The Number of Chandelier and Basket Cells Are Differentially Decreased in Prefrontal Cortex in Autism

Abstract

An interneuron alteration has been proposed as a source for the modified balance of excitation / inhibition in the cerebral cortex in autism. We previously demonstrated a decreased number of parvalbumin (PV)-expressing interneurons in prefrontal cortex in autism. PV-expressing interneurons include chandelier (Ch) and basket (Bsk) cells. We asked whether the decreased PV+ interneurons affected both Ch cells and Bsk cells in autism. The lack of single markers to specifically label Ch cells or Bsk cells presented an obstacle for addressing this question. We devised a method to discern between PV-Ch and PV-Bsk cells based on the differential expression of Vicia villosa lectin (VVA). VVA binds to N-acetylgalactosamine, that is present in the perineuronal net surrounding some cell types where it plays a role in intercellular communication. N-acetylgalactosamine is present in the perineuronal net surrounding Bsk but not Ch cells. We found that the number of Ch cells is consistently decreased in the prefrontal cortex of autistic (n = 10) when compared with control (n = 10) cases, while the number of Bsk cells is not as severely affected. This finding expand our understanding of GABAergic system functioning in the human cerebral cortex in autism, which will impact translational research directed towards providing better treatment paradigms for individuals with autism.

Keywords: autism; basket cells; chandelier cell; parvalbumin; prefrontal cortex.

Figure 1.

Prefrontal cortical tissue from BA9 stained with anti-PV antibodies (dark blue) and VVA lectin (brown). PV antibodies label the soma and proximal processes of PV-expressing interneurons. VVA binds to the carbohydrate N-acetylgalactosamine, which is present in the perineuronal net surrounding Bsk cells, but not Ch cells. (A, A’) Ch cells are PV+ / VVA−. Ch cells exhibit a dark blue PV-reaction product that fills the soma and proximal dendrites (black arrowheads). (B, B’) PV− / VVA+ cells present with a brown mesh-like puncta over the soma and proximal dendrites (red arrowheads). (C, C’) Bsk cells are PV+ / VVA+ and present with a dark blue PV-reaction product in the cytoplasm and proximal dendrites, and with a brown mesh-like puncta of VVA staining over the soma and proximal dendrites (white arrowheads). (D)  All 3 cell types were present in the prefrontal cortical areas we analyzed. Scale bar: 5 µm.

Figure 2.

PV-expressing cartridges were associated with the PV+/VVA-negative cells. Vertically oriented PV+ cartridges were not labeled with VVA. (A) Ch cell soma (black arrowhead) and cartridges (small arrows) were stained with dark blue anti-PV reaction product, but not with VVA lectin. VVA+ cells (brown) are visible, as well as VVA+ capillaries (asterisks), but VVA does not label the Ch cell body or cartridges. (B) Neurolucida reconstruction of PV staining in the image shown in panel A. (C, D) Golgi stained tissue showing the structure of boutons that comprise each PV-expressing Ch cell cartridge. Scale bar: A,B. 15 µm., C,D: 1 µm.

Figure 3.

VVA lectin labeled pyramidal neurons, interneurons, and astrocytes. (A) NeuN+ pyramidal cell (dark blue nucleus) with a VVA+ perineural net outlining the soma and proximal dendrites (brown puncta). (B) Some VVA+ cells express PV (arrow) while other VVA+ cells do not (arrowhead). (C) Some VVA+ cells express CB (arrow), while other VVA+ cells do not (arrowhead). (D) CR+ cells that express VVA (arrow) and that do not express VVA (arrowhead). (E, F) VVA+ cells (asterisk) associated with VVA+ capillaries. Scale bars: 15 µm.

Figure 4.

(A–C) The number of Ch cells in Brodmann Area (BA)46, BA47, and BA9 of prefrontal cortex is significantly lower in the autism cases (AU) than in control cases (CT). (D–F) The number of Bsk cells in BA46, BA47, and BA9 did not statistically differ between groups.