Numbers of Molecular Layer Interneurons

The numerical density of BC interneurons in the lower one-third of the molecular layer is shown in . In the control brains, the density ranged from 17,219 to 32,129 BCs/mm^{3} with a mean density ± SD of 23,751 ± 7,658 BCs/mm^{3}. The corresponding densities for the autistic brains were 14,222 to 28,293/mm^{3} with a mean density ± SD of 20,258 ± 5,980 BCs/mm^{3}. The numerical density of SC interneurons is shown in . In the control brains, the density ranged from 9,014 to 20,849 SCs/mm^{3} with a mean density ± SD of 14,794 ± 6,072 SCs/mm^{3}. The corresponding densities for the autistic brains were 8,164 to 22,586 SCs/mm^{3} with a mean density ± SD of 13,990 ± 6,164 SCs/mm^{3}. represents the density of the combined BC and SC (MLI) interneurons. In the control brains, the density ranged from 13,152 to 26,489 MLIs/mm^{3} with a mean density ± SD of 19,272 ± 6,853 MLIs/mm^{3}. The corresponding densities for the autistic brains were 11,193 to 25,440 MLIs/mm^{3} with a mean density ± SD of 17,124± 6,045 MLIs/mm^{3}. With the available number of cases, statistical analysis using the t-test did not detect a significant difference in the density of BC, SC or MLIs between autistic brains as compared with the control brains with p = 0.44, p= 0.84 and p= 0.62, respectively. Additionally, photographs from two autistic and two control cases () visually demonstrate the variability in molecular layer interneuron density within the autistic and control groups.

Correlations of Analyses of Molecular Layer Interneurons

Pearson correlation, used to examine the relationship between the #BCs/mm^{3} and the #SCs/mm^{3}, returned a value of r = 0.96 (p < 0.0001) (). Thus, as BC density increased, SC density also increased. Using the Pearson correlation, a statistically significant relationship was not identified when examining the relationship between the densities BCs and PCs or the relationship between the densities of SCs and PCs, with r = 0.21 (p = 0.57) and r = 0.35 (p = 0.34), respectively (). Analysis of the relationship between group (autistic, control) and the #BCs/mm^{3}, using the Pearson correlation, was not statistically significant with r = 0.28 (p = 0.44) (). Similarly, analysis of the relationship between group (autistic, control) and the #SCs/mm^{3} was not statistically significant with r = 0.07 (p = 0.84) ().

Number of Molecular Layer Interneurons Relative to Purkinje Cells

The number of BCs per PC is shown in . In the controls, the number of BCs per PC ranged from 16.0 to 34.9 with a mean ± SD of 22.7 ± 8.5 BCs/PC. In the autistic group, these numbers ranged from 10.6 to 124.5 with a mean ± SD of 39.2 ± 42.7 BCs/PC. Compared to the other cases, autistic case 4414 appears as an outlier with 124.5 BCs per PC (). This high number can be explained by the fact that autistic case 4414 had the lowest density of PCs (0.5 PC/mm, see ), but a normal density of BCs that fell within the control range (28,293BCs/mm^{3}, see ). The reduced PC numbers, coupled with normal BC numbers, yielded a high number of BCs per PC and shows that BCs were preserved in this case with decreased PCs. The number of SCs per PC is shown in . In the control cases, the number of SCs per PC ranged from 16.7 to 41.9 with a mean ± SD of 27.5 ± 11.1 SCs/PC. In the autistic cases, these numbers ranged from 12.3 to 195.3 with a mean ± SD of 56.3 ± 68.9 SCs/PC. Again, autistic case 4414 appears as an outlier with 195.3 SCs per PC () with this case having the lowest density of PCs (0.5 PC/mm, see ) coupled with a high density of SCs (22,586 BCs/mm^{3}, see ). Thus, the reduced PC numbers, coupled with high SC numbers, yielded a high number of SCs per PC and shows that SCs were preserved in this case with decreased PCs. The number of combined BCs and SCs (MLIs) per PC is shown in . In the controls, the number of MLIs per PC ranged from 32.7 to 76.8 with a mean ± SD of 50.1 ± 19.5 MLIs/PC. In the autistic group, these numbers ranged from 22.9 to 319.8 with a mean ± SD of 95.5 ± 111.5 MLIs/PC. With the available number of cases, statistical analysis using the t-test did not detect a significant difference in the number of BCs, SCs or MLIs per PC between autistic brains as compared with the control brains with p = 0.47, p = 0.44 and p = 0.45, respectively.

Effect of Time in Fixative and Post-mortem Delay

Pearson correlation, used to examine the relationship between density of MLIs (BCs and SCs) and years in formalin (2 control, 6 autistic), returned a value of r = 0.58 (p = 0.13). This data, although not statistically significant, suggests that 33% of the variability in the density of MLIs may be accounted for by the number of years in formalin (r^{2} = 0.33). It is important to note, however, that this is a positive correlation; as years in formalin increases, the #MLIs/mm^{3} also increases. Thus, longer time periods in formalin did not appear to have a detrimental effect on the number of molecular layer interneurons identified with PV immunohistochemistry. Pearson correlation, also used to examine the relationship between density of MLIs and PMI (2 control, 5 autistic), returned a value of r = 0.71 (r^{2} = 0.50) (p = 0.07). Again, this is a positive correlation; as PMI increases, the #MLIs/mm^{3} also increases. Thus, the longer PMI did not appear to have a detrimental effect on the MLIs.