3.1: INTRODUCTION.
The distribution o f nociceptin (NC) in the mature mammalian auditory pathway suggests that it m ay have a role in hearing. Furthermore, NC expression changes during development o f the auditory system, appearing to alter with the onset o f auditory function and exposure to a more complex acoustic environment. This evidence may suggest that the level o f NC expression in mature animals is dependent on auditory stimulation. If this is true, NC expression may be modified as a result o f loss or altered sensory input. By examining the expression o f NC under conditions where the auditory system is either exposed to acoustic overstimulation or to a unilateral loss o f afferent auditory input, it may be possible to further understand the role o f this peptide system in auditory fimction. Although both uni- and bi-lateral deafening have an impact, unilateral deafening causes an uneven deficit where effects are often more profound.
General Effects o f A ltered Auditory Input,
Sensory cortices are able to undergo extensive reorganisation in response to manipulations o f sensory input, either through excessive sensory stimulation or sensory deprivation (Merzenich et al., 1998). For example, removal o f one input to a nucleus in the brain may cause sprouting o f other afferents (Raisman, 1969; Schneider, 1973; Stewart and Vinsant, 1978; Tsukahara, 1981; Cotman et al., 1981).
In adult mammals, hearing loss appears to activate mechanisms that produce plastic changes in the central auditory system (Moore and Irvine, 1981; Rajan et al., 1993;
reside not only in the cortex, but also at the level o f the brainstem and even more peripheral structures (discussed later, Dostrovsky et ah, 1976; Devor and Wall, 1981, Sie and Rubel, 1992; Dodson et al., 1994).
Altered acoustic input can lead to changes in excitatory or inhibitory neurotransmission throughout the auditory pathway. These may culminate in physiological and structural changes via feedback mechanisms from the crossed olivocochlear bundle (COCB).
(I) Unilateral Deafening.
Experimental sensorineural hearing loss can paradoxically result in long term increases in the excitatory responses o f central auditory neurons o f undamaged contralateral pathways or o f intact parts o f the damaged cochlea (Kaas, 1999; Milbrandlt et al., 2000). The auditory system, like the central nervous system, adjusts to altered afferent input in ways that often compensate for these losses.
(i) Long Term Structural Effects.
Hair cell loss results in deafriess. Hair cell damage is followed by secondary degeneration o f more central neurones. One o f the first noticeable effects is a loss o f spiral ganglion cells (Spoendlin, 1971; Yiloski et al., 1974; Dodson, 1997; Takeno et al., 1998; Dodson and Mohuiddin, 2000) over a period o f weeks (Kellerhals et al., 1967; Spoendlin, 1971; Dodson et al., 1994; Dodson, 1997; Dodson and Mohuiddin, 2000). This is followed by reductions in spherical cell size (Hashisaki and Rubel, 1989; Sie and Rubel, 1992; Dodson et al., 1994), altered axonal projections and synaptic endings in the ventral cochlear nucleus (Benson et al., 1997; Bilak et al., 1997) and sustained dendritic atrophy in the MSO (Russell and Moore, 1999). Moore (1994) has also reported altered distribution o f efferent nerve fibres in the IC. If early in
development, cochlear damage can also result in neuronal loss and shrinkage in the ipsilateral lateral superior olive and associated structures (Moore, 1990, 1992; Sanes et al., 1992; Pasic et al., 1994). In general, all o f these changes are much more profound in neonates than adults (Trune, 1982; Nordeen et al., 1983; Moore, 1985).
This anatomical evidence o f neuronal loss and subsequent reorganisation is further supported by studies that have examined the expression o f growth-associated proteins. These proteins, such as GAP-43, are strongly expressed in growing axons during development, but are largely absent in mature animals. Following cochlear damage, GAP-43 is re-expressed in spiral ganglion neurones (Dodson and Mohuiddin, 2000) and fibres o f the CN, SOC and IC (Illing and Horvath, 1995, Illing et al., 1997).
(ii) Neurochemistry
Potashner et al. (2000) have shown that unilateral cochlear ablation induced persistent changes in synaptic glycine receptor activity, consistent with a deficiency in synaptic transmission in most regions o f the CN and LSO on the ablated side. On the intact side, persistent changes were consistent with the strengthening o f glycinergic inhibition in the LSO.
In circumstances where aberrant synapses in the CN and IC result, the pharmacologically immature state o f these synapses has been associated with non-N- methyl-D-aspartate (NMDA) receptors becoming less sensitive to their neurotransmitters (Zhou and Park, 1993).
ototoxic drugs has been shown to cause a down-regulation o f activity in GABA-ergic systems (Abbott et a l, 1999; Milbrandt et al., 2000; Mossop et al., 2000). Decreased levels o f GABA-synthesising enzymes (glutamic acid decarboxylase, GAD) in auditory nuclei (Raza et al., 1994; Mossop et al., 2000) have also been observed following cochlear damage. Moreover, cochlear destruction induces a complex series o f changes in the release, binding and reuptake at glutaminergic, glycinergic and GABAergic pathways in the auditory brainstem (Bledsoe et al., 1995; Potashner et al., 1997; Suneja et al., 1998a, b).
(iii) Physiological Effects.
Abnormal physiology is often found following deafening and can be due to changes in both structural anatomy and neurochemistry.
Damage to cochlear hair cells elevates auditory nerve fibre tuning and can result in broader tuning (Dallos and Harris, 1978). These changes have been associated with auditory threshold elevation, poor frequency selectivity and impaired speech discrimination (Salvi et al., 1983; Salvi and Ahroon, 1983).
Physiological studies by Moore and Kitzes (1985) and Me Alpine et al. (1997) show increased neuronal excitation in the IC and auditory cortex in adult mammals only hours after cochlear ablation. This may be due to rapid changes in the expression o f neurotransmitters and/or their receptors and intracellular signalling pathways (Mossop et al., 2000). These physiological changes may lead to the long-term rewiring o f the brainstem after cochlear damage observed by neuronal tracing studies (Nordeen et al., 1983; Moore, 1994). Furthermore, Nordeen et al. (1983) and Kitzes (1984) have shown that following cochlear removal in gerbils, the proportion o f excited neurones in the
ipsilateral IC was substantially increased. These neurones had lower thresholds, wider dynamic ranges, higher peak discharge rates and more sustained discharge patterns than those in binaurally intact control animals (Semple and Kitzes, 1985).
Again, these changes are likely to result from altered auditory neurotransmission. If hair cells tuned to particular frequencies are lost, the tonotopic organisation o f transmitted sound has been disturbed. Evidence from these studies suggests that the auditory system attempts to compensate for diminished neural input in the periphery. Cochlear ablation appears to result in abnormal physiology in the auditory pathway, inducing hyperexcitable states in several brainstem nuclei and the auditory cortex (Gerken, 1979; Salvi et al., 1992).
Similar phenomena have been observed following acoustic overexposure.
(II) Acoustic Overexposure.
(i) Anatomical Effects.
As with unilateral deafening, exposure to intense noise has been shown to induce plastic changes in subcortical nuclei o f the auditory system. M any o f these effects also originate in the cochlea, with anatomical damage o f inner and outer hair cells (Saunders and Flock, 1986), a reduction in the number o f OHCs (Spongr et al., 1992) and damage to the tectorial membrane (Saunders et al., 1991 review) all reported to result from excessive noise levels (over 120dB SPL). Thus, active micromechanical properties o f the cochlea are one o f the first mechanisms damaged by acoustic overexposure (Fuel et al., 1998). As well as preventing the normal transduction o f sound, this anatomical damage leads to secondary neurodegeneration in central auditory structures, which can further affect the transmission o f acoustic signals to the auditory cortex.
(ii) Neurochemical Effects
There is evidence for altered synaptic functions following acoustic overexposure or chronic electrical stimulation o f auditory fibres. Synapses between OC efferents and OHCs are altered following sound exposure; in particular an increase in the number and size o f synaptic vesicles has been shown (Dodson et al., 1986), suggesting that there m ay be increased expression and transmission o f efferent neurotransmitters. Intracochlear electrical stimulation is also known to affect neurotransmission at higher auditory centres.
Liao et al (2000) reported changes in neurotransmission, with changes in NMDA, AMP A and GABAa receptor expression following stimulation. Several other groups have also reported a down-regulation o f activity in GABAergic systems (Abbott et al., 1999; M ilbrandt et al., 2000; Mossop et al., 2000) and a decrease in protein levels o f GAD in auditory nuclei following overstimulation. (Raza et al., 1994; Mossop et al.,
2000).
(iii) Physiological Effects
As with unilateral deafening, these anatomical and neurochemical changes are reflected in altered physiological responses o f auditory neurones. Functional damage has been shown in OHCs using otoacoustic emission recordings (Kemp and Chum, 1980; Lonsbury-Martin et al., 1987; Kemp and Souter, 1988; Franklin et al., 1991; Ueda et al., 1997). 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 functional damage to the hearing organ.
There are also many changes in the physiology o f mid- and hind- brain auditory nuclei following overstimulation. A decrease o f inhibition and alterations o f the tonotopic map have been shown in the CN (Salvi et al., 2000). In the IC, enhancement o f auditory evoked potentials (Salvi et al., 1990; Popelar et al., 1994; Szczepaniak and Moller, 1995), an increase in both acoustically-evoked and spontaneous activity o f neurones (Lonsbury-Martin and Martin, 1981a,b), behaviourally measured hypersensitivity to electrical stimulation (Gerken et al., 1984) and alterations in temporal integration (Szczepaniak and Moller, 1995) have all been shown.
Some, if not all, o f these effects which result from altered afferent input can be modulated by the olivocochlear efferent system.
The Efferent System and A ltered Afferent Input,
Physiological activation o f the olivocochlear efferent pathways by sound can reduce the temporary cochlear desensitisation (temporary threshold shifts, TTSs) caused by loud tones (Cody and Johnstone, 1982; Handrock and Zeisberg, 1982; Rajan and Patuzzi, 1992; Rajan et al., 1996; Rajan and Johnstone, 1998; Rajan, 2001). The crossed olivocochlear (OC)) pathway provides protection during binaural loud sound, and uncrossed pathways protect when monaural or binaural loud sounds occur in background noise (Rajan, 1988, 1995a,b, 2000; Rajan and Johnstone, 1988).
Further support for the involvement o f the OC system in protecting the cochlea from acoustic over-exposure has come from experiments in which the auditory system becomes 'conditioned' to loud sounds by prior exposure to moderate noise (Clark et al., 1987; Subramaniam et al., 1991; Henderson et al., 1996; Canlon and Fransson, 1998;
Aims o f this Study.
The processes underlying these complex changes in auditory function are still not understood. However, by examining the expression o f neurochemicals in conditions o f altered input, it may be possible to speculate on their role in normal auditory processing.
To determine whether alterations in the expression o f nociceptin occurs in the cochlea and/or auditory brainstem as a result o f changes in afferent input, the two different models (described above) will be examined:
(i) Unilateral deafening.
3.2 M A TER IA LS AND M ETH O D S.
Animals.
Pigmented guinea pigs 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.
Unilateral Deafening.
Guinea pigs were unilaterally deafened 7 days after birth by intracochlear injection o f an aminoglycoside antibiotic (gentamicin) directly into the perilymph through the round window o f the left ear, following a technique that was shown to result in total hair cell destruction (Dodson et al., 1994; Dodson, 1997). Guinea pigs (weighing 100- 200g) were anaesthetised by intraperitoneal injection o f ketamine chlorhydrate (Ketalar; Parke-Davis and co., Pontypool, UK, 150mg kg'^ body weight) after premedication with an intramuscular injection o f buprenorphine (Vetergesic, Reckitt and Coleman, Hull, UK, 0.05mg kg’’ body weight). A post-auricular incision was made on the left side to expose the bulla. A hole was then bored in the bulla, allowing access to the round window, through which 60pl o f gentamicin (SOmg m l’’) were perfused into the perilymph. The hole was then filled with Carboxylate-cement (Durelon, Espe laboratories) and the skin sutured. Animals were then killed, as described below, after survival periods o f 6 hours, 1, 2 and 5 weeks (2 animals per group). This surgery was performed by D r Hilary Dodson (Institute o f Laryngology and Otology, University College London).