Abundant Expression of Calcineurin in Postsynaptic Elements of Excitatory Neurons in Mouse Somatosensory Cortex

Abstract

Calicineurin(CN) is a Ca²⁺/calmodulin-dependent serine/threonine protein phosphatase, and consists of catalytic subunit CNA and regulatory subunit CNB. CN plays an important role in intracellular signal pathway leading to the induction of long-term depression, a form of synaptic plasticity induced by NMDA-type glutamate receptor activation. In the brain, CN is composed of CNAα, CNAβ, and CNB 1 subunits, but their cellular expression and synaptic distribution have not been explored yet.
In the present study, I investigated expression of CNAα, CNAβ, and CNB l subunits in the layer 4 of the mouse somatosensory cortex, where whisker-related patterning, known as barrel, is formed. All three CN subunits exhibited similar cellular and subcellular distributions. Immunofluorescence visualized that the three CN subunits were distributed as tiny puncta or clusters in the cell body, dendrites, and neuropil. In double immunofluorescence for each CN subunit and MAP 2, a marker for neuronal cell bodies and dendrites, CNA α, CNAβ, and CNB 1 subunits were highly distributed on the surface of and inside MAP 2-positive dendrites. In addition, all CN subunits were positioned very close to VGLUT 1-positive excitatory terminals coming from the cortex. Low to moderate levels were found in dendrites of GAD 67-posotive inhibitory interneurons. In contrast, CNAα, CNAβ, and CNB 1 subunits showed no apparent overlap with VGLUT 2, VGAT or GLT 1, which are markers of excitatory terminals coming from the thalamus, inhibitory terminals, and astroglia, respectively. By immunoelectron microscopy using the silver-enhanced immunogold technique, metal particles representing CNAα, CNAβ, and CNB 1 subunits were detected at the highest level in spines and dendrites of excitatory neurons. Dendrites of inhibitory interneurons, which were identified by the lack of dendritic spines and the presence of asymmetrical synapse on dendritic shafts, were labeled about two times lower than those of excitatory neurons. Labeling in nerve terminal was detected but was low as compared to neuronal dendrites, and that in non-terminal portion of axons and glial elements were very low or negative.
This study shows that calcineurin is distributed in various neural elements with particular enrichment in postsynaptic elements of excitatory neurons. Because synaptic plasticity is induced following the activation of NMDA receptors on the postsynaptic membrane, the present findings provide the molecular-anatomical evidence that CN indeed constitutes intracellular signaling pathway downstream to NMDA receptors. Considering the essential role of NMDA receptors in barrel formation, CN enriched in postsynaptic elements of excitatory neurons appears to also play a role in activity-dependent synaptic refinement in the developing somatosensory cortex.