NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons

Abstract

The molecular repertoire of the “Ca2+-signaling toolkit” supports the specific kinetic requirements of Ca2+-dependent processes in different neuronal types. A well-known example is the unique expression pattern of calcium-binding proteins, such as parvalbumin, calbindin, and calretinin. These cytosolic Ca2+-buffers control presynaptic and somatodendritic processes in a cell-type-specific manner and have been used as neurochemical markers of GABAergic interneuron types for decades. Surprisingly, to date no typifying calcium-binding proteins have been found in CB1 cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons that represent a large population of GABAergic cells in cortical circuits. Because CB1/CCK-positive interneurons display disparate presynaptic and somatodendritic Ca2+-transients compared with other interneurons, we tested the hypothesis that they express alternative calcium-binding proteins. By in silico data mining in mouse single-cell RNA-seq databases, we identified high expression of Necab1 and Necab2 genes encoding N-terminal EF-hand calcium-binding proteins 1 and 2, respectively, in CB1/CCK-positive interneurons. Fluorescent in situ hybridization and immunostaining revealed cell-type-specific distribution of NECAB1 and NECAB2 throughout the isocortex, hippocampal formation, and basolateral amygdala complex. Combination of patch-clamp electrophysiology, confocal, and STORM super-resolution microscopy uncovered subcellular nanoscale differences indicating functional division of labor between the two calcium-binding proteins. These findings highlight NECAB1 and NECAB2 as predominant calcium-binding proteins in CB1/CCK-positive interneurons.

Neuronal Dystroglycan regulates postnatal development of CCK/cannabinoid receptor-1 interneurons

“…These include NECAB1, a calcium binding protein that specifically labels CCK+ IN cell bodies (Fig. 3 A) [ 52 ], and VGLUT3, a vesicular glutamate transporter enriched at CCK+ IN synapses ( Fig. 3 C ) [ 53 – 55 ].…”

Interneuron Types and Their Circuits in the Basolateral Amygdala

“…In some of these basket cells, VIP may also be expressed (Omiya et al, 2015 ; Rhomberg et al, 2018 ). Of note, a recent study has revealed that the Ca 2+ binding proteins NECAB1 and 2 are expressed in CCK/CB1, but not PV basket cells in cortical structures, including the BLA (Miczan et al, 2021 ). At the population level, half of the output synapses of CCK/CB1 basket cells target the perisomatic region (similarly to PV basket cells), whereas the other half contacts distal dendrites with a progressive decline in density toward the tip of dendrites ( Figures 2A,B ; Veres et al, 2017 ).…”

Neuronal Dystroglycan regulates postnatal development of CCK/cannabinoid receptor-1 interneurons

“…These include NECAB1, a calcium binding protein that specifically labels CCK+ IN cell bodies (Fig. 3A) (Miczan et al, 2021), and VGLUT3, a vesicular glutamate transporter enriched at CCK+ IN synapses (Fig. 3C) (del Pino et al, 2017; Pelkey et al, 2020; Somogyi et al, 2003).…”