GSK3β is present in biochemically separated nuclear fractions of normal mouse brain 
. However, through various pharmacological treatments and molecular methods it was determined in SH-SY5Y cells that the presence of GSK3β in the nucleus is transient 
, and its transit between the cytosol and the nucleus is highly dynamic 
. Thus, when isolated brain cell nuclei from healthy brain tissues are examined “en masse” by immunoblot analysis GSK3β can be detected in the nucleus, but GSK3β staining in individual healthy neuronal nuclei by microscopy may not be readily visible. Furthermore, GSK3β is known to accumulate in the nucleus upon activation of apoptosis signaling 
. Therefore nuclear GSK3β labeling in the brain may be more evident in dying neurons.
Another interesting finding was the noticeable GSK3β labeling of some postsynaptic densities. There is emerging evidence that GSK3β affects neuronal synaptic plasticity and is involved in synaptic activities 
. Previously, GSK3β had been detected in synaptosomal fractions 
and in the dendrites of cultured hippocampal neurons 
. Peineau et al. 
have reported that GSK3β mediates both N-methyl-D-aspartate receptor-dependent long-term potentiation and long-term depression. Furthermore, Zhu and colleagues 
have shown that GSK3β activation can impair the synapse ultrastructure in the tetanized CA3 region of the rat hippocampus, and inhibitors of GSK3β can restore the synapse to its normal morphology. Our report shows that GSK3β is present in some postsynaptic densities while others seem to be devoid of GSK3β, this evidence clearly indicates that GSK3β is located in the synapses and also that there is some degree of specificity of GSK3β signaling at different synapses, which requires further exploration.
Following our findings with the phospho-independent GSK3β antibody, it was surprising to see a marked difference of the pSer9GSK3β immunolabeling. For example, cell types, such as the microglia, which showed no evidence of the phospho-independent GSK3β staining, presented with clear and robust pSer9GSK3β staining. One possibility for the discrepant labeling could be that phosphorylation at serine-9 of GSK3β may partially block the immunoreactivity of the phospho-independent GSK3β antibody, thus regions with a high concentration of pSer9GSK3β may appear as devoid of GSK3β. Nonetheless, the presence of the serine-9-phosphorylated GSK3β within the microglia indicates that GSK3β must be present within these cell types. Previously, Yuskaitis and Jope 
reported that GSK3β signaling in microglia promotes the lipopolysaccharide-induced production of interleukin-6 and expression of inducible nitric oxide synthase, and regulates microglial migration, all of which can be blocked by GSK3β inhibitors. Our findings show that GSK3β signaling is resident within microglia in discreet areas on free ribosomes, the ER, and in the nuclei of some cells. However, under normal conditions GSK3β in resting microglia is mostly in its less activated, serine-9-phosphorylated state.
pSer9GSK3β-labeled neurons were also evident by light and electron microscopy, but this labeling was far less pronounced than the phospho-independent GSK3β labeling. The clearest pSer9GSK3β staining was in the superficial layers of the cortex, with diminishing staining in the deeper layers. Within the stained neurons there was pSer9GSK3β labeling on the ribosomes on the ER, and interestingly, inside the nuclei of some neurons. Previous findings by western blot analysis of total mouse brain homogenates 
noted a paucity of nuclear serine-9-phosphorylated GSK3β, but the present results indicate that there are clear regional variations in these levels.
Another well-known GSK3β phosphorylation site is at its tyrosine-216 residue. Active GSK3β is phosphorylated on tyrosine-216 
, and phospho-tyrosine-216 antibodies are sometimes used for labeling activated GSK3β signaling. Our attempts to immunohistochemically stain brain sections specifically for pTyr216GSK3β were unsuccessful as many of the antibodies that we tested also showed strong and equivalent reactivity to the tyrosine-phosphorylated GSK3α isoform, or yielded high background staining that was unusable for immunohistochemistry at the electron microscope. Nevertheless, the revelation that there are brain areas with strong basal pSer9GSK3β labeling does indicate that not all pools of GSK3β are equivalently activated, and that the activation state of GSK3β can vary widely throughout different brain regions, cell types, and cellular subfractions.
In conclusion, our light microscopy study of GSK3β mostly corroborated previous immunohistological analyses of GSK3β distribution in the brain. However, no previous study had analyzed in detail the subcellular localization of GSK3β in the brain. The presence of GSK3β within various intracellular compartments was previously deduced through biochemical studies; however, the presence of GSK3β in these compartments was not confirmed by visualization of the protein in these structures. The present study now visually demonstrates in detail the location of GSK3β at the subcellular level in neurons and astrocytes, confirming previous findings of biochemical studies. The specific intracellular distribution of GSK3β within these brain cells and at selective neuronal synapses opens some new avenues of exploration of this kinase.