Evidence for the cholinergic nature of C‐terminals associated with subsurface cisterns in α‐Motoneurons of rat

Abstract

C-terminals can be distinguished at the ultrastructural level from other types of nerve endings on motoneurons by their prominent and regularly occurring postsynaptic specializations termed subsurface cisterns (SSC). We have previously shown (Yamamoto et al., 1991) that an antibody directed against a sequence within the gap junction protein connexin32 immunolabels these motoneuronal SSCs and can therefore serve as a immunohistochemical tool to visualize indirectly the location of C-terminals on motoneurons at the light microscope level. Here we have used this anti-SSC antibody in combination with antibodies against choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) to determine whether C-terminals on motoneurons contain these cholinergic enzyme markers. In sections at all major spinal cord levels and in several cranial motor nuclei examined, motoneuronal cell bodies and their proximal dendrites were studded with large ChAT-immunoreactive (ChAT-IR) boutons. Boutons having a similar distribution and appearance on motoneurons were also immunolabeled for AChE. In addition, motoneurons were surrounded by a dense plexus of AChE-immunoreactive (AChE-IR) varicose fibers and fine preterminal axons. In double-labeled sections, AChE-IR boutons corresponded to those immunolabeled for ChAT. In sections processed for simultaneous immunofluorescence detection of ChAT and SSCs, ChAT-IR boutons were very often found in apposition to immunolabeled SSCs. In sections processed for simultaneous labeling of AChE and SSCs. AChE-IR boutons were again frequently seen abutting labeled SSCs. These results provide the first strong evidence at the LM level that a large proportion, if not the entirety, of C-terminals are cholinergic and show that these terminals consist in part of relatively large varicosities along highly varicose axons that form en passant type contacts on motoneurons. At the same time, our results substantially narrow possibilities regarding the as yet undetermined source of C-terminals, which can now be considered to originate from cholinergic neurons, such as those located in the brainstem and/or the spinal cord.

C-terminals on motoneurons: Electron microscope localization of cholinergic markers in adult rats and antibody-induced depletion in neonates

“…8 In agreement with electron microscopy histochemical results showing high concentrations of acetylcholinesterase (AChE) reaction product at motoneuronal C-terminal subsurface cistern complexes, 10,34 a high degree of co-localization was found between immunofluorescence labelling for subsurface cisterns and AChE. 24 These results, together with our observations that large ChAT-containing boutons on motoneurons correspond to those containing AChE, suggested that most, if not all, C-terminals are cholinergic. In order to substantiate this conclusion and to confirm data derived from indirect light microscopic analyses using the antisubsurface cistern antibody, the immunohistochemical localization of the cholinergic markers ChAT and AChE in terminals on motoneurons in the facial nucleus and lumbar spinal cord of adult rats was examined by light microscopy.…”

“…40 We found by light microscopy double immunolabelling methods that subsurface cisterns co-localize with choline acetyltransferase (ChAT)-labelled boutons on motoneurons at most cranial and spinal levels, 24 which is consistent with electron microscopic observations that C-terminals on motoneurons in the hypoglossal nucleus are cholinergic, based on their immunolabelling for ChAT. 8 In agreement with electron microscopy histochemical results showing high concentrations of acetylcholinesterase (AChE) reaction product at motoneuronal C-terminal subsurface cistern complexes, 10,34 a high degree of co-localization was found between immunofluorescence labelling for subsurface cisterns and AChE.…”

“…39,40 The possible significance of this antibody cross-reaction at subsurface cisterns has been discussed in detail 39,40 in the context of earlier speculated functions of subsurface cisterns in calcium mobilization 23 and the morphological similarity of subsurface cisterns to the sarcoplasmic reticulum terminal cistern/Ttubule complex in muscle 33 as well as with reference to the currently unknown anatomical origin of C-terminals. 24 Irrespective of molecular or functional considerations, we have attempted to develop this anti-subsurface cistern antibody as a marker for C-terminals, and thus as a potential tool to examine at the light microscopic level the distribution, transmitter nature and origin of these structures, which previously could only be identified at the ultrastructural level.…”

Beginning at the end: Repetitive firing properties in the final common pathway

“…Early on, Conradi and Skoglund (1969) presented immunohistochemical and electronmicroscopic data revealing large terminals on spinal motoneurons. These C-boutons were later shown to be cholinergic (Nagy et al, 1993;Li et al, 1995). They were shown to develop in the early post-natal period in mice (Wilson et al, 2004) and to be in apposition to both post-synaptic type 2 muscarinic (m 2 ) receptors and delayed rectifier K V 2.1 channels (Hellström et al, 2003;Muennich and Fyffe, 2004;Wilson et al, 2004).…”

Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion

“…The somata and proximal dendrites of MNs are contacted by large cholinergic varicosities named ''C boutons'' (3)(4)(5)(6)(7)(8)(9)(10)(11). It has been known since 1972 (12) that C boutons originate from spinal cord neurons, but the location of these cells remains unknown (10).…”