Lack of Molecular-Anatomical Evidence for GABAergic Influence on Axon Initial Segment of Cerebellar Purkinje Cells by the Pinceau Formation

Abstract

The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABA A R␣1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells. GAD and VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABA A R␣1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored.

Localization and expression of CaBP1/caldendrin in the mouse brain

“…Given the strong inhibitory modulation of Ca v 2.1 by CaBP1 (Lee et al, 2002), CaBP1/CD may be important for shaping presynaptic Ca 2+ signals that regulate precise timing of Purkinje cell inhibition. Although morphological evidence argues against a role for the pinceaux in the release of GABA onto Purkinje neurons (Iwakura et al, 2012), it has been proposed that the pinceau mediates a “field effect” whereby electrical activity in the basket cell terminal influences the membrane potential of the ensheathed Purkinje neurons (Korn and Axelrad, 1980). Although we did not observe gross motor impairment in C-KO mice, further analyses will be required in these mice to define a potential role of CaBP1/CD in controlling cerebellar output.…”

Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1

“…Approximately 10%-20% of BC axonal terminals cover the AIS at the pinceau; the majority are separated from PC AIS by astrocyte processes (40) (Figure 8B). This unique anatomic configuration of astrocytes at the pinceau is thought to play a crucial role in BCinduced ephaptic inhibition (40).…”

“…This unique anatomic configuration of astrocytes at the pinceau is thought to play a crucial role in BCinduced ephaptic inhibition (40). To study the neuronal-glial interface, we costained the cerebellar sections with GFAP and calbindin.…”

Enrichment of GABARAP Relative to LC3 in the Axonal Initial Segments of Neurons

“…Although the latter structure is unique, recent precise morphological and immunohistochemical analyses have shown that typical synaptic contact at the AIS of cerebellar Purkinje cells is very rare. Moreover, the alpha1 subunit of the GABA A receptor, a subunit in Purkinje cells, did not form detectable clusters along the AIS, indicating that Purkinje cells do not receive GABA-mediated synaptic inhibition from basket cells on the AIS [36].…”