Summary of the method used to quantify spinophilin-immunoreactive puncta. (A) Schematic of a coronal section through Heschl's gyrus and the planum temporale showing the location of BA 41 (light gray) and BA 42 (dark gray). Contours (clear zones) for quantification in deep layer 3 are shown. (B) Representative sampling grid systematic randomly overlayed on a contour. (C) An enlarged view of an optical disector from the sampling grid shown in B. The solid and dashed lines show the boundaries of the counting frame, represented at the top and bottom of the section's z-axis thickness. The zone of uniform spinophilin-immunoreactive puncta density sampled in the middle 50% of the z-axis thickness (see also Figure 5) is shown by the red band. (D) Photomicrograph of BA 42 from a normal comparison subject processed for Nissl substance. The distinctive cytoarchitectonic features of BA 42 are evident: broad, densely granular layers 2 and 4; large pyramidal neurons in deep layer 3; and a relatively hypocellular layer 5. (E) Confocal micrograph (XY Montage) of an adjacent section processed for spinophilin immunoreactivity. Intense labeling is evident throughout all cortical layers, with little to no label in the white matter.

Absence of photobleaching and quenching effects on quantification of spinophilin-immunoreactive puncta. (A) Counts of spinophilin-immunoreactive puncta at a single site during continuous fluorescent excitation. Counts were obtained using a 100×, 1.4NA oil immersion objective. All puncta within a 3.5 × 3.5 μm counting frame throughout the entire section thickness were counted at approximately 5 min intervals. (B) Counts of spinophilin-immunoreactive puncta, similarly obtained at each of multiple sites within a section. Each section (represented by a separate line) was sampled during a single session with continuous fluorescent excitation.

Spinophilin-immunoreactive puncta densities in deep layer 3 of BA 41 (A) and BA 42 (B). In all panels, densities are shown as number of spinophilin-immunoreactive puncta/μm³. Each circle represents a pair with the comparison subject's mean puncta density value represented on the x axis and the schizophrenia subject's value on the y axis. Squares represent pairs with schizoaffective subjects [pairs 1, 6, 13]. Shaded circles and squares indicate pairs in which the subject with schizophrenia/schizoaffective disorder died due to suicide. Filled triangles denote group means. Markers below the diagonal reference line indicate pairs for which the schizophrenia or schizoaffective subject had a lower mean puncta density than the matched comparison subject.

Schematic summary of auditory cortical circuits and alterations observed in subjects with schizophrenia. Auditory sensory processing is primarily initiated via projections (red) from the ventral subdivision of the medial geniculate nucleus of the thalamus to layers 4 and 3c of BA 41, the primary auditory (core) cortex (Pandya et al. 1994; Pandya and Rosene, 1993; Molinari et al. 1995; Hashikawa et al. 1995). Excitation within the core spreads through layer 3 via ascending projections from layer 4 (black) and axon collaterals from layer 3 (thin green) pyramidal cells, synapsing predominantly onto dendritic spines of layer 3 pyramidal cells (Mitani et al. 1985; Ojima et al. 1991; Wallace et al. 1991; Lee et al. 2004; Watts and Thomson, 2005). Feedforward projections from core to lateral belt (thick green) arise predominantly from layer 3 pyramidal cells (Pandya and Sanides, 1973; Jones et al. 1995; Galaburda and Pandya, 1983; Fitzpatrick and Imig, 1980). Feedback projections (blue) from lateral belt arise predominantly from layer 5 and terminate in layer 1 of core (Jones et al. 1995; Galaburda and Pandya, 1983; Pandya and Sanides, 1973; Pandya and Rosene, 1993). The same arrangement of feedforward and feedback projections exists between lateral belt and parabelt (not shown), which together comprise BA 42. Layers assessed in prior studies of subjects with schizophrenia are indicated by the dashed lines, with findings summarized as shown. MGN_V, ventral subdivision of the medial geniculate nucleus; MGN_D, dorsal subdivision of the medial geniculate nucleus.

Spinophilin immunoreactive puncta in human and anti-psychotic exposed monkey. (A) Labeling of dendritic spines by the anti-spinophilin antibody utilized for studies of monkey tissue in mature primary neuronal culture. A dendrite with spines labeled with phalloidin can be clearly seen to colocalize spinophilin. Axon terminals labeled by antibody to synaptophysin appose some of these spines (arrows). (B) Confocal projection micrograph of spinophilin immunoreactivity in human cortex showing labeled puncta from deep layer 3 of Figure 2E at higher magnification. (C) Spinophilin-immunoreactive puncta, labeled using the same antibody as in (A), shown in deep layer 3 of monkey auditory cortex.

Frequency distribution of spinophilin-immunoreactive puncta within the cut section thickness of 40 μm (z-axis) for all subjects. Spinophilin-immunoreactive puncta were quantified at each site and the z axis depth of each puncta recorded. Total puncta counted in bins representing 12.5% of the z-axis depth at each site are shown (tissue surface, 0%; surface of the slide, 100%). The vertical reference lines indicate the z-axis position of the top and bottom of the disector, respectively, i.e. spinophilin-immunoreactive puncta density was calculated based on counts obtained from the zone between these lines. It can be clearly seen that puncta counts were uniform throughout this zone, indicating consistent immunolabeling and the absence of cutting artifacts.

Correlations of spinophilin-immunoreactive puncta densities in deep layer 3 of BA41 and BA42 with axon terminal density (A and B) and pyramidal cell somal volume (C and D) in the same locations. Filled circles represent comparison subjects, Squares represent subjects with schizophrenia.