Distribution of M-channel subunits KCNQ2 and KCNQ3 in rat hippocampus

Abstract

Neuronal M-channels are low threshold, slowly activating and non-inactivating, voltage dependent K(+) channels that play a crucial role in controlling neuronal excitability. The native M-channel is composed of heteromeric or homomeric assemblies of subunits belonging to the Kv7/KCNQ family, with KCNQ2/3 heteromers being the most abundant form. KCNQ2 and KCNQ3 subunits have been found to be expressed in various neurons in the central and peripheral nervous system of rodents and humans. Previous evidence shows preferential localization of both subunits to axon initial segments, somata and nodes of Ranvier. In this work, we show the distribution and co-localization of KCNQ2 and KCNQ3 subunits throughout the hippocampal formation, via immunostaining experiments on unfixed rat brain slices and confocal microscopy. We find intense localization and colocalization to the axonal initial segment in several regions of the hippocampus, as well as staining for non-neuronal cells in the area of the lateral ventricle. We did not observe colocalization of KCNQ2 or KCNQ3 with the presynaptic protein, synaptophysin.

Fig. 1

Shown is a 40X magnification image of a fixed whole-brain slice stained with hematoxylin and eosin (H&E), showing the hippocampal region studied in this paper. The approximate locations of each Z-stack set of images taken in the figures shown hereafter in this paper are indicated by the white boxes, with the figure numbers indicated.

Fig. 2

3-D reconstructions showing expression of KCNQ2 and KCNQ3 in the CA1 region of the hippocampus. Unfixed rat brain slices were stained for either KCNQ2 (Q2, A) or KCNQ3 (Q3, B) (red) and synaptophysin (green). Both KCNQ2 and KCNQ3 subunits are present in the CA1 region of the hippocampus. The tubular shape of the staining and the localization in the proximity of the pyramidal cell layer (stratus pyramidale, Sp) indicate the localization of the channel subunits to the axonal initial segment (AIS) of the neurons. No colocalization of either KCNQ2 or KCNQ3 with synaptophysin could be observed. Stratum oriens (So) and stratum radiatum (Sr) are indicated. Together with KCNQ3, DAPI staining (blue) was used in order to visualize the cell nuclei. Scale bar 20 μm.

Fig 3

3-D reconstructions showing expression of KCNQ2 and KCNQ3 in the CA3 region of the hippocampus. Unfixed rat brain slices were stained for either KCNQ2 (A) or KCNQ3 (B) (red) and synaptophysin (green). Both subunits are expressed at the AIS of pyramidal neurons (arrows). Asterisks indicate diffuse expression of KCNQ2 at the neuronal somata within the stratum pyramidale (Sp). No colocalization of either KCNQ2 or KCNQ3 with synaptophysin could be observed. Stratum lucidum (Sl) and stratum oriens (So) are indicated. In (B), cell nuclei were visualized by staining with DAPI (blue). Scale bar 20 μm.

Fig 4

3-D reconstructions showing expression of KCNQ2 and KCNQ3 in dentate gyrus of the hippocampus. Unfixed rat brain slices were stained for either KCNQ2 (A) or KCNQ3 (B) (red) and synaptophysin (green). Intense staining for synaptophysin is present in stratum multiforme (Sm) and stratum moleculare (Smo), whereas neuronal cell bodies are concentrated within granule cell layer (stratum granulare, Sg). Both anti-KCNQ2 (A) and anti-KCNQ3 (B) antibodies label the neuronal AIS. In addition, KCNQ3 expression is observed in the cell somata of a few neurons, indicated by the asterisks in (B). Blue shows nuclear staining by DAPI. No presynaptic localization of either KCNQ2 or KCNQ3 could be observed. Scale bar 20 μm.

Fig 5

3-D reconstructions showing colocalization of KCNQ2 and KCNQ3 throughout the hippocampus. Unfixed rat brain slices were stained for KCNQ2 (green) and KCNQ3 (red). (A) The CA1 region in hippocampus shows the presence of both KCNQ2 and KCNQ3 at the level of the AIS of pyramidal neurons as well as their colocalization (merged image). (B) Similarly, in the CA3 region, although less densely expressed, KCNQ2 (green) and KCNQ3 (red) subunits colocalize to the AIS (yellow in merged image). (C) Shown is the dentate gyrus, with expression of M-channel subunits KCNQ2 (green) and KCNQ3 (red) at the AIS of neurons in the granule cell layer. Merged images reveal colocalization of both subunits. In all merged images, blue indicates DAPI staining of the cell nuclei. Stratum radiatum (Sr), stratum pyramidale (Sp), stratum oriens (So), stratum lucidum (Sl), stratum granulare (Sg) stratum multiforme (Sm) and stratum moleculare (Smo) are indicated. Scale bar 20 μm.

Fig 6

3-D reconstructions showing the lateral ventricle stained for KCNQ2, KCNQ3 and synaptophysin. (A) Double-staining reveals the lack of both KCNQ2 (red) and synaptophysin (green), in this region. DAPI staining (blue) indicate the presence of cell somata within the ventricle. (B) Anti-KCNQ3 antibody shows intense staining at the membrane of the cells contained in the lateral ventricle, most probably cells of the choroid plexus. No specific staining for synaptophysin was observed, whereas cell nuclei were visualized by DAPI staining (blue). (C) A different slide was co-stained for both M-channel subunits. Again, KCNQ3 (red) shows intense membrane localization, whereas no expression of KCNQ2 could be observed. Scale bar 20 μm.