**4.1.6 ABR and CAP for characterising auditory temporal resolution**

Boettcher et al. (1996) analysed the CAP and ABR responses to two successive 50 ms broadband noise pulses at 60 and 80 dBSPL as a function of the time interval (gap; 2, 4, 8, 16 and 32 ms) between the two noise pulses in young and old gerbils. This design corresponds to the gap detection paradigm in psychoacoustic studies, where the duration of the smallest detectable gap is used as a measure of auditory temporal resolution. The CAP and ABR analysis compared the onset responses to the first and second noise pulse as a function of gap duration. ABR thresholds for tone pips between 1 and 16 kHz were elevated by 10-15 dB in the group of 10 old (33-38 months) as compared to 9 young (4-8 months) gerbils, indicating only a moderate degree of peripheral hearing loss. Consistent with previous CAP (Hellstrom & Schmiedt, 1990) and ABR (Boettcher et al., 1993a) studies, the amplitudes of the potentials evoked by the noise bursts were reduced in old gerbils. In both age groups, the response amplitude to the onset of the second noise pulse decreased with decreasing gap duration, while the response amplitude to the first pulse was independent of gap duration. To compare the recovery of the response with increasing gap duration between the two age groups, despite the reduced response amplitude in old gerbils, the ratio of the response to the second burst divided by the response to the first burst was used. The ratio was smallest for the 2 ms gap duration. For the CAP, the response to the onset of the second

The Mongolian Gerbil as a Model for

resolved.

neurons in PVCN.

the Analysis of Peripheral and Central Age-Dependent Hearing Loss 79

electron microscopy revealed only 8% of the lesions in association with myelinated axons. Thus the contribution of glia and neurons to the formation of microcysts is currently not

McGinn & Faddis (1987) showed that ligature of the external auditory ear canal in 12 day old gerbils before the onset of hearing suppressed the development of the lesions. Gerbils kept in acoustic isolation between 1 and 3 months of age developed fewer lesions compared to controls exposed to 74-80 dB ambient noise (McGinn et al., 1990). Czibulka & Schwartz (1991) found that the number and size of microcysts decreased between 1 and 3 years of age and the degree of lesions in old gerbils was related to hearing status: the number and the size of lesions were largest in the normal hearing old gerbils, while hearing loss was associated with smaller and fewer lesions. Thus, the activity of auditory nerve fibres terminating in the CN is an important factor for the formation of spongiform lesions and it has been suggested that lesions may be the result of excitotoxicity due to transmitter release by the auditory nerve fibres (Czibulka & Schwartz, 1991; McGinn et al., 1990; Faddis & McGinn, 1997). However, it remains an enigma why lesions are prominent in PVCN, yet typically spare AVCN, which also receives massive input from auditory nerve fibres. Possible functional consequences of spongiform lesions in old gerbils are not yet known.

In addition to spongiform lesions, evidence for neuronal degeneration in the CN was observed by electron microscopy (Ostapoff & Morest, 1989) and through use of aminocupric silver impregnation (McGinn & Faddis, 1998). Czibulka & Schwartz (1991) found a significant reduction in the size of PVCN neurons, but no significant reduction in the number of neurons in the PVCN of gerbils between the age of 1 and 3 years. Ostapoff & Morrest (1989) argued that at most 5-6% of the PVCN neurons may be lost due to microcysts. Thus, these studies suggest that age is not associated with a prominent loss of

The cross sectional area of DCN, PVCN and AVCN was determined for 11 young and 18 old gerbils in sections at defined positions along the rostro-caudal extension of the CN (Stehle, 2010; Gleich et al., 2007c). Comparing the group means of young and old gerbils revealed a significant reduction of the cross-sectional area by 12% in old as compared to young gerbils only for AVCN. The data showed a much higher inter-animal variability of AVCN crosssectional area in old as compared to young gerbils. A subgroup of 8 old gerbils had crosssectional areas below those from young gerbils (representing an average reduction of almost 25%) while cross-sectional area of the other 10 old gerbils varied within the range observed for young gerbils. Counts of the GABA- and glycine-immunoreactive neurons in the CN subdivisions (Stangl et al., 2009) revealed only for the GABAergic neurons in the AVCN a significant reduction (mean 35%) in old as compared to young gerbils. The analysis of the size (cross sectional area) of inhibitory neurons as a function of age showed only for GABAergic cells of the PVCN a significant reduction (mean 16%) in old gerbils. These data demonstrate distinct and specific age-dependent changes in the CN subdivisions of the gerbil. The shrinkage of AVCN (presumably due to a loss of neuropil) in approximately half of the old gerbils was not associated with a comparable shrinkage in DCN and PVCN. A loss of GABAergic cells was only observed for AVCN, while the size of GABAergic cells was only reduced in the PVCN of old gerbils. Presently, the functional consequences of these

structural age-dependent changes in the CN of old gerbils are unknown.

noise pulse for the 32 ms gap had not fully recovered and the ratio was around 0.6 in young and old gerbils. Recovery of ABR wave ii and iv functions with increasing gap duration was more complete as compared to the CAP in young and old gerbils. The comparison of amplitude ratio as a function of gap duration showed no clear systematic difference between both age groups. Thus, despite an absolute difference of response amplitude, the recovery of the response amplitude to the second noise burst with increasing gap duration was similar in young and old gerbils.

The latency for the first noise pulse in the CAP and ABR responses was very similar in young and old gerbils, despite the elevated ABR thresholds to tone pips and the reduced response amplitudes in old gerbils. For the CAP, response latency to the second noise pulse was very similar in old and young gerbils and showed only a small decrease (≈ 0.1 ms) with increasing gap duration. In contrast to the CAP, response latency to the second noise burst showed a higher degree of variation with gap duration for wave ii (> 0.2 ms) and for wave iv (> 0.28 ms). Compared to young gerbils, the response latency of ABR wave ii was elevated for the 2 ms gap and that of wave iv for the 2 and 4 ms gaps in old gerbils. The variation of response latency as a function of gap duration did not differ between young and old gerbils for the CAP. Thus, while response latencies at the level of the cochlea (CAP) did not differ between age groups, the elevated latencies for short gaps in the ABR response (most pronounced for wave iv) argue for altered processing at the brainstem level that is not related to peripheral deficits. Boettcher et al. (1996) proposed that the latency shifts at the level of the brainstem without corresponding shifts in the periphery could be related to a loss of inhibition in the central auditory pathway of aged subjects.

#### **4.2 Structural changes**

"Healthy" ageing is associated with shrinkage of the brain that is predominantly due to shrinkage of neurons, loss of synapses, reduction of synaptic spines, reduction of the length of myelinated axons and, to a lesser degree, loss of neurons (Fjell & Waldhovd, 2010). The pattern of age-dependent structural changes varies greatly between different brain regions. In the following, we will present data on age-dependent changes of auditory brainstem nuclei in the gerbil.

#### **4.2.1 The cochlear nucleus (CN)**

Spongiform lesions begin to develop in the CN of young gerbils at the age of a few weeks or months and increase in size and number as the gerbil reaches 1-2 years of age (Czibulka & Schwartz, 1991; McGinn & Faddis, 1987; Ostapoff & Morrest, 1989; Statler et al., 1990). Lesions first become prominent in the PVCN and auditory nerve root and can spread to the deep layer of DCN and the caudal region of AVCN. In 1-2 year old gerbils, microcysts also developed in the superior olive, including LSO, the lateral lemniscus and ventral IC, while other non-auditory regions of the brain remained free of lesions (Ostapoff & Morest, 1989). Using immunostaining with antibodies against GFAP and S100, Czibulka & Schwartz (1993) concluded that up to 80% of the microcysts arise from astrocytes and only few lesions occur in dendrites or axons. In contrast, based on ultrastructural analysis and immunostaining with antibodies to MAP2, GFAP and S100, Faddis & McGinn (1997) concluded that their data "did not support a major role for astrocytes in lesion formation", and transmission

noise pulse for the 32 ms gap had not fully recovered and the ratio was around 0.6 in young and old gerbils. Recovery of ABR wave ii and iv functions with increasing gap duration was more complete as compared to the CAP in young and old gerbils. The comparison of amplitude ratio as a function of gap duration showed no clear systematic difference between both age groups. Thus, despite an absolute difference of response amplitude, the recovery of the response amplitude to the second noise burst with increasing gap duration was similar

The latency for the first noise pulse in the CAP and ABR responses was very similar in young and old gerbils, despite the elevated ABR thresholds to tone pips and the reduced response amplitudes in old gerbils. For the CAP, response latency to the second noise pulse was very similar in old and young gerbils and showed only a small decrease (≈ 0.1 ms) with increasing gap duration. In contrast to the CAP, response latency to the second noise burst showed a higher degree of variation with gap duration for wave ii (> 0.2 ms) and for wave iv (> 0.28 ms). Compared to young gerbils, the response latency of ABR wave ii was elevated for the 2 ms gap and that of wave iv for the 2 and 4 ms gaps in old gerbils. The variation of response latency as a function of gap duration did not differ between young and old gerbils for the CAP. Thus, while response latencies at the level of the cochlea (CAP) did not differ between age groups, the elevated latencies for short gaps in the ABR response (most pronounced for wave iv) argue for altered processing at the brainstem level that is not related to peripheral deficits. Boettcher et al. (1996) proposed that the latency shifts at the level of the brainstem without corresponding shifts in the periphery could be related to a

"Healthy" ageing is associated with shrinkage of the brain that is predominantly due to shrinkage of neurons, loss of synapses, reduction of synaptic spines, reduction of the length of myelinated axons and, to a lesser degree, loss of neurons (Fjell & Waldhovd, 2010). The pattern of age-dependent structural changes varies greatly between different brain regions. In the following, we will present data on age-dependent changes of auditory brainstem

Spongiform lesions begin to develop in the CN of young gerbils at the age of a few weeks or months and increase in size and number as the gerbil reaches 1-2 years of age (Czibulka & Schwartz, 1991; McGinn & Faddis, 1987; Ostapoff & Morrest, 1989; Statler et al., 1990). Lesions first become prominent in the PVCN and auditory nerve root and can spread to the deep layer of DCN and the caudal region of AVCN. In 1-2 year old gerbils, microcysts also developed in the superior olive, including LSO, the lateral lemniscus and ventral IC, while other non-auditory regions of the brain remained free of lesions (Ostapoff & Morest, 1989). Using immunostaining with antibodies against GFAP and S100, Czibulka & Schwartz (1993) concluded that up to 80% of the microcysts arise from astrocytes and only few lesions occur in dendrites or axons. In contrast, based on ultrastructural analysis and immunostaining with antibodies to MAP2, GFAP and S100, Faddis & McGinn (1997) concluded that their data "did not support a major role for astrocytes in lesion formation", and transmission

loss of inhibition in the central auditory pathway of aged subjects.

in young and old gerbils.

**4.2 Structural changes** 

nuclei in the gerbil.

**4.2.1 The cochlear nucleus (CN)** 

electron microscopy revealed only 8% of the lesions in association with myelinated axons. Thus the contribution of glia and neurons to the formation of microcysts is currently not resolved.

McGinn & Faddis (1987) showed that ligature of the external auditory ear canal in 12 day old gerbils before the onset of hearing suppressed the development of the lesions. Gerbils kept in acoustic isolation between 1 and 3 months of age developed fewer lesions compared to controls exposed to 74-80 dB ambient noise (McGinn et al., 1990). Czibulka & Schwartz (1991) found that the number and size of microcysts decreased between 1 and 3 years of age and the degree of lesions in old gerbils was related to hearing status: the number and the size of lesions were largest in the normal hearing old gerbils, while hearing loss was associated with smaller and fewer lesions. Thus, the activity of auditory nerve fibres terminating in the CN is an important factor for the formation of spongiform lesions and it has been suggested that lesions may be the result of excitotoxicity due to transmitter release by the auditory nerve fibres (Czibulka & Schwartz, 1991; McGinn et al., 1990; Faddis & McGinn, 1997). However, it remains an enigma why lesions are prominent in PVCN, yet typically spare AVCN, which also receives massive input from auditory nerve fibres. Possible functional consequences of spongiform lesions in old gerbils are not yet known.

In addition to spongiform lesions, evidence for neuronal degeneration in the CN was observed by electron microscopy (Ostapoff & Morest, 1989) and through use of aminocupric silver impregnation (McGinn & Faddis, 1998). Czibulka & Schwartz (1991) found a significant reduction in the size of PVCN neurons, but no significant reduction in the number of neurons in the PVCN of gerbils between the age of 1 and 3 years. Ostapoff & Morrest (1989) argued that at most 5-6% of the PVCN neurons may be lost due to microcysts. Thus, these studies suggest that age is not associated with a prominent loss of neurons in PVCN.

The cross sectional area of DCN, PVCN and AVCN was determined for 11 young and 18 old gerbils in sections at defined positions along the rostro-caudal extension of the CN (Stehle, 2010; Gleich et al., 2007c). Comparing the group means of young and old gerbils revealed a significant reduction of the cross-sectional area by 12% in old as compared to young gerbils only for AVCN. The data showed a much higher inter-animal variability of AVCN crosssectional area in old as compared to young gerbils. A subgroup of 8 old gerbils had crosssectional areas below those from young gerbils (representing an average reduction of almost 25%) while cross-sectional area of the other 10 old gerbils varied within the range observed for young gerbils. Counts of the GABA- and glycine-immunoreactive neurons in the CN subdivisions (Stangl et al., 2009) revealed only for the GABAergic neurons in the AVCN a significant reduction (mean 35%) in old as compared to young gerbils. The analysis of the size (cross sectional area) of inhibitory neurons as a function of age showed only for GABAergic cells of the PVCN a significant reduction (mean 16%) in old gerbils. These data demonstrate distinct and specific age-dependent changes in the CN subdivisions of the gerbil. The shrinkage of AVCN (presumably due to a loss of neuropil) in approximately half of the old gerbils was not associated with a comparable shrinkage in DCN and PVCN. A loss of GABAergic cells was only observed for AVCN, while the size of GABAergic cells was only reduced in the PVCN of old gerbils. Presently, the functional consequences of these structural age-dependent changes in the CN of old gerbils are unknown.

The Mongolian Gerbil as a Model for

were quite pronounced.

**4.2.5 The inferior colliculus (IC)** 

**potential functional consequences** 

functional age-dependent pathology.

**5. Psychoacoustic / behavioural measurements** 

the Analysis of Peripheral and Central Age-Dependent Hearing Loss 81

GABA and glycine (Dalles, 2009, Gleich, 2006; 2007). The number of MSO neurons was independent of age: there was no loss of MSO neurons in old gerbils. However, the cross sectional area of MSO neurons and the cross sectional area of MSO both decreased by 10% and 20% respectively in old as compared to young gerbils. The shrinkage of MSO in old gerbils is a combination of the shrinkage of MSO neurons and a reduction in the innervation density of MSO (loss of neuropil). Age-dependent structural changes in the gerbil MSO

The analysis of age-dependent changes of the gerbil IC (Gleich et al., 2011) revealed a significant shrinkage of the IC cross-sectional area (13%) in old as compared to young gerbils. Although the mean number and cross-sectional areas of GABAergic cells in the IC were slightly smaller in old as compared to young gerbils, the difference between both groups was not significant in the sample of 7 young and 18 old gerbils analysed. The agedependent changes in the GABAergic system of the gerbil IC appeared less pronounced than those previously described in rat (Caspary et al., 1995). This might be explained by

differences in the degree of peripheral hearing loss of old rats and gerbils.

**4.2.6 Variation of age-dependent structural changes between auditory nuclei and** 

The structural changes in the different auditory nuclei discussed above (loss of neurons, shrinkage of neurons and shrinkage of the whole nucleus due to loss of innervation) vary considerably. The effect of age appeared least in DCN and LSO and most for AVCN and MSO. Unfortunately, the functional consequences of the age-dependent structural changes in a specific nucleus on auditory processing are typically not well understood except for MSO and LSO, where it has been shown that they process two distinct aspects of binaural sound analysis: MSO analyses inter-aural time differences while LSO analyses inter-aural level differences (see review in Irvine, 1992), two separate cues that can be used for localisation or lateralisation of a sound source. The limited age-dependent pathology in LSO and the more pronounced pathology of MSO suggest that lateralisation of a sound source in old gerbils should be less affected when based on inter-aural level difference and more affected when based on inter-aural time difference. Unfortunately, behavioural data in gerbils addressing this question are not available. However, Babkoff et al. (2002) showed that for a sample of 78 human subjects aged 21-88 years, tested by the presentation of click trains via head phones, lateralisation based on inter-aural level difference did not change with age while the inter-aural time difference for correct lateralisation increased with age. The correlation of the degree of age-dependent structural changes in LSO and MSO of the gerbil and the effect of age on lateralisation based on inter aural level- and inter aural timedifference in humans is an example for a potential causal relationship of structural and

The first behavioural audiogram of the gerbil was determined by Ryan (1976) using a shock avoidance procedure. Subsequently, Sinnott et al. (1997) developed a go-nogo procedure

#### **4.2.2 The medial nucleus of the trapezoid body (MNTB)**

The neurons of the MNTB are glycinergic. They convert the excitatory input from the contra-lateral VCN to inhibitory glycinergic projections predominantly to MSO and LSO. A light microscopic analysis of glycine immunoreacted sections showed that spongiform lesions, like those previously described for the CN, were very prominent in the MNTB of 3 year old gerbils, but were almost absent in 1 year old gerbils (Gleich & Strutz, 2002). Thus, spongiform lesions in MNTB develop with a delay of approximately 1-2 years compared to the CN. Spongiform lesions in old gerbils showed a gradient along the MNTB, decreasing from caudal towards rostral. The volume of MNTB was independent of age, as there was no shrinkage of the MNTB in old gerbils. In addition, there was no significant loss of glycinergic neurons in old as compared to young gerbils. In young and old gerbils, there was a systematic gradient of MNTB neuron size: MNTB neurons were largest in the ventrolateral and smallest in the dorso-medial part of MNTB. According to the tonotopic organisation of MNTB, low-frequency neurons appeared larger on average than highfrequency neurons. Comparing soma size of young and old gerbils revealed a homogenous reduction of cross-sectional area by approximately 20% throughout the MNTB in old gerbils, without any indication that the shrinkage of neurons varied with the tonotopic organisation of MNTB. The reduced size of MNTB neurons in old gerbils may lead to a reduced glycinergic input into MSO and LSO (and other nuclei receiving input from MNTB) and consequently affect processing of binaural stimuli.

#### **4.2.3 The lateral superior olive (LSO)**

The light microscopic analysis of GABA and glycine immunostained sections through the LSO in gerbils revealed that this nucleus was rather resistant to age-dependent changes (Gleich et al., 2004). Although Ostapoff & Morest (1989) had reported the presence of microcysts in the LSO of 1-2 year old gerbils, we found no or only small lesions in the LSO of 7 gerbils over 3 years of age. Only 4 old gerbils showed more-prominent lesions that were mainly restricted to the medial (high frequency) limb of LSO, although all 11 old gerbils in this sample had prominent lesions in the MNTB. Thus, LSO appeared more resistant to the formation of spongiform lesions than the MNTB. Neither the rostro-caudal extension, nor the cross-sectional area of LSO varied with age, demonstrating that the LSO did not shrink in old gerbils. In addition, the number of neurons in Nissl stained sections, as well as the number of GABA- and glycine-immunoreactive neurons did not change with age: there was no loss of neurons in the LSO of old gerbils. The density of inhibitory neurons showed the same gradient along the tonotopic representation of the LSO in young and old gerbils: GABAergic and glycinergic neurons were more prominent in the low as compared to the high-frequency limb. The comparison of the size of inhibitory neurons revealed that the cross sectional area of GABAergic and glycinergic LSO neurons was not affected by age in the lateral low-frequency limb, while there was a significant reduction (≈ 30%) in the medial high-frequency limb. Overall, the LSO showed only limited age-related changes that were restricted to the high-frequency limb.

#### **4.2.4 The medial superior olive (MSO)**

The neurons of the MSO do not express GABA or glycine, but MSO was well recognised in sections through the gerbil brainstem that were immunostained with antibodies against GABA and glycine (Dalles, 2009, Gleich, 2006; 2007). The number of MSO neurons was independent of age: there was no loss of MSO neurons in old gerbils. However, the cross sectional area of MSO neurons and the cross sectional area of MSO both decreased by 10% and 20% respectively in old as compared to young gerbils. The shrinkage of MSO in old gerbils is a combination of the shrinkage of MSO neurons and a reduction in the innervation density of MSO (loss of neuropil). Age-dependent structural changes in the gerbil MSO were quite pronounced.
