In Situ Analysis of the Mu-Opioid Receptor Splice Variants in Adult Rat Brain

10.26686/wgtn.16967281 ◽

2021 ◽

Author(s):

◽

Kylie M Price

Keyword(s):

Rat Brain ◽

Opioid Receptor ◽

Splice Variants ◽

Distribution Patterns ◽

Mu Opioid Receptor ◽

Mu Opioid ◽

Adult Rat ◽

Adult Rat Brain ◽

Exon 1

<p>The original intention of this study was to exploit the specificity of circularisable ligation probes (CLiPs) in a unique approach of in situ genotyping the mu-opioid receptor (MOR) splice variants. CLiPs were designed to target a PCR generated MOR-1 template in vivo. The ligation results were consistent with circularised CLiPs, however due to the inherent limitations of this method the more conventional technique of fluorescent in situ hybridisation (FISH) was substituted for CLiPs to analyse to distribution of MOR splice variants in rat brain. Utilising FISH, the aim was to produce RNA probes (riboprobes) approximately the same size as the target specific region of CLiPs (~60-70 nt) to analyse the distribution patterns of MOR splice variants in rat brain. Five short (70-222 nt) riboprobes were generated to exons 1, 3, 4 and 9, and the 5' UTR + exon 1 of the Rattus norvegicus MOR gene (Oprm) to be utilised in FISH. The exon 1, 4 and 5' UTR + exon 1 riboprobes were shown to localise to MOR mRNA in brain structures previously reported to express MORs. These riboprobes also localised to mRNA within the Purkinje cells of the adult rat cerebellum, where it is generally accepted that only DOR is expressed in the rat cerebellum. MOR mRNA was visualised in many structures in the rat brain, including the dendate gyrus, inferior olive and spinal trigeminal nucleus. Riboprobes generated to the 5' UTR + exon 1 and exon 4 showed differential distribution patterns, the functional significance of this discovery is unknown, however these results implicate a role for FISH in tracking the distribution patterns of untranslated and translated mRNA. The use of novel new short riboprobes represents a technically difficult yet feasible technique for mapping MOR mRNA distribution in adult rat brain.</p>

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Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: Evidence for region-specific processing

The Journal of Comparative Neurology ◽

10.1002/(sici)1096-9861(20000403)419:2<244::aid-cne8>3.0.co;2-r ◽

2000 ◽

Vol 419 (2) ◽

pp. 244-256 ◽

Cited By ~ 107

Author(s):

Catherine Abbadie ◽

Ying-Xian Pan ◽

Gavril W. Pasternak

Keyword(s):

Rat Brain ◽

Opioid Receptor ◽

Splice Variants ◽

Mu Opioid Receptor ◽

Differential Distribution ◽

Mu Opioid ◽

Carboxy Terminal

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Purification and partial amino acid sequence of a mu opioid receptor from rat brain.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)74335-x ◽

1993 ◽

Vol 268 (35) ◽

pp. 26447-26451

Author(s):

C M Eppler ◽

J D Hulmes ◽

J B Wang ◽

B Johnson ◽

M Corbett ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Rat Brain ◽

Opioid Receptor ◽

Mu Opioid Receptor ◽

Partial Amino Acid Sequence ◽

Mu Opioid

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Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

Journal of Neurophysiology ◽

10.1152/jn.1992.68.3.756 ◽

1992 ◽

Vol 68 (3) ◽

pp. 756-766 ◽

Cited By ~ 158

Author(s):

T. M. Perney ◽

J. Marshall ◽

K. A. Martin ◽

S. Hockfield ◽

L. K. Kaczmarek

Keyword(s):

In Situ Hybridization ◽

Rat Brain ◽

K Channel ◽

Rnase Protection ◽

Hybridization Histochemistry ◽

Adult Rat ◽

In Situ Hybridization Histochemistry ◽

Adult Rat Brain ◽

Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

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