In all cases and controls, the massive callosal fiber tracts were readily discernable from bordering cingulum bundle and subcortical white matter (). The ventral portion of the cingulate cortex in cases and controls showed the expected bipartition with the six-layered cortex of BA24, bordering upon the less laminated BA33, which is defined by “merging” of prominent pyramidal cell laminae of lower layer III and layer V (32
NeuN+ neuronal density is increased in cingulate white matter in schizophrenia and bipolar disorder
In sections immunostained for NeuN, the gray/white matter border was readily discernable, particularly at low magnification (). The NeuN+ neurons were defined by robust immunoreactivity in the nucleus and more lightly stained cytoplasm and processes, which were overall more readily discernable in gray matter than in white matter (). In the gray matter, a large majority of neurons were NeuN+, resulting in a Nissl-type staining patterns ().
In controls, NeuN+ neuronal density was highest in the most superficial white matter (4.25 cells/mm2), and subsequently declined in the deeper white matter to densities of 0.794 cells/mm2 (). Both schizophrenia and bipolar groups were observed to have greater overall mean NeuN+ neuronal densities compared to controls (2.87 ± 0.50 and 2.29 ± 0.44 cells/mm2 versus 1.28 ± 0.13 cells/mm2; mean ± standard error). Diagnosis had a significant effect on overall mean NeuN+ density (K = 14.0; p = 0.001). The increased NeuN+ densities observed in schizophrenia and bipolar groups were both significantly different from control density (Q = 13.6, p < 0.01 and Q = 7.24, p < 0.01), but the two patient groups were not significantly different from one another (Q = − 2.51, p > 0.05). When each compartment was analyzed individually, diagnosis was found to have a significant effect on NeuN+ density in all five compartments (K = 6.32, p = 0.042; K = 10.5, p = 0.005; K = 6.83, p = 0.033; K = 11.8, p = 0.003; K = 12.4, p = 0.002), with fold increases ranging from 1.17 to 3.75 (). The increased NeuN+ densities observed in schizophrenia were significantly different from controls in all compartments (Q = 8.83, p < 0.01; Q 7.93, p < 0.01; Q = 8.43, p < 0.01; Q = 10.8, p < 0.01; and Q = 9.90, p < 0.01). NeuN+ densities in the bipolar cohort were significantly different from controls in compartments II–V (Q = 8.90, p < 0.01; Q = 4.42, p < 0.01; Q = 6.87, p < 0.01; and Q = 9.66, p < 0.01).
Increased NeuN+ neuronal density in white matter in schizophrenia and bipolar disorder
Notably, 7 out of 22 schizophrenia subjects and 3 of 15 bipolar subjects demonstrated overall NeuN+ densities more than two standard deviations above the control mean (); this increased proportion of cases exceeding the 95th percentile of control mean was significant for both schizophrenia (χ2 = 12.3, p < 0.0001) and bipolar groups (χ2 = 5.7, p = 0.017). We conclude that approximately 25% of cases diagnosed with bipolar disorder or schizophrenia show a more robust increase in cingulate white matter neuron densities. Importantly, when these 10 clinical cases were removed from the analyses, differences between the remaining schizophrenia, bipolar and the control subjects for each of the five compartments were not significant when corrected for multiple comparisons. No demographic or other factor was identifiable that could distinguish between the 10 cases with white matter neuron density above the 95th percentile of controls and the remaining patients. For example, 3/7 (4/7) affected schizophrenia subjects were male (female), while all 3 affected bipolar subjects were female. Approximately one half of these cases were from the left and from the right hemisphere, and ages were not significantly different from the cohort means.
Also of note, NeuN+ neurons were present in the deeper white matter in only 24/45 of control subjects, but were found in 12/15 of bipolar subjects and in 20/22 schizophrenia subjects. These differences in distribution were significant for both schizophrenia (χ2
= 10.3, p = 0.001) and bipolar groups (χ2
= 8.4, p = 0.004). These findings also resonate with several previous reports suggesting elevated neuronal numbers in deeper white matter (8
). Among the 4 cases that were not on antipsychotic medication prior to death, 3 cases (75%) were found to harbor NeuN+ neurons in white compartments III–V. Therefore, the much higher proportion of subjects with NeuN+ neurons in deeper white matter in the two disease cohorts is unlikely to be due to antipsychotic medication. No association was observed between NeuN+ densities and age (r = 0.050) or PMI (r = −0.029), and there was no robust correlation with age-of-onset (r = −0.36, R2
= 0.14), albeit information about this clinical parameter was available only for a subset of cases. Additionally, there was no significant difference in NeuN+ density between left and right hemispheres (U = 154.5; p = 0.480). Furthermore, there were no significant differences between genders within the 3 diagnostic categories (control
: U = 65.5, p = 0.552; schizophrenia
: U = 36.0, p = 0.335; bipolar
: U = 18.0, p = 0.087). Finally, among 5 factors tested by ANCOVA (diagnosis, hemisphere, PMI, age, gender) only diagnosis had a significant effect on overall NeuN density (F = 8, p = 0.0007). Therefore, the observed increases in white matter NeuN+ neuronal density in the clinical samples of our cohort are most likely related to the disease itself and not due to other factors.
NRG+ neuronal density is unaltered in cingulate white matter in schizophrenia and bipolar disorder
Next, we wanted to study additional white matter neuron populations. To this end, we noticed that while immunostaining with the anti-Neuregulin α (NRG) antibody (see Methods) resulted in robust labeling of a subpopulation of bi- or multipolar neurons residing in cortex and white matter (), none of the NeuN+ neurons in white matter () expressed NRG. Therefore, the white matter neuronal population expressing NeuN does not overlap with NRG+ cells, which is consistent with previous reports that NeuN is expressed in some—but not all—subpopulations of neurons (33
NeuN and NRG1α(NRG) immunoreactivity define two non-overlapping neuronal subpopulations
We determined the density of NRG+ neurons in both gray and white matter in a subset of 10 bipolar, 16 schizophrenia, and 26 control subjects. (We had previously observed increased NeuN+ white matter neurons in these clinical cases, similar to the ones reported above for the larger cohorts (data not shown).) For each of the 3 diagnostic groups, NRG+ density was lower in the cortical gray matter of cingulate as compared to the white matter (), which is consistent with a previous report demonstrating that NRG+ neurons are less common in gray matter compared to white in adult human brain (27
). In white matter, both schizophrenia and bipolar groups demonstrated similar mean NRG+ densities compared to controls (1.34 ± 0.099 and 1.31 ± 0.18 cells/mm2
versus 1.26 ± 0.13 cells/mm2
), and there was no significant effect of diagnosis on overall, or compartment-specific, NRG+ density (K = 1.8, p = 0.406) (). These findings further confirm that the observed increase in NeuN+ density in cingulate white matter bipolar disorder and schizophrenia is specific for that neuronal population.
NeuN+ neuronal density in cingulate gray matter is unaltered in schizophrenia and bipolar disorder
NeuN+ densities in gray matter were typically 50–150 fold higher than in white matter, but there was no significant correlation between the two (bipolar
r = 0.0005; schizophrenia
r = 0.004; control
r = 0.0319; all subjects
r = 0.0013) (Supplemental Figure 1
). We conclude that the variation in neuronal densities in cingulate white matter is dissociated from any density changes of the overlying cortex.
Size of NeuN+ neurons in cingulate white matter is unaltered in schizophrenia and bipolar disorder
To rule out that the observed increase in NeuN+ density in bipolar disorder and schizophrenia could be an artifact due to a larger size of the NeuN-immunoreactive cells, we determined the average area of these cells in bipolar, schizophrenia, and control subjects, and no significant differences between diagnostic groups were found (0.092 ± 0.006; 0.099 ± 0.003; and 0.101 ± 0.003 mm2) (F = 1.09; p = 0.367).
NeuN+ neuronal density is also increased in dorsolateral prefrontal cortex (BA9) white matter in schizophrenia
In the control subjects of the current study, the average density of NeuN+ neurons in cingulate white matter near the sulcal bottom ranged from 0.79 to 4.31 cells/mm2
. This contrasts with a previous study conducted on the middle banks of dorsolateral prefrontal cortex that reported NeuN+ densities of up to 40 cells/mm2
in sections of similar thickness (14
). These differences are not unexpected, because the number of interstitial white matter neurons tend to decline towards sulcal fundi and, moreover, the thickness of the neuronal layer in the embryonic interstitial zone (future white matter), is thinner underneath the cingulate in comparison to the lateral cortex (23
Therefore, we wanted to determine whether neuronal densities show regional differences in frontal lobe white matter and whether increased NeuN+ densities in the cingulate could be extrapolated to other areas. Indeed, NeuN+ densities in white matter beneath the sulcal bottom of the superior frontal gyrus (BA9) of 5 μm thick sections (see Methods) ranged from 0.21–2.48 cells/mm2 in control subjects. If adjusted for differences in section thickness, NeuN+ neuron density in BA9 is 5-fold higher when compared to the cingulate. NeuN+ densities in the 6 schizophrenia cases showed a significant increase in BA9, compared to controls matched for age, gender and autolysis time (); these same 6 subjects also demonstrated increased NeuN+ densities in cingulate cortex as compared to the 6 controls (mean = 4.01 cells/mm2 versus 1.28 cells/mm2). Therefore, altered NeuN+ densities in psychosis potentially affect widespread portions of the frontal lobes.
NeuN+ nuclei are increased in cingulate white matter in schizophrenia
Next, we wanted to confirm that the elevated two-dimensional (2D) NeuN+ cell density can be replicated with a three-dimensional counting method. However, optical dissectors are not ideal for white matter neuron quantifications because neuronal numbers are low overall and unevenly distributed (35
). To bypass these limitations, we calculated the total number of NeuN+ and NeuN− nuclei extracted from a standardized column of white matter tissue from frozen, unfixed anterior cingulate (see Methods) (). These additional studies were conducted on 6 schizophrenia cases that—in comparison to 5 matched controls—showed a 6.6-fold increase in overall NeuN+ densities as determined by the 2D approach (schizophrenia
: 3.59 ± 0.70 cells/mm2
: 0.55 ± 0.41 cells/mm2
, Mann-Whitney U = 31, p < 0.05). As shown in , these 6 schizophrenia cases also showed a significant, 7.4-fold increase in NeuN+ nuclei per mm3
white matter tissue (schizophrenia
: 376 ± 253 nuclei/mm3
: 51 ± 38 cells/mm3
; Mann-Whitney U = 0, p = 0.006). Across the 11 subjects, there was a good linear correlation between the 2D and 3D counts (R
= + 0.72). In contrast to the observed increase in NeuN+ nuclei, the schizophrenia group did not show increased numbers of overall nuclei (neuronal and non-neuronal) (). This increase in NeuN+ nuclei—in the absence of an increase in DAPI+ nuclei—makes sense given that less than 4% of the white matter nuclei population is neuronal (), and, thus, an increase in neuronal nuclei will not necessarily be reflected by an increase in all nuclei. From these findings, we draw two conclusions: First, increased numbers and densities of NeuN+ white matter neurons in our clinical cases are apparent when employing two different methodologies. Second, this alteration is highly specific, because overall numbers of cells in the white matter remained unaltered.
Isolation and quantification of nuclei from cells residing in white matter
Iba1 immunoreactivity is unaltered in cingulate white matter in schizophrenia and bipolar disorder
Next, we wanted to explore whether the observed alterations in NeuN+ density in white matter were related to a change in the microglia population, because a recent study in multiple sclerosis suggests a potential link between microglial activation—a hallmark of the inflammatory response in brain—and increased white matter neuron densities (30
). To this end, we graded immunoreactivity for the microglial marker, Iba1 (see Methods), on a scale of 0–4 in all schizophrenia and bipolar subjects, together with a subset of controls (Supplemental Figure 2A
). We found no significant difference in Iba1 immunoreactivity between groups, nor was it associated with NeuN+ density in white matter (Supplementary Figure 2B, C
). We conclude that increased presence of NeuN+ neurons in white matter of a subset of schizophrenia and bipolar subjects is not associated with microglial activation.
NeuN+ neuronal density in white matter decreases during development
Interstitial white matter neurons of adult brain are thought to be vestiges of the subplate, a transient structure of the developing brain that shows a progressive involution starting in the 3rd
). Previous studies utilized Nissl, Golgi and acetylcholinesterase staining to determine developmental changes in subplate neuronal densities subjacent to visual, somatosensory, motor, and prefrontal cortices (23
). While there is general consensus that neuronal density in subcortical white matter declines during the first year of postnatal life, the specific timing of this event varied depending upon the cell markers used.
Presently, it is not known whether NeuN-immunoreactive white matter neurons undergo changes in numbers or densities during development. Therefore, we monitored the temporal course of NeuN+ neuronal density in postmortem specimens of a new postmortem cohort ranging in age from 40 weeks of gestation to 51 years of age (). To avoid variability of frontal lobe anatomy as potential confound, we focused on white matter space underneath the lobe’s rostral pole, which is unambiguously identifiable in both young and old brain. There was a steep decline in NeuN+ densities during the first year of life, after which the density plateaued and remained relatively constant throughout childhood, adolescence, and adulthood (). The mean NeuN+ density for the 10 subjects > 1 year (14.6 ± 6.42 cells/mm2) demonstrated a 76.7% decrease as compared to the mean density of 4 subjects < 1 year (62.7 ± 29.6 cells/mm2); this difference was significant (U = 0, p = 0.005; 1 degree of freedom).
White matter NeuN+ neuronal density demonstrates an age-associated decline during development