Nociceptin and the OP4 receptor in the auditory system

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N O C IC E PTIN AN D THE OP4 R E C E PTO R IN THE

A UDITOR Y S Y S T E M

Wendy J N oble

A thesis presented to the University o f London f o r the degree o f

D octor o f Philosophy.

k

UCL

Institute o f Laryngology and Otology University College London

London, UK.

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Acknowledgements

/ would like to express my sincere thanks to my supervisors Dr. Hilary Dodson and Professor Andrew Forge fo r giving me the opportunity to pursue this PhD in their laboratory and fo r all their time and effort during the course o f my studentship. In particular, I would like to thank H ilary fo r her tireless effort - fo r passing on her expertise in this line o f work from the very fir st day I started. For her humour, patience, optimism and friendship over the last fe w years - it has all been greatly

appreciated and will never be forgotten.

I am very grateful to my supervisor at Pfizer, Dr. Christine Williams fo r the opportunity to study in deepest, darkest Kent. Her knowledge and help with those experiments, and valued input in all other aspects o f the work were invaluable. The near heart attack in her back garden after dinner nearly set this back a fe w years, but thanlfully I survived! Thanks also to Jo and Bernie fo r their hospitality in Broadstairs, you made me fe e l very welcome and I hope I can repay the fa vou r some day. (You can

keep the ghost cat down there with you though!)

For advice and assistance with all molecular biology work, my thanks to Dr. Stephano Casalotti. From the endless PCR runs in the beginning, to the disappointments with in situ, you were always there and always optimistic. Additional thanks fo r your input when this thesis was being written. Good luck to Italy in 2004!

Many thanks to Graham Neville fo r all the technical help and fo r making the lab an enjoyable place to work. The latest story on bringing up children was always guaranteed to raise a smile on the worst o f days! You've only about another 15 years to go.

For all their help and expertise with the in vivo Physiology, I am extremely grateful to Drs D avid McAlpine and Neil Ingham.

Thanks to all the girls in the lab - Jill, Victoria, Nerissa, Regina and Ruth fo r making the Institute a good place to be. Special thanks to Jill fo r being a good friend, fo r sharing the laughs and the bad days and fo r making me hope that Rovers have had a good game. I ’m afraid my heart will always lie with the mighty Liverpool F C though

(and Hibs o f course, dad).

Thanks to Dr. Karen D u ff and Dr. M ark B um s in New York fo r their understanding and advice when I was finishing the writing.

To my parents, I am indebted fo r all your support (financial and otherwise). From the bottle o f sauce delivered to the other end o f the country to the paper delivered to the other side o f the world, I am so grateful fo r everything you have done. You have always had fa ith in me, never let me give up on anything and have always been there when I have needed you. I appreciate it all and love you more than I ’m sure I tell you (so y o u ’ve got this in writing to remind me).

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ABSTRACT.

The opioid receptor- like OP4 receptor and its endogenous ligand, nociceptin (NC),

have recently been isolated. Evidence suggests that the NC system m ay modulate

sensitivity o f the auditory system (Nishi et al., 1997).

The work presented in this thesis was undertaken to elucidate the role o f the NC system

in auditory function. NC is expressed in both central and peripheral auditory structures.

Furthermore, levels o f NC expression appear to correlate with the development and

maturation o f the auditory system, both in precocial and altricial animals, suggesting

that nociceptin expression may be related to auditory stimulation. To explore this

possibility further, NC expression was examined in models o f altered afferent input.

Results obtained suggest that NC is affected by the level o f acoustic stimulation and

may act via the olivocochlear efferent system to protect the cochlea from damage.

Evidence also suggests that NC is involved in auditory plasticity as long-term

alterations in NC expression were noted following unilateral deafening.

A differential regional and developmental pattern o f OP4 receptor splice variant

expression was observed, suggesting that the level o f receptor expression may be

related to development o f the auditory system and raising the possibility that different

variants m ay play specific roles in each tissue.

Pharmacological properties o f the OP4 receptor were determined in an OP4 receptor-

transfected CHO cell line. Peptides identified in this study were then used in in vivo

physiological experiments. These have shown that NC antagonists are able to reduce

the discharge rate o f binaurally-stimulated inferior colliculus neurones in response to

pure tones.

In conclusion, it appears that the NC system may play an important regulatory role in

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neurotransmitters to modulate auditory sensitivity. These effects may be a result o f NC

receptor activation at the level o f the organ o f Corti and/or in more central auditory

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1

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CHAPTER 1: GENERAL INTRODUCTION.

The auditory pathway is a highly integrated communication network, which transmits

incoming auditory information to the auditory cortex where we perceive and interpret

sound. The transduction o f sound from acoustic into neural signals occurs in the

cochlea. The transduced information travels via the cochlear nerve, is then subdivided

and projects tonotopically through the various nuclei that constitute the auditory

pathway (see below). All o f these nuclei have a role in modifying and analysing

incoming auditory information and are also subject to descending, efferent influences

from higher centres. Efferent feedback mechanisms can modulate sensitivity

throughout the system and are crucial in normal hearing, as well as in the development

and maintenance o f neural connections (Brugge et al., 1978; Favre and Sans, 1978;

Lenoir et al., 1980; Ginzberg and Morest, 1983).

The transduction and transmission o f auditory signals involves inhibitory and

excitatory mechanisms mediated by neurochemicals. Neurones communicate via the

release o f neurotransmitters, which can initiate action potentials in adjacent neurones.

In this way, auditory information is passed from one part o f the system to another.

Other chemicals react to environmental and intrinsic cues to regulate and adapt neural

transmission. These neuromodulators may also function as neurotransmitters, and can

act to excite or inhibit the release o f other neurochemicals (Fex and Altschuler, 1981;

Hoffrnan et al., 1983, 1984, 1985; Eybalin et al., 1984; Eybalin and Pujol, 1984;

Altschuler et al., 1984a,b, 1985a,b; Drescher and Drescher, 1991, 1992; Scholtz et al.,

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Nociceptin, an opioid-like neuropeptide may play a role as an auditory neuromodulator,

regulating the responsiveness o f the system to auditory stimulation. Despite its

structural homology to other opioids, nociceptin binds specifically to its own receptor,

OP4. In OP4 receptor knockout mice, Nishi et al (1997) described a significant

elevation in the threshold o f the auditory brainstem response following exposure to

intense noise (105dB SPL, 60 minutes). Furthermore, there was a significant delay in

recovery in comparison to normal animals. This would suggest that the nociceptin

system plays an important role in modulating auditory sensitivity and as such is an

important area o f research.

Before it is possible to consider a specific role o f nociceptin in the auditory system, it is

necessary to have an understanding o f auditory function. The anatomy, physiology and

neurochemistry o f the auditory pathway from the cochlea to the inferior colliculus will

be described below, together with a general description o f the nociceptin system and its

possible involvement in auditory function.

The Inner Ear.

The inner ear is a fluid-filled bony structure embedded deep in the temporal bone o f the

skull. It contains the semicircular canals, the vestibule and the cochlea. The cochlea

consists o f a coiled canal, divided longitudinally into three scalae, spiralling around the

central modiolus. The scala media contains the potassium rich endolymph. It is

separated fi"om the scala vestibuli above by Reissner’s membrane and from the scala

tympani below by the basilar membrane together with the osseous spiral lamina

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The ossicles in the air-filled middle ear transfer sound vibrations from the tympanic

membrane, causing a displacement o f fluids in the cochlea. The pattern o f displacement

exhibited by the mammalian cochlear duct takes the form o f a travelling wave with

several unique features (von Bekesy 1951). This wave is unidirectional, originating at

the base and travelling towards the apex o f the cochlea. It exhibits a peak o f maximum

displacement at a specific location along the cochlear duct that is strictly determined by

the fi-equency o f the acoustic stimulus; high frequency stimulation causes maximum

displacement near the base o f the cochlear duct, lower frequencies at progressively

more apical locations. This allows the generation o f a cochlear frequency-place map,

which underlies the ability o f mammals to analyse a spectrum o f sounds. This

cochleotopic and tonotopic organisation is maintained throughout the auditory

pathway.

The organ o f Corti.

Acoustic transduction within the cochlea takes place in the organ o f Corti, a complex

epithelial ridge that protrudes into the scala media and rests on the basilar membrane

throughout the length o f the cochlea duct (Figures 1.2a and 1.2b).

Two main categories o f cell are found in the organ o f Corti. There are the cochlear hair

cells, sensory receptors which transduce acoustic into neural signals. In contrast, the

supporting cells do not respond to acoustic stimulation but provide support,

surrounding and underpinning the auditory receptors.

Cochlear hair cells are capped, on their apical surface, by several rows o f stereocilia.

H air cells can be divided into two morphological and functional types, inner hair cells

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shaped with a centrally located nucleus. The contractile outer hair cells may contribute

to mechanical feedback processes, playing an important role in maintaining normal

frequency selectivity and hearing sensitivity. (Kiang et al., 1970; Ryan and Dallos,

1975; Cody and Russell, 1985). These are cylindrical in shape, with a nucleus located

at the base o f the cell.

There is typically one row o f IHCs and between three and five rows o f OHCs, with a

human ear containing approximately 15000 hair cells. A shearing motion between the

tectorial membrane and the reticular lamina stimulates the hair cells. This movement

brings about a deflection o f the stereocilia bundles at the apical surfaces o f the hair

cells, resulting in the generation o f a receptor potential. The stereocilia o f the OHCs are

embedded within the tectorial membrane, whereas IHC stereocilia are mechanically

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F ig u r e 1.1 T h e M a m m a lia n C o c h le a

a)

Rjeissner’s membrme

Oïgan o f Corti

Basilar mertibrarie

Spiral ^ g b o n

m odiolus

b)

Scala vestibuli

Scala media

Spiral ganglion

Scala tympani

Reissner’s membrane

Stria vascularis

Spiral ligament

Organ of Corti

Basilar membrane

Fig. 1.1a: A midmodiolar section through the guinea pig cochlea. The scala vestibuli (SV), scala media (SM) and scala tympani (ST) can be seen, separated from each other by the basilar membrane and Reissner’s membrane. The three scalae spiral together to form four turns. The organ o f Corti can be seen sitting on the basilar membrane. Also labelled are the modiolus (Mo), stria vascularis (St. V) and the spiral ligament (Sp. L). Taken from Slepecky(1996, Fig 2.1).

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F ig u r e 1.2. T h e O r g a n o f C o r ti.

a)

Outer hair

c e lls C e lls o f H ensen

T ectorial m em brane

Inner hair c ell

O

b)

T ectorial m em brane

Supporting (Deiters’ cells)

\

Irrher hair cell Outer hair cells

mm

Fig. 1.2a: Cross sections of the organ of Corti. One row of inner hair cells and three rows of outer hair cells can be seen, together with a variety of supporting cells. The entire structure sits on the basilar membrane and is covered by the fibrous tectorial membrane. The stereociliary bundles of the outer hair cells can be seen to make contact with the tectorial membrane. Taken from Pickles (1988, Fig. 3 .ID).

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Cochlear Innervation.

Inner hair cells are the primary transducers o f sound waves, whereas outer hair cells are

involved in regulating the responsiveness o f this sensory organ (Kiang et al, 1970;

Ryan and Dallos, 1975; Cody and Russell, 1985). The difference in function is made

more apparent if we consider their pattern o f innervation.

Approximately 20 radial afferent fibres contact each IHC. These Type 1 afferent

neurones are bipolar, their peripheral process projecting to the hair cells and the central

process to the cells o f the cochlear nucleus in the brainstem. Outer hair cells are

contacted by few, convergent. Type II afferents, each neurite branching to innervate ten

or more OHCs. Therefore, the great majority (approximately 93%) o f the cochlear

spiral ganglion neurones innervate the IHCs only, whereas about 7% innervate OHCs

(Spoendlin, 1972; Morrison, Schindler & Wersall, 1975). The IHC afferents are

myelinated central to the habenula and are o f larger diameter than the unmyelinated

OHC afferents, suggesting they conduct nerve impulses more rapidly. Axons from both

types o f afferent arise from the type I and type II ganglion cells lying in Rosenthals

canal. Both types o f afferent run via the cochlear nerve to the cochlear nucleus. An

efferent pathway parallels the ascending pathway throughout.

The olivocochlear bundle comprises the efferent innervation o f the cochlea. The nerves

o f this system can be anatomically divided into two components (W arr and Guinan,

1979; Warr, 1992). The somata o f the lateral olivocochlear efferent system (LOC) are

found in the lateral superior olive (LSO). They project mainly to the ip si lateral cochlea

where their thin, unmyelinated axons synapse with radial auditory dendrites below

inner hair cells. Medial olivocochlear efferent fibres (MOC) originate from neurones

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their axons projecting to the contralateral cochlea (the crossed olivocochlear bundle),

the remaining 30% to the ipsilateral ear. There, they synapse directly with the basal

pole o f the outer hair cells (Fig 1.3).

Figure 1.3: The Olivocochlear Efferent System,

Neurotransmitters

Figure 1.3: The olivocochlear efferent system. The main efferent pathways from the auditory brainstem nuclei to the organ o f Corti are shown. As can be seen there are two main divisions o f this system. The lateral efferent system arises from the LSO and projects to the ipsilateral cells. The medial efferent system arises mainly from die MSO and projects to the contralateral cochlea, where the fibres innervate the outer hair cells. The main neurotransmitters involved are also shown.

VCN: ventral cochlear nucleus, LSO: lateral superior olive, MSO: medial superior olive, ICP: inferior cerebellar peduncle, IHC: inner hair cell, OHC: outer hair cell.

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Auditory Brainstem N uclei

The Cochlear Nucleus

The cochlear nuclear complex (CNC) is the first nucleus o f the central auditory

pathway where the neural integration o f acoustic information occurs. It comprises the

anteroventral (AVCN), posteroventral (PVCN) and dorsal (DCN) cochlear nuclei and

plays a role in encoding binaural differences in the time and intensity o f acoustic

stimuli. The cochleotopic projection o f afferent fibres in different nuclear divisions

leads to an orderly arrangement o f the characteristic frequencies o f the neurones they

innervate. Axons tuned to high frequencies are located dorsally, with low frequency

axons occurring more ventrally.

In the ventral cochlear nucleus there are three major classes o f neurones: spherical,

octopus and multipolar cells. Spherical cells are found in the rostral pole o f the AVCN.

These are contacted axosomatically by one or more ascending auditory nerve fibres,

which form a chalice like structure, the end bulb o f Held. Spherical cells project to the

ipsilateral superior olive and then to the medial superior olivary complex o f both sides.

There are three layers in the dorsal cochlear nucleus: the molecular, granular and deep

layers. Small intrinsic excitatory neurones, the granule cells, send axons from the

granular layer into the outer molecular layer. In the granular layer, fusiform or

pyramidal cells receive input from afferent cochlear nerve fibres. They send projections

to the central nucleus o f the contralateral inferior colliculus, and are believed to have an

important role in binaural processing (Kitzes, 1978; Moore, 1991). Giant or multipolar

cells in the deep layer also project to the inferior colliculus, although some project to

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connections with the pyramidal cells, the granule cells and each other. They also

project axons to the inferior colliculus and the AVCN.

The Auditory Midbrain,

The Superior Olivary Complex (SOC).

The superior olivary complex is located in the midbrain at the level o f the lower pons.

In mammals, there are three main nuclei: the lateral and medial nuclei o f the superior

olive (LSO and MSO) and the medial nucleus o f the trapezoid body (MNTB). The LSO

and M SO are surrounded by the smaller cell groups o f the pre- and peri-olivary nuclei

(Rasmussen et al., 1946; Elverland, 1978).

The LSO receives both ipsi- and contra-lateral connections from the VCN and

indirectly from the VCN via the MNTB. The region as a whole is predominantly

responsive to direction o f a sound source.

The M SO comprises bipolar neurones arranged in parallel layers. The medial dendrites

receive contralateral input from the AVCN, the lateral dendrites receiving the

ipsilateral input. This is the first level o f the auditory system at which there is

integration o f information from the two ears onto individual neurones. This integrated

information is transferred to other cells within the SOC for further processing and to

higher centres, the lateral lemniscus and inferior colliculus, as well as back down to the

CNC and cochlea. The main role o f the MSO is to respond to disparities in interaural

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The Inferior Colliculus (IC).

The IC can be subdivided into a central nucleus (IC) an external cortex (EC) and a

dorsal cortex (DC).The central nucleus has a lamellar organisation. It is the main link in

the ascending auditory pathway and is reciprocally connected with the contralateral

central nucleus. The cochleotopic organisation o f the auditory pathway is maintained in

the IC, with high CF units found ventromedially and low ones dorsolaterally. The IC is

multi-sensory, receiving ascending input from somatosensory afferents together with

auditory input from the central nucleus and lower auditory centres. Almost all

brainstem auditory nuclei terminate in the inferior colliculus en route to the thalamus

and auditory cortex. Each region o f the IC is connected with its contralateral

counterpart, enabling information to be integrated across the parallel ascending

pathways.

Function o f the Cochlea,

There are three main elements o f cochlear function: the sound-induced motion o f the

basilar membrane, the transduction o f this motion by the inner hair cells into a neural

signal, and the neuro-mechanical feedback introduced by the outer hair cells. It is

possible that the nociceptin system may be involved in this feedback response in some

way.

The Olivocochlear Efferent System,

The organisation o f the olivocochlear efferent system has been described in some detail

previously. In this section, the main function o f this system will be discussed.

The absolute sensitivity o f the ear is determined by the minimal acoustic energy

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determined, primarily, at the level o f the outer hair cells by fibres o f the olivocochlear

efferent system (Guinan, 1996 review).

Both the inner hair cell and its afferent fibres are susceptible to acoustic

overstimulation, which results in an elevated threshold to sound. Threshold shifts can

be temporary or permanent and are known to correlate with structural changes in the

stereocilia, cuticular plate, hair cell body, synaptic region o f the hair cell and post-

synaptic afferent nerve terminals (Saunders et al, 1985 review). Electrophysiological

evidence (Galambos, 1956) has determined that stimulation o f the efferent system can

alter the structure o f OHCs and their efferent terminals and also inhibits afferent fibre

activity. Furthermore, Dodson et al. (1986) electrically stimulated the afferent pathway

on one side via a round window electrode. The structural changes in OHCs and efferent

terminals observed in both cochleae were considered to have occurred through

feedback mechanisms via the crossed olivocochlear bundle (COCB).

Electrical stimulation o f the olivocochlear bundle has been shown to raise thresholds o f

primary afferent neurones within 100ms (Galambos, 1956; Wiederhold and Kiang,

1970; Guinan and Gifford, 1988a,b). Furthermore, this 'protective' mechanism is

blocked by systemically applied strychnine, a known blocker o f cholinergic efferent

synapses. This fast effect is thought to arise from the hyperpolarisation o f OHCs by

calcium influx through nicotinic receptors. This acts to decrease their amplification of

basilar membrane motion and hence decreases stimulation o f IHCs (Reiter and

Liberman, 1995). The OHCs aid the sharp tuning o f the IHCs via what is believed to be

an amplification o f the travelling waves along the basilar membrane (Ashmore and

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be responsible for the increased timing to the basilar membrane. Thus, the medial OC

efferents would exert their effect mechanically. Sridhar et al (1995) reported an

additional slower response o f sound-evoked nerve activity that is also efferent-

modulated. They propose that the slow effect is attributable to release o f calcium from

the subsurface cistemae o f the OHC, causing calcium-gated potassium channels to

open and leading to hyperpolarisation o f the hair cell on a much larger time-scale

(Sridhar et al, 1997). Reiter and Liberman (1995) believe that protection against

temporary threshold shifts caused by high-level tones is correlated with the OC slow

effect. Efferent stimulation also reduces the level o f otoacoustic distortion products

(Mountain, 1980; Siegel and Kim, 1982). Otoacoustic emissions (OAEs) are low-

intensity sounds, produced specifically by the cochlea and, most probably, by the

cochlear outer hair cells as they undergo fast, reversible length changes. Changes in

their amplitude as a result o f acoustic overstimulation reflect structural and fimctional

damage to the hearing organ.

In summary, the olivocochlear system provides a mechanism by which the inner ear

can (i) improve the detection o f signals in noise: olivocochlear stimulation suppresses

continuous background noise, (ii) be protected fi*om noise damage, due to changes

elicited in the mechanical response o f the cochlea and (iii) control the mechanical state

o f the cochlea. This may be used to adjust for the effects o f changes in the cochlea

during aging, thus maintaining a relatively constant mechanical state in the cochlea.

Auditory Neurotransmitters

Neurones transmit their spikes to other neurones over their axon and contact other

neurones through synapses. M ost synapses release neurotransmitters into the cleft

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presynaptic cell. The neurotransmitter binds to specific receptors in the postsynaptic

cell, causing specific ion channels to open. The type o f neurotransmitter involved

depends on the type o f synapse, with its effect determined by the specific class o f

receptor and its cellular effectors (discussed in more detail in Chapter 5).

The principal inhibitory neurotransmitters in the auditory system are y-aminobutyric

acid (GABA) and glycine, which open chloride channels in the post-synaptic cell,

eliciting inhibitory post-synaptic potentials (IPSPs) and in most cases hyperpolarising

the cell.

In the mammalian cochlea, glutamate is the excitatory afferent neurotransmitter.

Glutamate is both spontaneously released by IHCs, and released in response to

stimulation. Released glutamate binds to both N-methyl-D-aspartate (NMD A) and non-

NMDA receptors in type I auditory neurones (Puel, 1995). These fibres, which

innervate the modiolar IHC surface, are contacted by a relatively large number o f

lateral efferent terminals.

Neurochemically, the lateral and medial olivocochlear systems are quite distinct. It is

reported that the medial efferent system is nearly exclusively cholinergic, with

calcitonin gene-related peptide (CGRP) and GABA most probably used as additional

neurotransmitters and/or neuromodulators (Eybalin and Pujol, 1987, Fig 4). At the level

o f lateral efferent terminals, acetylcholine (ACh), GABA and CGRP have again been

proposed, with the additional involvement o f dopamine and the opioid peptides

enkephalins and dynorphins (Fex and Altschuler, 1981; Usami et al, 1988a,b; Gil-

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(co)-release different neurotransmitters depending on certain physiological and/or

pathological conditions (Drescher et al, 1983; Altschuler et al, 1984a, b, c).

Endogenous Opioid Neuropeptides,

Although substantial evidence indicates that ACh is the main neurotransmitter for

medial and lateral OC efferents, these fibres also use neuroactive opioid peptides.

The term ’opioid' is used to describe all substances that are opium- or morphine- like in

action, and retain their capacity to modulate sensory input via their binding to

designated (opioid) receptors (Sahley and Nodar, 1994). These substances include

synthetic and natural drug alkaloids as well as the neuroactive endogenous enkephalin-

and dynorphin-related peptides (Simon and Hiller, 1978 a,b). Three main classes o f

opioid receptor have been identified, p, ô and k , which are activated by morphine-,

enkephalin- and dynorphin- related peptides respectively (Evans et al., 1992; Kieffer et

al., 1992; Chen et al., 1993; Minami et al., 1993).

Further attempts to clone additional opioid receptor subtypes led to the isolation o f

cDNA encoding a homologous protein with a high degree o f sequence similarity to the

opioid receptors, but did not bind any existing opioid peptides with high affinity. This

'orphan' opioid receptor has now been identified from human (O R L l, Mollereau et al,

1994), mouse (MOR-C, Pan et al, 1995) and rat genomic libraries (LC132, X O R l, C3,

ROR-C, oprl and H yp8, Bunzow et al, 1994; Fukuda et al, 1994). In accordance with

recent UIPHAR recommendations, the term OP4 will be used to describe the receptor

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In 1995, two groups simultaneously reported the isolation o f the natural ligand for OP4.

This peptide neurotransmitter was biologically related to the opioids, but heightened

sensitivity to pain when injected into the brains o f mice. Hence, M eunier et al (1995)

named it nociceptin (NC). As the receptor was found before the ligand, Reinscheid et al

(1995) referred to the peptide as orphanin FQ, the peptide for the ‘orphan receptor’, and

because its amino acid sequence begins with phenylalinine (F) and ends with glutamine

(Q) (discussed in more detail in chapter 5).

Nociceptin,

Like the endogenous opioid ligands, NC is derived from a larger precursor,

prepronociceptin (preproNC). The nucleotide sequence o f the preproNC gene has

similar sequence characteristics and organisation to the opioid peptide precursors,

particularly preproenkephalin and preprodynorphin, suggesting these peptides may

share a common evolutionary ancestor. However, despite the structural similarities

between the opioid and NC systems, they are pharmacologically distinct. As a general

rule, NC does not bind to opioid receptors and opioid ligands have no affinity for the

OP4 receptor (Table 1.1). However there have been recent studies suggesting that

opioid peptides may bind with specific subtypes o f the nociceptin receptor (Mathis et

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Table 1.1: Opioid Receptors and their Ligands.

R ecepto r N am e _{O Pi} _{O P}2 O P3 O P4

Previous name Ô K O RLl

Coupling Gi/o Gi/o Gi/o Gi/o

Natural ligands Leu-enkephalin

Met-enkephalin

Dynorphin

Dynorphin (1-8)

P-endorphins

endomorphins

nociceptin

Synthetic

agonists

DPDPE Spiradoline DAMGO NC(1-13)NH2

Selective

antagonists

Natrindole Nor-BNI CTOP [Nphe‘]N C

(l-13)NH2

Non-s elective

antagonists

Naloxone Naloxone Naloxone Naloxone

insensitive

Table 1.1: G protein-coupled opioid receptors and their specificity. Opioid ligands do

not bind to the OP4 receptor, just as nociceptin does not bind to any opioid receptors.

(Data from Corbett et al, 1982; Zadina et al, 1997; Guerrini et al, 1998; Calo et al,

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Effect o f the Nociceptin System on Neurotransmitter release.

In the central nervous system, nociceptin inhibits the release o f neurotransmitters such

as dopamine (Murphy et al., 1996), glutamate (Nicol et al., 1998), GABA (Meis and

Pape, 2001), acetylcholine (Neal et al., 1997), noradrenaline (Schlicker et al., 1998)

and serotonin (Sbrenna et al., 1999). Similarly, in the periphery, NC inhibits the release

o f neurotransmitters from sympathetic (Nicholson et al., 1998), parasympathetic (Calo

et al., 1996) and peptidergic nerves (Giuliani et al., 1997; Rizzi et al., 1999 a,b). NC

has been shown to inhibit dopamine release in anaesthetised rats when administered

intracerebroventrically ((icv), M urphy et al., 1996). However, it has also been shown to

increase dopamine release in the striatum o f conscious rats (Konya et al., 1998),

indicating its effects on neurotransmitter release are not clear-cut. NC also has a

biphasic effect on enkephalin release. Low doses (1-lOnM) inhibit, whereas high doses

(lOO-lOOOnM) stimulate release.

In various preparations, NC has been reported to inhibit neurotransmitter release due to

a reduction in excitability o f the presynaptic neuronal elements and/or a reduction in

presynaptic neurotransmitter secretion (Calo et al., 2000). NC may act by inhibiting

Ca^^ channels within the nerve terminals, activating presynaptic K^ channels or through

direct modulation o f the release apparatus, usually thought to result from the

spontaneous exocytosis o f transmitter-containing vesicles occurring in the absence o f

Ca^"^ influx (Miller, 1998). Meis and Pape (2001) have reported that NC inhibits the

release o f GABA and glutamate in the rat lateral amygdala by such presynaptic

mechanisms.

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suggests that nociceptin, like the classical opioids may be a very important

neuromodulator. In the auditory system, little has been published regarding the

distribution o f nociceptin or its receptor. However, it has been shown in other systems

that nociceptin and the opioids are found in parallel, yet distinct fibre routes. This may

also be true in the auditory pathway (discussed in more detail in chapter 2).

Involvem ent o f Opioids in M odulating Auditory Function,

Neuroactive enkephalins and dynorphins typically produce inhibition within the

nervous system (Sahley and Nodar, 1994). Opioid receptors can exist pre- or post-

synaptically (Cherubini and North, 1985; Reisine and Bell, 1993; Hiller et al., 1997).

Evidence for the existence o f opioid receptors within the mammalian cochlea has been

determined mainly from stereospecific binding studies (Hoffinan, 1986) and the

observation that inhibition o f receptor-linked adenylate cyclase in the cochlea is

reversible by naloxone, a specific opioid antagonist (Eybalin et al, 1987 a,b).

There is a growing amount o f evidence to suggest that opioid-containing OC neurones

are involved in modifying auditory function. Sahley and Nodar (1994, 1996) present

results that indicate that dynorphin peptides contained within lateral efferent OC

neurones act via k - opioid receptors to improve auditory sensitivity to low intensity

stimuli. Interestingly, interactions between opiates and cochlear acetylcholine receptors

have been shown in Xenopus auditory organs by Lioudyno et al. (2000). There is

evidence that opioids may also be acting at the level o f the auditory brainstem

(Mansour et al, 1994; Kungel and Friauf, 1995; Code, 1996; Code and McDaniel,

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neurotransmitters such as acetylcholine to exert their effect on hearing sensitivity

(Sahley and Nodar, 1996).

Nociceptin and Auditory Function,

Further evidence for an opioid-mediated effect on hearing has been shown using

nociceptin-receptor knockout mice. Nishi et al (1997) reported that after exposure to

intense sound homozygous ORL-1 knockout mice showed a significant increase in

auditory brainstem response as compared to baseline readings (average: 37dB SPL) and

with heterozygous mutants and wild-type controls. This threshold shift returned to

normal levels within 3 days and was therefore deemed temporary.

The auditory system is able to adjust following injury or sensory receptor loss to

function in ways that can often compensate for these losses. For example, auditory

ftmction is impaired by unilateral deafening, noise-induced hearing loss or by nerve

damage resulting from acoustic over-exposure. Under such circumstances, the 'plastic'

properties o f the auditory system allow the reorganisation o f neurones formerly

involved in areas that have been damaged (discussed in more detail in Chapters 2). This

reorganisation o f neurones is not fully understood, and can often result in unwanted

side effects, such as mislocalisation, hyperacusis and tinnitus (Kaas, 1995).

Opioid peptides may be involved in such physiological states as hyperacusis and

tinnitus. Opioids are involved in the overall biological response to stress (Akil et al,

1984) and can also enhance the regulatory functions o f other neurotransmitters (Lewis

et al, 1987 a,b). During stress reactions opioid peptides, specifically dynorphins, may

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NM DA receptors in the cochlea. Consequently, the spontaneous activity generated

during periods o f quiet may be altered or amplified, producing symptoms o f tinnitus.

Nociceptin is known to be heavily involved in pain pathways and mechanisms

throughout the CNS. A pathophysiology has been suggested between chronic pain and

chronic tinnitus (Tonndorf, 1987; Moller, 1997, 2000). In these models, both pain and

tinnitus consist o f neural sensitisation, involving a reduction in threshold and/or an

increased neural excitability to stimuli. In this case, intense sound would exacerbate

tinnitus and repeated exposure to such stimuli in tinnitus sufferers may lower auditory

tolerance to all sounds (Gabriels, 1996; Jastreboff, 1996).

As has been discussed above, initial evidence supports a role for the nociceptin system

in the modulation o f auditory function (Nishi et al., 1996). This is supported by

localisation studies, which have shown that nociceptin is highly expressed in auditory

brainstem nuclei (Anton et al., 1996; Boom et al., 1999; M ollereau et al., 2000,

Kakimoto et al., 2001). There has been no further published information regarding the

possible involvement o f nociceptin in auditory function.

The results presented in this thesis describe research undertaken to elucidate the

possible role o f nociceptin and its receptor in the auditory system. This has been

achieved with several studies, each concerned with one particular aspect o f the

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2 Distribution o f Nociceptin

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CHAPTER 2: DISTRIBUTION OF NOCICEPTIN DURING DEVELOPMENT.

2.1 INTRODUCTION.

During development, neurones are often functional before they become fully mature, a

process dependent on both excitatory and inhibitory influences (Kaas, 1996). For

example, release and binding o f main auditory neurotransmitters such as ACh have

been determined in several brainstem auditory nuclei prior to maturity. If nociceptin

plays a role in auditory function, it may also contribute to these developmental

processes. Therefore, by examining NC immunoreactivity during development, insights

may be provided into possible functions o f NC in the auditory system.

Two species o f animal have been used in this study. Guinea pigs are precocial, like

man they begin to hear in utero and undergo a sudden exposure to a more complex

acoustic environment at birth. At this time, there is a major shift fi*om fluid and tissue

conducted sound to air-home noise, thus involving exposure to a very different sound

spectrum. Conversely, the altricial mouse is bom in an immature, deaf state, resembling

the auditory system o f a human embryo o f 15 weeks (Kikuchi and Hilding, 1965). With

the onset o f auditory function not occurring until postnatal day 1 2 (P I2), this species

allows for the study o f early developmental processes.

Development o f the mammalian auditory system.

An understanding o f the major events occurring during maturation o f the auditory

system is cmcial to the interpretation o f the results o f any developmental study. A brief

description o f development o f the neural circuitry o f the brainstem and cochlea,

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In both mice and guinea pigs, various levels o f the auditory system develop

simultaneously. Some processes occurring during development are intrinsic to the

tissue while others depend on the functioning o f synaptic inputs. During the initial

stages o f maturation the auditory neurones are generated, neurites migrate to their adult

locations, axons are sent out, dendrites begin to differentiate and synaptogenesis

begins. Programmed cell death also occurs during this time. In this way, the adult

pattern o f circuitry is established at an early stage o f development.

The Cochlea (i) Anatomy

There are two types o f adult mammalian cochlear hair cells. Inner hair cells (IHCs)

primarily transduce incoming auditory stimuli into neural signals. Outer hair cells

(OHCs) are believed to be involved in feedback mechanisms, protecting the inner ear

from acoustic overstimulation. These different functions are reflected in their pattern o f

innervation, with IHCs having predominantly afferent innervation, and OHCs with a

dense medial olivocochlear efferent innervation. This is not, however, always the case.

As soon as IHCs begin to differentiate they are contacted with nerve endings o f radial

type I spiral ganglion neurones, as well as lateral olivocochlear fibres. Around the onset

o f hearing, spiral ganglion neurones become myelinated (Figure 2.1) and there is an

increase in the number and size o f afferent nerve endings at the basal pole o f IHCs

(Romand et al, 1980; D odson and Mohuiddin, 2000). The efferent fibres decrease in

number, with the remaining efferent terminals contacting afferent dendrites. It has been

suggested that this early efferent innervation may act to protect the afferent fibres from

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W hen OHCs begin differentiation they are contacted exclusively by spiral afferents

(Romand, 1983). However, a short time after the onset o f cochlear function there are

very few afferent endings around them, with the cell contacted by many efferent

terminals. The adult appearance o f efferent synapses has been described as one o f the

final events in the maturation o f cochlear sensory and neural structures (Pujol, Carlier

and Lenoir, 1980).

(ii) Neurochemistry.

Auditory neurotransmitters such as ACh and GABA are both present in the cochlea

well before the onset o f cochlear function (Lenoir, Shnerson and Pujol, 1980; Whitlon

and Sobkowicz, 1989; Merchan-Perez et al., 1990), perhaps suggesting a role for these

efferent neurotransmitters in cochlear synaptogenesis (Pujol et al, 1979). It has been

hypothesised that, as in other systems, these neurotransmitters may play a role in the

formation and maintenance o f ACh synapses (Pujol and Puel, 1999). Finally, opioid

peptides, such as met-enkephalin have been detected in the inner spiral bundle around

the time o f the onset o f cochlear function (Pujol and Hilding, 1973). This presynaptic

localisation implicates opioid peptides in efferent neurotransmission processes. Such

early involvement o f neurotransmitters is believed to provide trophic support for

postsynaptic neurones and may contribute to the stabilisation o f specific neuronal

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Figure 2.1: Myelination o f Guinea Pig Spiral Ganglion Cells

During Development

%

2 m icroiis

Fig. 2.1. Arrows indicate site of myelination. There is no myelination o f spiral ganglion cells at gestational day 40 (040, a). At 06 0 , the approximate time of onset o f hearing, loosely packed myelin is apparent around ganglion cells (b). As the guinea pigs age,

the myelin compacts and appears darker (c & d).

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The Brainstem (i) Anatomy

Even in highly altricial species, the brain stem circuitry at birth has patterns o f

connections characteristic o f the adult. Both the radial afferent and spiral afferent

fibres, comprising the auditory nerve, enter the brain stem while the neurones o f the

future cochlear nucleus are still being generated. Similarly, axons o f the cochlear

nucleus (CN) reach the superior olivary complex (SOC) and inferior colliculus (IC) at a

time when genesis o f the neurones that will populate these higher auditory centres is

still underway (Altman and Bayer, 1980; Repetto-Antoine and Meininger, 1982;

Kandler and Friauf, 1993). M any o f these new connections already have a clear

tonotopic organisation along the presumptive hearing axis (Moore, 1982; Henkel and

Brunso-Bechtold, 1993).

Gleich et al. (1998) observed that following the onset o f hearing the antero ventral

cochlear nucleus almost doubles in size. Further studies by Dodson (1986) have shown

increases in spherical cell size in this region, both before and after the onset o f cochlear

function in guinea pigs (Figure 2.2). Kudo et al (1988, 1990) demonstrated that within

the lateral superior olive (LSO), neurones projecting ipsilaterally and contralaterally are

generated at different times, ipsilateral connections being generated earlier. In the

medial superior olive (MSO), the dendrites develop their characteristic bipolar

orientation some time before the onset o f hearing (Henkel and Brunso-Bechtold, 1990).

Other evidence suggests that early connections are made between the brainstem and

cochlea via the olivocochlear efferent fibres. Even after the onset o f cochlear function,

it appears that an ongoing process o f change and rearrangement o f these neurones

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(ii) Neurochemistry.

Electron microscopy and physiological techniques have shown that spherical cells in

the cochlear nucleus have synaptic contact with both the endbulbs o f Held and the

inhibitory GABA-positive terminals before hearing begins (Kane and Habib, 1978;

Brugge and O'Connor, 1984; Wu and Oertel, 1987; Schweitzer et al, 1993). Studies

have also shown that both GABA receptors and glutamate-responsive receptors are

functional at this stage. Thus, excitatory and inhibitory receptors are functional before

the circuitry is completely operational (Walsh et al., 1987, 1990, 1993).

In the adult, several immunohistochemical studies have shown the presence o f

endogenous opioids within these efferent neurones (Wamsley et al, 1980). Both

proenkephalin and prodynorphin gene products are found prominently in the lateral

efferent terminals innervating eighth nerve dendrites under mature IHCs (Fex and

Altschuler, 1981; Hoffman et al, 1983, 1984, 1985; Altschuler et al, 1984, 1985;

Eybalin et al, 1984a,b 1987; Drescher et al., 1992; Scholtz et al, 1998). Further studies

provide evidence that these two opioid peptide families co-exist within the same

neurones (Altschuler et al, 1986; Abou-Madi, 1987). The distribution o f opioid

peptides within the medial efferent system is less clear. Recently Ryan et al (1991)

found no enkephalin-ir in lateral OC neurones o f the gerbil, with h alf o f the medial OC

population positively labelled, suggesting there may be two, biochemically distinct

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Figure 2.2: Increase in Guinea Pig Spherical Cell Size During Development.

Fig. 2.2: Spherical Cells in the antero ventral cochlear nucleus. Spherical cells increase in size between 0 4 0 (a) and 0 6 0 (b). There is no further increase in size before birth (c). (EM Plates were provided by Dr. Hilary Dodson, ILO).

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Postnatal Development,

The most obvious maturational change after hearing has begun is the rearrangement

and growth o f dendrites, together with the axonal arbors that innervate them. There also

appears to be an environmental cause for the refinement o f axonal connections from the

CN to areas such as the IC. There is much evidence to suggest that an intact cochlea is

important for the normal development o f such axons (Sanes and Takacs, 1993; Moore

and Kitzes, 1985; Moore and Kowalchuk, 1988; Moore, 1990, 1994) and the

maintenance o f cell size. In this way, at the onset o f hearing, the auditory system o f all

mammalian species has reached essentially the same stage (Morey and Carlile, 1990),

with the basic structures in place and basic patterns o f circuitry established.

Distribution o f Nociceptin in the Auditory Pathway,

There is relatively little information concerning the distribution o f nociceptin in these

structures. Nociceptin mRNA expression in the brainstem was examined by Boom et

al (1999). M oderate to high levels o f prepronociceptin mRNA were expressed in the

inferior colliculus, lateral lemniscus, periolivary nuclei and superior olivary complex.

Houtani et al (1996) also described a moderate to high distribution o f NC in the IC,

superior olive and periolivary region. More recently, Kakimoto et al (2001) identified

nociceptin mRNA positive cells in several auditory brainstem nuclei, such as the dorsal

and ventral nucelei o f the lateral lemniscus, the periolivary nuclei and the inferior

colliculus. The distribution does appear to be quite similar to that o f the opioids. As yet,

there is no published data showing NC expression in the cochlea.

Opioid Activity in the Auditory System.

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enkephalin-containing olivocochlear neurones discharge with noise stimuli.

Furthermore, Sahley et al (1996a, b) used a selective k-opioid receptor antagonist, Nor-

Bni, to show the reversibility o f opioid- mediated neural effects in the cochlea. The

same group has also shown the non-selective opioid antagonist, naloxone, has similar

effects in blocking the opioid receptor- mediated actions o f (-) pentazocine in the

cochlea. Pentazocine alone was shown to improve auditory sensitivity (Sahley and

Nodar, 1994).

Results obtained from localisation studies can often be used as an indicator for

physiological actions. For example, the NC system is known to be involved in pain

perception mechanisms and Boom et al (1999) reported that NC mRNA expression was

detected in areas implicated in descending antinociceptive pathways, such as the

gigantocellular nucleus and raphe magnus nucleus. Similarly, Boom et al (1999)

observed high NC mRNA expression in association with auditory relay nuclei such as

the inferior colliculi, lateral lemniscus nuclei, medioventral preolivary nucleus and the

superior olivary complex. It was observations similar to these that prompted Nishi et al

(1997) to investigate the hearing ability o f NC receptor knockout mice. Following

exposure to intense noise there was a significant increase in the threshold o f the

auditory brainstem response in NC knockout mice compared to that in controls. These

experiments provided the first evidence that the NC system is involved in modulating

auditory function.

Aims,

Although, evidence shows that NC is found in more central auditory nuclei, so far there

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the expression o f NC in the auditory pathway during development. The aims o f this

study were to determine:

1. The distribution o f NC in the adult mouse and guinea pig.

2. Changes occurring in distribution during development that could be associated with

hearing function in

a) the altricial mouse, which has postnatal development o f hearing,

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2.2 MATERIALS AND METHODS.

Animals.

CBA/ca mice (2-lOg) and pigmented guinea pigs (40-400g) bred in the Animal Facility

o f Biological Services, University College London, were used. Animals were housed in

a room maintained at 23 ± 2°C and a 12-hour light/dark cycle (light on at 6:30 A.M.).

Food and water were available ad libitum. The treatment o f animals was in accordance

with the Animals (Scientific Procedures) Act 1986.

Developmental and maturational changes in NC expression were examined in guinea

pigs at gestational day 40 and 60 (G40, G60), the day o f birth (PO) and at three

postnatal weeks (mature). The distribution o f NC in mice was examined from

embryonic day 15.5 (E15.5) and at 2-day intervals until 4 postnatal weeks. Two

animals were used per group.

Tissue Preparation fo r Light Microscopy and Immunofluorescence.

(i) Postnatal Animals

Guinea pigs and mice were deeply anaesthetised with an intraperitoneal injection o f

sodium pentobarbital (Reckitt and Coleman, Hull, UK, 0.05mg kg'^ body weight).

Once the rib cage was completely exposed, bone scissors were used to cut each side o f

the ribcage, lateral to the midline, allowing the mid-portion o f the ribcage to be raised,

exposing the underlying organs. A needle was inserted into the left ventricle o f the

heart. This was held in place using a butterfly clip. A small incision was made in the

Nociceptin and the OP4 receptor in the auditory system