Localization of mRNAs Encoding Acetylcholinesterase and Butyrylcholinesterase in the Rat Spinal Cord by Nonradioactive In Situ Hybridization

Abstract

In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in α-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. α-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.

Unbiased Expression Mapping Identifies a Link between the Complement and Cholinergic Systems in the Rat Central Nervous System

“…Previous data demonstrate that in the rat spinal cord BChE is mainly expressed in motor neurons (51), corroborated in the present study by the finding of BChE immunoreactivity in axotomized motor neurons. Unlike many other molecules involved in synaptic transmission, the expression of BChE was increased in the ventral horn on the lesioned side and was also higher in DA compared with PVG strains both in naive and injured animals.…”

Cholinesterases are down-expressed in human colorectal carcinoma

“…4) contrasts with the higher BuChE activity in gut. Although the data regarding the number of copies of the BuChE mRNA can be affected by some bias on the RT step, a lack of relationship between the content of ChE transcripts and the level of activity has also been observed in rat spinal cord neurons, which show a greater amount of AChE than BuChE transcripts, despite the prevalence of BuChE activity in them [30]. The scant amount of BuChE mRNA in gut does not necessarily mean that the same occurs with the mature protein, considering the different steps at which the protein content can be regulated [31].…”

Acetylcholinesterase and agrin: Different functions, similar expression patterns, multiple roles

“…Timed-pregnant Wistar rats at 15 or 21 days of gestation and adult rats (1-3 months old) were sacrificed by exposure to CO 2 narcosis. Further steps of the preparation of the spinal cord sections for the RT PCR analyses and in situ hybridization were performed as described [21]. Human skeletal muscle myotubes were used to assess alternative effects of agrin.…”

“…AChE-T (splice variant T) and ColQ mRNA expression was measured by competitive RT-PCR; primers sequences for AChE-T and ColQ and the detailed protocols are provided in [21] and [6], respectively. The same technique was used for determination of agrin mRNA transcripts 0, 8 and 19.…”

“…Primers spanned alternative splicing site Z for agrin mRNA enabling us to detect all splice variants at this position; the following primers were used: forward 5 0 -CATCGAATACCTCAATGCTGTGATTGAG-3 0 -exon 31, reverse 5 0 -GCAGAACGTGCAGATTACATGGCC-3 0 -exon 34. The motor neuron origin of AChE and agrin mRNAs was verified by non-radioactive in situ hybridization using digoxigenine-labeled RNA probe as described before [21]. AChE probe extended exons 4 and 6 of rat AChE-T cDNA (nucleotides 1471-1801).…”