DISCUSSION.

The results presented in this chapter have provided some useful and interesting information regarding the expression o f the nociceptin receptor in the auditory system. One o f the most exciting results was the discovery that the OP4 receptor (and possibly NC) undergoes splicing in the auditory pathway. Furthermore, especially in the case o f OP4, these splicing events appear to be developmentally regulated and are tissue- specific. The significance o f these results will be discussed further below.

The distribution o f OP4 mRNA as determined by rt-PCR is not ideal. It is impossible to

say if protein is also expressed in the regions mRNA was detected. There is some controversy over whether expression patterns o f mRNA and protein are comparable. After mRNA exits the nucleus, protein synthesis may either take place within the soma o f the same cell, or where the protein is targeted. Nevertheless, the results presented previously may provide some information regarding the synthesis and production o f nociceptin and its receptor.

As mentioned previously, it was not possible to carry out a quantitative investigation, however every effort has been made to standardise each experiment. All RNA was diluted to the same concentration. Positive and negative controls were used in each experiment. Furthermore, each experiment was performed in duplicate on 5-10 separate occasions. Because o f the qualitative nature o f this study, it is difficult to comment on changes in levels o f OP4 expression at different ages. However, it is possible to remark on changes observed in the ratio o f splice variant expression in the same tissues at different developmental stages. It is also possible to draw conclusions regarding the

The Expression o f OP4 mRNA in the Auditory System.

T he results show ing expression o f OP4, and splice variants o f the OP4 receptor, during developm ent have revealed several interesting phenom ena.

Firstly, the expression pattern o f OP4 variants changes during developm ent. A t each tim e point it seem s that the total R N A produced in each tissue is equal, w ith differences occurring in the ratios o f transcripts detected. On the day o f birth, 0P4e is m ore highly expressed than OP4 in all tissues. A t P6 and onw ards, this pattern w as retained only in the frontal cortex and cerebellum (not shown). In the auditory tissues, OP4 becam e the m ore prom inent transcript, being produced in increasingly higher am ounts in com parison to OP4e

P ostnatal day 12 is a critical stage o f developm ent, w here the anim al is reacting to a m ore com plex acoustic environm ent for the first time. A t this tim e, it w ould be expected that any m ajor changes observed in the pattern o f transcripts expressed would reflect the auditory system adapting to respond to this state o f increased stim ulation.

O ne m ajor difference clearly observed at P I2, and thereafter is that the ratio o f 0P4:0P4e has increased in the auditory areas. If, as has b een suggested, nociceptin acts on the OP4 receptor to m odulate sensitivity o f the auditory system , the upregulation o f OP4 m R N A m ay reflect m ajor changes taking place in the auditory pathw ay at this tim e. Further differences can be seen in the cortex, w here OP4 and 0P4b transcripts are detected for the first tim e and 0P4a m R N A appears to be transiently dow nregulated in the inferior colliculus. This could suggest that OP4 m ay be involved in som e kind o f protective m echanism , protecting the ear from acoustic overstim ulation, or m ay rem ove

an excitatory influence on the brainstem. This hypothesis may be strengthened by the observation that 0 ?4a is present in the adult inferior colliculus, by which time the

system will have been able to adjust to the different conditions.

Finally, in the adult, the cochlear nucleus does not seem to express 0P4e mRNA and the inferior colliculus is expressing five nociceptin receptor splice variants, OP4, 0P4a,

0P4b, OP4C and 0P4e. The observation that OP4C is expressed only in this tissue in the

adult m ay suggest an involvement in the link between auditory and somatosensory systems, which is not mature until adulthood.

0 P4a and OP4C are only expressed in auditory tissues and it is possible that they may

have a specific role in the auditory system. In contrast OP4, 0P4b and Op4e are widespread and may play a general neuromodulatory role in the central nervous system.

In general, these results are in agreement with those obtained by Pan et al. (1998) who describe differential regional expression o f OP4 transcripts in mouse brain. They report a relative abundance in the whole brain o f 0P4> 0P4a> OP4C. They also report that there

is a low level o f expression o f 0P4b in mouse brain. This is in general agreement with these results, although a conclusion cannot be made for whole mouse brain. It does seem that the least abundant transcripts were generally 0P4b and OP4C, with OP4, 0P4a

and 0 P4e being more widespread, although differences between tissues and the overall

proportion o f each tissue in normal mice brain makes this hard to judge.

brain, with 0 ?4c and 0 P4d expressed exclusively in the brain and no other tissue

examined. It is possible that 0P4d was detected in this study, but as this variant differs from OP4 by only 10 bases, a lObp deletion, it is unlikely that it would have been distinguished in these preparations. Finally, Xie et al. (1999) report that OP4 mRNA splice variants are differentially expressed in sensory and sympathetic ganglia. Their data show that OP4 receptor mRNA is more highly expressed than the 0P4e variant in rat brain and dorsal root ganglion, whereas, expression o f the longer 0 P4e variant is

more highly expressed in sympathetic ganglia. In the case o f 0P4e, the alternative splicing which creates a truncated OP4 may be an important negative control mechanism to regulate the level o f OP4 mRNA (Xie et al., 2000).

The differential expression o f variants that have been detected among the regions implies region-specific splicing and argues strongly for a functional relevance. This will be discussed in more detail below.

The Expression o f Prepronociceptin mRNA in the Auditory System.

Previous studies o f mouse prepronociceptin (ppNC) transcripts revealed alternative splicing at the 3 -end o f exon 3 yielding two precursor proteins with different C- terminal sequences (Saito et al., 1996). The longer transcript, named N23K, corresponds to the cDNA clones isolated during the search for the endogenous 0 P 4 receptor ligand (M eunier et al., 1995; Reinscheid et al., 1995), and gives rise to a predicted precursor protein o f 23kDa. In the shorter transcript, named N27K, a stop codon in exon 3 is removed, resulting in a longer predicted precursor protein with an extended C-terminal sequence (Aijomand and Evans, 2001). It is likely that the results in Figure 4.8 show transcripts o f N23K (890bp) and the shorter N27K (~800bp) as

these lengths roughly correlate with those reported in mouse (Saito et al., 1996), rat and human (Aijom and and Evans, 2001).

Aijom and and Evans (2001) were able to detect two additional splice variants o f the ppNC gene, both o f which were shorter in length, being devoid o f exon 2. It is possible that the band o f approximately 300bp, observed in this study, corresponds to these shorter transcripts.

Several attempts were made to examine the expression o f ppNC mRNA during development in the mouse, however results were variable and inconclusive and have therefore not been shown. It is possible that the animals were stressed immediately before sacrifice and more ppNC RNA transcribed. This would not necessarily explain why expression was variable in purely auditory tissues. However due to the nature o f rt-PCR experiments, whole tissue blocks are used, which would contain sympathetic nerves and blood vessels, and it is impossible to separate ppNC contained within these areas from that o f the whole preparation. It is unlikely that similar circumstances would be seen with immunohistochemistry due to the time delay between mRNA production and protein synthesis. It is also possible that the variation observed between concurrent rt-PCR experiments were the result o f technical difficulties, although every effort was made to control for variations.

Studies conducted by Saito et al. (1997) reported that murine ppNC splice variants were expressed during neuronal differentiation and that both transcript and protein levels diminished with maturation. However, Aijomand and Evans (2001) report that ppNC splice variants were present as early as embryonic day 13 in rat brain and that

N27K, was consistently less abundant than the longer N23K transcript at each time point examined. In contrast, in the results presented here, it appeared that the shorter transcript was more abundant. These findings may be accounted for by species differences between the mouse and the rat.

In conclusion it appears that more work is needed to establish whether these NC transcripts are differentially expressed during development and what their function may be. It does appear that the transcripts may be differentially expressed in brain tissues o f the mouse. Even within the auditory system, the pattern o f transcript distribution differed between peripheral and central areas, and again between exclusively auditory areas (the cochlear nuclear complex) and those which also have a somatosensory input (the inferior colliculus). W hether or not this is significant remains to be determined.

It has been possible to draw similarities between the expression o f NC mRNA and the distribution o f NC protein in the auditory system. W ith immunohistochemistry, NC was localised in the efferent terminals and spiral ganglion o f the cochlea. Despite some technical problems, rt-PCR has suggested the presence o f ppNC mRNA in the cochlea, although it is not possible to say where exactly in the cochlea it is expressed.

Alternative RNA Splicing in the Nervous System,

It is evident that even a small change in the coding region o f mRNA can lead to a substantial switch in protein function, and that alternative splicing is used extensively as a way o f increasing diversity, particularly in the nervous system (Stamm et al., 2000; Claverie, 2001). This hypothesis has been suggested by numerous experiments which have looked at individual gene splicing, but the most recent evidence has come from the human genome project. Rough estimates have suggested that the number o f protein

encoding genes in the human genome is much less than expected (approximately 30,000: Consortium 2001; Venter et al., 2001). Although there are a number of different ways in which to generate the much larger number o f proteins expressed in the mammalian brain (including post-translational modifications, RNA editing and multiple start sites o f transcription), the major mechanism for generating isoform diversity is alternative splicing.

In the central nervous system interest has centred on the NMDA receptor due to its importance in synaptic plasticity and neuronal development. In the case o f NMDA R1 receptors, which are selectively localised at the postsynaptic membrane in mammalian brain, evidence indicates that alternative splicing specifies this localisation.

Recent work has shown that inserts in specific NMDA receptor variants are capable o f directing the receptor to the plasma membrane. However, the insert alone is insufficient to direct this localisation itself (Ehlers et al., 1995; Zukin and Bennett, 1995). In the case o f this receptor, the insert contains a phosphorylation site, as well as a high affinity binding site for calcium/calmodulin. The inclusion o f this insert thus impacts on the cellular signalling pathways that originate with the receptor (Ehlers et al., 1996).

There is some evidence that suggests that OP4 splice variants may have different functional properties. Mathis et al. (1997) conducted a pharmacological study on the binding properties o f the OP4 receptor in tissue obtained from mouse brain or from transfected cell lines. Saturation studies in mouse brain membranes revealed both a high and low affinity binding site in mouse brain, compared to only a single site in the transfected cell line. They conclude by suggesting the presence o f heterogenous,

Further studies by this group (Mathis et al., 1999) identified a high-affmity binding site with a selectivity profile distinct from the OP4 receptor and all the traditional opioid receptors. The most striking difference was that whereas opioids show no or very low affinity binding to the OP4 receptor, several opioid peptides such as dynorphin A analogues bound to this new receptor with relatively high affinity. Furthermore, this novel receptor had a unique regional distribution, being detected almost exclusively in a number o f brainstem structures (Mathis et al., 1999). It is possible that the receptor described above may be 0 P4a or OP4C, splice variants, which were only detected in

auditory tissues (Table 4.1).

In conclusion, it has been possible to show that both nociceptin and its receptor undergo alternative splicing. Saito et al. (1996) have shown that two alternative splice variants o f the ppNC gene (N23K and N27K) are functional and are able to promote neurite outgrowth. Additional NC splice variants are believed to lack a signal peptide sequence, causing alterations in their cellular trafficking and processing (Aijomand and Evans, 2001).

In the case o f OP4, there is evidence to suggest that the alternative splicing results in several proteins with different functions. At this time, further investigations are needed to determine whether all the transcripts can be functionally expressed and then it may be possible to gain an insight into their binding properties and functional characteristics. It may be, as has been suggested by Mathis et al., (1999), that each receptor splice variant has different binding selectivity, and could be activated in a number o f diverse conditions. If it is the case that some o f the OP4 alternative splice variants are exclusively involved in modulating auditory sensitivity, the discovery o f

peptides, which are selective for these receptor subtypes, could have important therapeutic implications.

The results presented here have shown that the OP4 transcript is more highly expressed in the auditory tissues examined in comparison to non-auditory CNS areas. The next chapter will describe a study undertaken to describe the basic pharmacological properties o f this receptor.

5