Reduced Rate of Neural Differentiation in the Dentate Gyrus of Adult Dysbindin Null (Sandy) Mouse

Genetic variations in the gene encoding dysbindin has consistently been associated with schizophrenia and bipolar disorder, although little is known about the neural functions carried out by dysbindin. To gain some insight into this area, we took advantage of the readily available dysbindin-null mouse sandy (sdy−/−) and studied hippocampal neurogenesis using thymidine analogue bromodeoxuridine (BrdU). No significant differences were found in the proliferation (4 hours) or survival (1, 4 and 8 weeks after the last BrdU injection) of progenitors in the subgranular regions of the dentate gyrus between sdy−/− and sdy+/+ (control) mice. However, 4 weeks after the last BrdU injection, a significant reduction was observed in the ratio of neuronal differentiation in sdy−/− when compared to that of sdy+/+ (sdy+/+  = 87.0±5.3% vs. sdy−/−  = 71.3±8.3%, p = 0.01). These findings suggest that dysbindin plays a role during differentiation process in the adult hippocampal neurogenesis and that its deficit may negatively affect neurogenesis-related functions such as cognition and mood

Neuropathological Similarities and Differences between Schizophrenia and Bipolar Disorder: A Flow Cytometric Postmortem Brain Study

Recent studies suggest that schizophrenia (SCH) and bipolar disorder (BPD) may share a similar etiopathology. However, their precise neuropathological natures have rarely been characterized in a comprehensive and quantitative fashion. We have recently developed a rapid, quantitative cell-counting method for frozen unfixed postmortem brains using a flow cytometer. In the present study, we not only counted stained nuclei, but also measured their sizes in the gray matter of frontopolar cortices (FPCs) and inferior temporal cortices (ITCs) from patients with SCH or BPD, as well as in that from normal controls. In terms of NeuN(+) neuronal nuclei size, particularly in the reduced densities of small NeuN(+) nuclei, we found abnormal distributions present in the ITC gray matter of both patient groups. These same abnormalities were also found in the FPCs of SCH patients, whereas in the FPCs of BPD patients, a reduction in oligodendrocyte lineage (olig2(+)) cells was much more common. Surprisingly, in the SCH FPC, normal left-greater-than-right asymmetry in neural nuclei densities was almost completely reversed. In the BPD FPC, this asymmetry, though not obvious, differed significantly from that in the SCH FPC. These findings indicate that while similar neuropathological abnormalities are shared by patients with SCH or BPD, differences also exist, mainly in the FPC, which may at least partially explain the differences observed in many aspects in these disorders

Measurement of glyoxalase activities

Glyoxalase I catalyses the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal and glutathione to S-D-lactoylglutathione. The activity of glyoxalase I is conventionally measured spectrophotometrically by following the increase in A240 for which the change in molar absorption coefficient Δε240=2.86 mM⁻¹·cm⁻¹. The hemithioacetal is pre-formed in situ by incubation of methylglyoxal and glutathione in 50 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min. The cell extract is then added, the A240 is monitored over 5 min, and the initial rate of increase in A240 and hence glyoxalase I activity deduced with correction for blank. Glyoxalase I activity is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the formation of 1 μmol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase II catalyses the hydrolysis of S-D-lactoylglutathione to D-lactate and glutathione. Glyoxalase II activity is also measured spectrophotometrically by following the decrease in A240 for which the change in molar absorption coefficient Δε240=-3.10 mM⁻¹·cm⁻¹. It is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the hydrolysis of 1 μmol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase I and glyoxalase II activity measurements have been modified for use with a UV-transparent microplate for higher sample throughput

Asymmetry of the nuclei densities in the FPC.

<p>(A) 7-AAD(+), (B) NeuN(+), (C) olig2(+), and (D) NeuN(−)/olig2(−) nuclei. Left-right asymmetry rates (%) were calculated using the following formula: (the mean nuclei density in the left hemisphere)/(the mean nuclei density in the right hemisphere) ×100–100 (%). The positive % indicates left-greater-than-right asymmetry in the nuclei densities and the negative % vice versa. * <i>P</i><0.05, **<i>P</i><0.01, two-factor factorial ANOVA (diagnostic group×brain hemisphere).</p

Distribution of FS values of NeuN(+) nuclei (10,000 nuclei) in the gray matter tissue.

<p>(A) FPC, (B) ITC, (a) Abnormal distributions of FS values of NeuN(+) nuclei. (b) Abnormal reductions of the small NeuN(+) nuclei counts (FS 200 – 300) in SCH and BPD. * <i>P</i><0.05, **<i>P</i><0.01, unpaired <i>t</i>-test. mean±s.e.m.</p

Densities of nuclei in 1 mg of gray matter tissue.

<p>(a-d) FPC and (e-h) ITC. (a, e) 7-AAD(+), (b, f) NeuN(+), (c, g) olig2(+), and (d, h) NeuN(−)/olig2(−) nuclei. *P<0.05 by unpaired <i>t</i>-test.</p

Validation of forward scatter (FS) values of nuclei isolated from frozen unfixed human postmortem brains.

<p>(A) Appearance of a human NeuN(+) (upper) or olig2(+) nucleus (lower) in PBS (280 mOsm). Scale bar, 20 µm. (B) FCM measurement of 7-AAD(+) nuclei. Arrows indicate two major populations of 7-AAD(+) nuclei. (C) Stability of isolated NeuN(+) nuclei in PBS. No difference was found in the FS distributions of NeuN(+) nuclei analyzed at 4 h and 18 h after homogenization, suggesting that the size of NeuN(+) nuclei remained quite stable in PBS. (D) Distribution of 7-AAD(+) (red) and NeuN(+) (green) nuclei areas (µ m²) as determined by microscopic measurements. The nuclei populations were delineated every 50 µm². (E) Correlation between the areas of 7-AAD(+) and NeuN(+) nuclei (µ m²) as measured microscopically. The arrow (200 µm² of NeuN(+) area) indicates a point that corresponds approximately to FS 400. Beginning with this point, the area of the NeuN(+) nuclei became gradually larger than that of the 7-AAD(+) nuclei, although the areas of the 7-AAD(+) and NeuN(+) nuclei correlated to a significant degree (<i>r</i> = 0.917, <i>P</i><0.001).</p

Densities of olig2^weak(+) and olig2^strong(+) nuclei in 1 mg of gray matter tissue from BPD FPC. (A) olig2^weak(+), and (B) olig2^strong(+) nuclei.

<p>*<i>P</i><0.05 by unpaired <i>t</i>-test.</p