Response Properties of Neurons in the Inferior Colliculus of the Monaurally Deafened Ferret to Acoustic Stimulation of the Intact Ear

David McAlpine, Russell L. Martin, Jennifer E. Mossop, and David R. Moore

University Laboratory of Physiology, Oxford University, Oxford OX1 3PT, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

McAlpine, David, Russell L. Martin, Jennifer E. Mossop, and David R. Moore. Response properties of neurons in the inferior colliculus of the monaurally deafened ferret to acoustic stimulation of the intact ear. J. Neurophysiol. 78: 767-779, 1997. Response properties of neurons in the central nucleus of the inferior colliculus (ICC) were investigated after unilateral cochlear removal at various ages during infancy. Nineteen ferrets had the right cochlea surgically ablated, either in adulthood or on postnatal day (P) 5, 25, or 40, 3-18 mo before recording. Adult ablations were made on the same day as ("acute," n = 3), or 2-3 mo before ("chronic," n = 3), recording. Two ferrets were left binaurally intact. Single-unit (n = 702) and multiunit (n = 1,819) recordings were made in the ICC of barbiturate-anesthetized ferrets ipsilateral (all ages) or contralateral (P5 and acute adult only) to the intact ear. In binaurally intact animals, tonal stimulation of the contralateral ear evoked excitatory activity at the majority (94%) of recording loci, whereas stimulation of the ipsilateral ear evoked activity at only 33% of recording loci. In acutely ablated animals, the majority of contralateral (90%) and ipsilateral (70%) loci were excited by tonal stimulation of the intact ear. In chronically ablated animals, 80-90% of loci were excited by ipsilateral stimulation. Single-unit thresholds were generally higher for low-best frequency (BF) than for high-BF units, and higher in the ipsilateral than in the contralateral ICC. Analysis of covariance showed highly significant differences between all of the ipsilateral and contralateral groups, but no effects of age at ablation or survival time following ablation, other than that the group ablated at P25 had higher mean ipsilateral thresholds than the groups ablated at P5 or, acutely, in adulthood. Cochlear ablation at P5, 25, or 40 resulted in a significant increase in dynamic ranges of ipsilateral ICC unit rate-intensity functions relative to acutely ablated animals. Dynamic ranges of units in the contralateral ICC of P5-ablated ferrets were also significantly increased compared with those of acutely ablated animals. Cochlear ablation at P5, 25, or 40 resulted in a significant increase in single-unit spontaneous discharge rates in the ICC ipsilateral but not contralateral (P5 only) to the intact ear. These data show that unilateral cochlear removal in adult ferrets leads to a rapid and dramatic increase in the proportion of neurons in the ICC ipsilateral to the intact ear that is excited by acoustic stimulation of that ear. In addition, the data confirm that, in ferrets, cochlear removal in infancy leads to a further increase in responsiveness of individual neurons in the ipsilateral ICC. Finally, the data show that responses in the ICC contralateral to the intact ear are largely but not completely unchanged by unilateral cochlear removal.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Maturation of central auditory system neurons and their projections is known to be influenced by manipulations of sensory input. Levi-Montalcini (1949) first showed that removal of the chick otocyst during the embryonic period resulted in a progressive shrinkage and loss of neurons throughout the deafferented nucleus magnocellularis. Subsequent investigations in a variety of species have confirmed and extended these findings (chick: Born and Rubel 1985; Parks 1979; mouse: Trune 1982; gerbil: Hashasaki and Rubel 1989; ferret: Moore 1990a). In addition to changing target neuron number and size, cochlear removal produces an immediate cessation in the production of action potentials in the nucleus magnocellularis (Tucci et al. 1987) and in its mammalian homologue, the anteroventral cochlear nucleus (CN) (Koerber et al. 1966). Studies in which tetrodotoxin was applied to the round window of the cochlea (Born and Rubel 1988; Pasic and Rubel 1989) suggest that the cessation of activity triggers the cascade of cellular events in the nucleus magnocellularis and anteroventral CN that rapidly follow cochlear removal. These events include a decrease of cytoplasmic protein synthesis (Born and Rubel 1988; Sie and Rubel 1992; Steward and Rubel 1985), a disaggregation of ribosomes (Rubel et al. 1991), a biphasic change in ribosomal RNA (Garden et al. 1994), and an increase, followed by a decrease, of oxidative metabolic enzymes (Durham et al. 1993) and mitochondrial volume (Hyde and Durham 1994). In mature animals, cochlear removal does not result in neuron loss in the CN, although a metabolic downregulation of neurons appears to persist as a consequence of cochlear removal at any age (Moore 1990a; Rubel et al. 1990; Tierney et al. 1997).

In addition to somatic changes in CN neurons, unilateral cochlear removal leads to restructuring of more central auditory pathways. Neuron loss and shrinkage following cochlear removal have been reported in the ipsilateral lateral superior olive and contralateral medial nucleus of the trapezoid body of young gerbils (Pasic et al. 1994; Sanes et al. 1992) and ferrets (Moore 1990b, 1992). In addition, increases have been found in the number of CN neurons retrogradely labeled by injections of horseradish peroxidase in the ipsilateral inferior colliculus (IC), following removal of the contralateral cochlea in gerbils (Nordeen et al. 1983) and ferrets (Moore 1994; Moore and Kowalchuk 1988). An exuberance of projections on the side of the intact ear has also been shown in the gerbil by anterograde techniques. Moore and Kitzes (1985) found an increased density of axon terminals from CN neurons in the ipsilateral IC, and aberrant termination zones around and within each of the principal superior olivary complex (SOC) nuclei on both sides of the brain have recently been reported (Kitzes et al. 1995; Russell and Moore 1995). However, some brain stem projections may not be changed by cochlear removal. Retrogradely labeled neuron numbers in the gerbil medial superior olive (Nordeen et al. 1983) and the ferret lateral superior olive (Moore et al. 1995b) following horseradish peroxidase injections in the IC on the side of the intact ear appear to remain stable.

These anatomic studies suggest that removal of one cochlea, although leading to largely degenerative changes on the side of the deafened ear, also leads to generative effects. The possibility of enhanced responses in neurons in the central auditory system to acoustic stimulation of the intact ear has also been investigated. Using multiunit recording and systematic sampling, Nordeen et al. (1983) and Kitzes (1984) observed that the proportion of ipsilaterally excited IC recording loci was substantially increased, from ~30% in adult gerbils raised with both cochleas intact to 90% in adult gerbils that had one cochlea removed on postnatal day (P) 2. Subsequently, Kitzes and Semple (1985) showed that ipsilaterally excited single units in the IC had lower thresholds, wider dynamic ranges, higher peak discharge rates, and more sustained discharge patterns in P2-operated than in binaurally intact control animals. These ipsilaterally evoked responses were qualitatively and quantitatively similar to the more robust responses normally evoked by stimulation of the ear contralateral to the recording site (e.g., Semple and Kitzes 1985).

Similar observations of increased ipsilateral excitability have also been made in the primary auditory cortex (AI) of the cat following unilateral cochlear removal during the first postnatal week (Reale et al. 1987). The proportion of AI responsive loci on the side of the intact ear was significantly increased (from 65 to 95% of loci) and the mean single-unit threshold was significantly reduced compared with values in animals raised with both cochleas intact. Response properties of AI units on the same side as the removal to stimulation of the intact ear (contralateral stimulation) were unaffected by the lesion. A small number of animals receiving cochlear removal as adults and surviving for 24 h (Reale et al. 1987), or for many months (Nordeen et al. 1983), did not appear to exhibit such marked changes.

To determine whether the sparsity of changes in IC responsiveness following cochlear removal in maturity was due to the persistence of the ventral CN on the side of the removal, Moore and Kitzes (1986) lesioned the left CN of adult gerbils and recorded response properties in the right IC to stimulation of the right ear. The ventral CN provides a large number of direct and indirect projections to the IC. Response properties of IC units in either acutely or chronically operated animals were unaffected by the lesion, suggesting that physiological changes following cochlear removal are age dependent and not simply related to CN degeneration.

Two major issues remain unaddressed by these studies. First, most studies have examined the effects of cochlear removal at only a single age in infancy. However, in some anatomic studies researchers have discovered that small variations in age at the time of removal can greatly affect the outcome. For example, Moore (1990a) reported that the reduction in the number of CN neurons was restricted to removals performed early in the postnatal period (<P24). The reduction in CN neuron soma area, on the other hand, was about equal at all ages tested (P5-180). The early cessation of the sensitive period for CN neuron loss also contrasted with the longer period of sensitivity and more gradual reorganization of the CN-to-IC projection following unilateral cochlear removal. Removal at P24 produced a gradual increase in the number of CN neurons retrogradely labeled by tracer injections in the ipsilateral IC on the intact side of the brain. This increase was significant by 30 days after the removal (Moore and Kowalchuk 1988), but continued beyond 3 mo after the removal (Moore 1994). Later removals (at P90) were also found to produce an enhanced CN-to-IC projection if very long (2.5 yr) survival times were allowed (Moore 1994). No single-unit studies of the effects of cochlear removal at different ages have been performed. Such studies are of interest because they should show some of the functional consequences of the various anatomic changes cited above and thereby provide further insight into the mechanisms underlying the response of the auditory system to deafness. Thus one aim of this study was to investigate the effects of unilateral cochlear removal, performed at a variety of postnatal ages, on the response properties of units in the ferret central nucleus of IC (ICC).

The second issue is the extent to which the adult auditory system is affected physiologically by cochlear removal. Some previous studies have not examined adult removal at all, or have obtained unclear results. It has also been common practice to compare long-term, unilaterally deafened animals with unlesioned adults, thus making the assumption that short-term deafening in adults was ineffective. There is now abundant evidence (e.g., Gilbert 1996; Irvine and Rajan 1995; Kaas 1991) showing that adult sensory systems are affected by peripheral perturbations and sensory experience in adulthood. The second aim of this study was to examine the effects of unilateral cochlear removal in adulthood on the responses of units in the ICC and to compare cochlear removal in infancy with cochlear removal in adulthood.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Twenty-one pigmented ferrets (Mustela putorius furo) were used in this study. All but three of the animals were born in the Departmental animal house after timed matings. Each experimental animal (n = 19) had the right cochlea surgically destroyed, either in the postnatal period (P5, 25, or 40) or in adulthood. Animals that had the cochlear surgery as neonates were raised to maturity before recordings were made. Animals that had the cochlear surgery as adults were operated either on the day recordings were begun ("acute"), or >3 mo before recordings were made ("chronic"). Data were obtained from nine different groups (normal contra, normal ipsi, acute contra, acute ipsi, chronic ipsi, P5 ipsi, P25 ipsi, P40 ipsi, and P5 contra) defined by whether the animal was binaurally intact, the age at which the cochlea was removed, and the ICC (relative to the intact ear) in which recordings were made. Data from some normal and acute adults and P5-ablated animals were analyzed in two groups.

Cochlear removal

Infant ferrets were anesthetized with Saffan (Glaxovet; 0.5-2.0 ml/kg im) and animals in the adult groups were anesthetized with pentobarbital sodium (Sagatal; 40 mg/kg ip). The right cochlea was surgically destroyed as described previously (Moore 1990a; Moore and Kowalachuk 1988). Briefly, the cochlea was exposed through the ventral bulla and macerated with jewelers' forceps and/or aspirated with a Pasteur pipette. All chronically operated animals (including all the infants) recovered from the surgery and spent a further 3-18 mo in the animal house before ICC recording.

Stimulus presentation and measurement

Two methods were used to generate the stimuli used in this study. In earlier experiments, tones (100 Hz-30 kHz) were generated with the use of an analog system (see Moore et al. 1983). Later experiments employed tonal stimuli generated, shaped, and gated by a PC-based programmable digital stimulus system. The Auditory Laboratory Digital Stimulus System (ALDISS) consisted of a fast digital-to-analogue conversion card housed in a PC and a number of controlling routines written in Turbo Pascal and native assembler languages. The digital-to-analogue conversion was carried out by the QDA2 board supplied by Tucker-Davis Technologies (TDT, Gainesville, FL). The QDA2 provided an output bus to control a TDT attenuator (PA3) with programmable attenuation (resolution 0.1 dB). Antialiasing was achieved with the TDT FLT2 filter (corner frequency 30 kHz).

All auditory signals were amplified by a Quad 240 Power Amplifier, before presentation via a calibrated Sennheiser HD424X earphone coupled to an earpiece sealed to the transected ear canal. Stimuli were 310 ms in duration (including 10-ms rise-fall time) and were presented at 1.1-s intervals. A calibrated probe tube, inserted into the earpiece, was attached to a B&K 1/2-in. condenser microphone and preamplifier. This was coupled to a B&K band-pass filter and measuring amplifier system situated outside the acoustic chamber. This system allowed measurement of sound pressure level (re 20 µPa) close to the point of delivery.

Recording procedures

Adult ferrets were prepared for IC recordings by the general methods described by Moore et al. (1983). After induction of anesthesia with Sagatal (40 mg/kg ip), the left brachial vein was exposed and cannulated. Further anesthetic (Sagatal 1:5 in saline iv) was administered as indicated by reflex withdrawal to a pinch of the forepaw.

Each animal was placed in a stereotaxic frame and the head was stabilized by means of a head holder attached to the skull with screws and dental acrylic. The left (and, in the binaurally intact adults, the right) pinna was transected, and the external auditory canal was cut close to where it entered the skull. This enabled placement of the stimulus delivery system to within a few millimeters of the tympanum. A craniotomy was performed to expose the caudal cerebral cortex overlying the IC. The dura was removed, and the cortical tissue covering the midbrain was aspirated to expose the dorsal surface of the IC. Glass-coated, platinum-plated tungsten electrodes (Merrill and Ainsworth 1972) were advanced through the nucleus, in a dorsal-to-ventral trajectory, by a microdrive under remote control. Multiple penetrations were made in each animal.

Electrical activity was amplified (×1,000) and filtered (band-pass 300 Hz to 20 kHz), displayed on an oscilloscope, and passed to an audio monitor. Multiunit neural activity was monitored in response to tonal stimulation. At each locus, spaced at 100-µm intervals, the presence or absence of evoked and spontaneous spike activity was noted, as well as the response type (onset or sustained). When single units were isolated, the unit's best frequency (BF) was determined. Spike discharges were discriminated and delivered to a 1401 laboratory interface (Cambridge Electronic Design) as a series of logic pulses. Discharge rates were gathered over a range of stimulus intensities, extending below that of the audiovisually determined threshold and incrementing in either 5- or 10-dB steps, to construct discharge rate-versus-intensity (R/I) functions. Peristimulus time histograms of the spike data at each stimulus intensity were available on-line. Analysis of evoked and spontaneous spike counts was performed off-line.

Spontaneous discharge rates for single units were calculated as the firing rate occurring between 550 and 860 ms after the stimulus trigger, and were averaged across all stimulus presentations. As a result, spontaneous rate for each unit was, in the majority of cases, calculated as the average of between 100 and 200 samples, each 310 ms in duration.

At the end of an electrode penetration, a small electrolytic lesion (5-µA negative current for 5 s) was made to enable histological verification of the position of electrode tracks.

Histology

On completion of an experiment, animals were perfused through the heart with 0.1 M phosphate-buffered saline and a solution of either mixed aldehydes in phosphate-buffered saline or 10% formal-saline. The brain and the right temporal bone (where lesioned) were removed and trimmed. For the formalin-fixed brains, 40-µm frozen frontal sections were cut through the brain stem, mounted, and Nissl stained. Camera lucida drawings were made of sections containing electrode tracks or lesions. All recording locations reported in this paper were in the ICC. Within the ICC, dorsoventral penetrations were widely spaced across the nucleus and no systematic attempt was made to sample from, or to exclude, any particularpart of the ICC. After decalcification, wax-embedded sections(10 µm) of the right temporal bone were made in a midmodiolar plane to determine the success of the cochlear lesion (see Moore and Kowalchuk 1988).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The data presented in this paper were derived from 1,819 recording loci in ICC. Single-unit recordings were made at 702 of these sites, and the remainder included a mixture of evoked activity (both clear but undiscriminated spike activity and "swish"---audible, sustained increases in activity without visually obvious spikes) and loci at which no excitatory drive was recorded. The undiscriminated spike activity and swish were used to examine the proportion of recording loci at which excitatory drive could be evoked. For all other analyses, only discriminated, single-unit responses were considered.

The presentation of these results is divided into three sections. The first section examines the effect of cochlear removal in adulthood by comparing neural responses in the ICC to stimulation of the intact ear (acute contra, acute ipsi, and chronic ipsi groups) with responses in binaurally intact animals (normal contra and normal ipsi). The second section examines the effect of cochlear removal at different ages on responses in the IC ipsilateral to the intact, stimulated ear by comparing, where appropriate, the infant removal groups (P5 ipsi, P25 ipsi, and P40 ipsi) with the adult ipsi groups. The third section examines the effect of early (P5) cochlear removal on contralaterally evoked IC responses by comparing the P5 contra group with contralaterally evoked responses in the normal contra and acute contra groups.

Some of the results presented here have been published previously in preliminary reports (see Moore et al. 1993).

IC responses ipsilateral and contralateral to the intact ear following acute and chronic unilateral cochlear removal in the adult period

RECORDING LOCI. The proportion of recording loci at which acoustic stimulation produced excitatory activity is shown in Fig. 1. Recording loci were characterized as excitatory if tonal stimulation elicited a qualitative increase in spike activity, even if this was limited to the period just after the stimulus onset. Data for the normal contra and normal ipsi groups (Fig. 1A) were obtained from 251 loci. Of these, the vast majority (94%, 235 of 251) was excited by stimulation of the contralateral ear, but only a third (33%, 83 of 251) were excited by stimulation of the ipsilateral ear, and this difference was significant (chi 2 = 194.08, df = 1, P < 0.001). After cochlear removal, the proportion of recording loci at which ipsilateral stimulation elicited an excitatory response increased dramatically and significantly in both the acute ipsi (70%, 178 of 254; chi 2 = 69.25, df = 1, P < 0.001) and chronic ipsi (92%, 123 of 134; chi 2 = 121.10, df = 1, P < 0.001) groups. In addition, the proportion of excitatory loci was significantly greater (chi 2 = 23.77, df = 1, P < 0.001) in the chronic ipsi group than in the acute ipsi group, suggesting that the length of time between cochlear removal and recording had an effect on IC responsiveness. By contrast, acute cochlear removal had little or no effect on the proportion of contralaterally evoked excitatory loci (acute contra: 90%, 131 of 146). The single-unit data confirmed these multiunit observations. Thus acute, unilateral cochlear removal produced a rapid (within hours) enhancement of driven activity in the IC ispilateral to the intact ear.


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1. A: proportion of excited inferior colliculus (IC) recording loci for binaurally intact animals (normal contra and normal ipsi), and for 3 adult-ablated groups (acute contra, acute ipsi, and chronic ipsi). B: proportion of excited IC recording loci for neonatal contra and ipsi groups. P, postnatal day.

DISCHARGE PATTERNS. Discharge patterns of single units that were excited during a BF-tone stimulus were characterized as either "onset" or "sustained" according to the form of their peristimulus time histogram (Fig. 2). Examples of onset units are shown in Fig. 2, A and E. The sustained category included units that displayed both more phasic (Fig. 2, B and C) and more tonic (Fig. 2D) discharge patterns, but with at least some discharges above the spontaneous level occurring after the onset epoch. Many spontaneously active onset units were inhibited after the stimulus onset, usually for the duration of the stimulus (Fig. 2E). A small number of offset units that were excited only at the termination of the stimulus were sampled, but were not included in any further analysis.


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 2. Examples of peristimulus time histograms (20-ms bins) evoked by 20 repetitions of 310-ms tone burst at best frequency (BF) classified as onset (A and E) and sustained (B-D). Black bar beneath each histogram: duration of stimulus.

The distribution of onset and sustained units in the normal and acute contra and acute and chronic ipsi groups is shown in Fig. 3A. The vast majority of units (90%, 46 of 51) in the normal contra group showed sustained discharge patterns, which accords with previous reports (see Semple and Kitzes 1985). However, in the acute contra group, only 67% (74 of 110) of units contralateral to the stimulated ear showed sustained discharge patterns, and this difference was significant (chi 2 = 9.65, df = 1, P < 0.002). Nevertheless, the proportion of sustained units in the acute contra group was still significantly higher (chi 2 = 17.52, df = 1, P < 0.001) than that in the acute ipsi group, where only 38% of excited units showed sustained discharge patterns. A similar proportion (28%) of units in the chronic ipsi group showed sustained responses. Because of the poor responsiveness of units in the normal ipsi group, we did not obtain sufficient data (n = 16) from this group to enable quantitative comparison with the other groups. However, those units that were recorded tended to be predominantly onset.


View larger version (34K):
[in this window]
[in a new window]
 
FIG. 3. Proportion of onset and sustained IC single units for comparisons of (A) normal contra and ipsi, acute contra and ipsi, and chronic ipsi groups; (B) acute and chronic ipsi and 3 neonatal ipsi groups; and (C) adult contra and P5 contra groups. See text for details of onset/sustained criteria.

SINGLE-UNIT THRESHOLDS. Figure 4A shows single-unit thresholds as a function of BF for the normal (bullet ) and acute (open circle ) contra groups, and the chronic (down-triangle) and acute (×) ipsi groups. For all groups, there was a clear relationship between BF and threshold. Low-BF units had the highest thresholds, and, as BF increased, thresholds initially decreased (up to ~10 kHz) and then increased. These threshold-frequency relations followed the general shape of the behavioral audiogram for the ferret (see Kelly et al. 1986) and are consistent with a previous report from this laboratory of ferret ICC thresholds (Moore et al. 1983). To compare thresholds between the groups, it is therefore necessary to control for both threshold and sampling differences with respect to BF. To perform this control, an analysis of covariance (S-Plus, v0.3.3, StatSci, Seattle, WA) was used. Initially, a cubic model was found to fit the normal contra data. This was then tested against all the other groups (including the neonatally ablated animals), and was found to fit them if a logarithmic transformation was performed on the threshold data. Finally, the significance of the difference between the threshold intercept of this model, applied to each group, was tested. The results (Table 1) showed that the mean threshold of neurons did not differ significantly in either the two contra groups or the two ipsi groups, whereas both contra groups had significantly lower thresholds than both ipsi groups.


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 4. Single-unit thresholds plotted as function of BF for (A) normal and acute contra, and acute and chronic ipsi groups; (B) acute, P5, 25, and 40 ipsi groups, and (C) acute and P5 contra groups. See keys for details.

 
View this table:
[in this window] [in a new window]
 
TABLE 1. Results of the analysis of covariance comparing mean thresholds in each group

R/I FUNCTIONS. The relation between unit discharge rate and stimulus intensity was compared between the acute contra and acute ipsi groups. Units were divided into four categories depending on the shape of their R/I functions: 1) monotonic saturating---units whose discharge rate at intensities greater than that required to evoke the maximum rate did not fall below 75% of the maximum; 2) monotonic nonsaturating---units whose discharge rate at the highest intensity examined was >10% greater than for the next highest intensity; 3) nonmonotonic---units that showed a reduction in discharge rate of >= 25% of the maximum at intensities greater than that needed to evoke the maximum; and 4) "others"---units whose R/I functions showed fluctuations in discharge rate >10% of the maximum at intensities below that required to evoke the maximum. Figure 5A shows the proportion of units with R/I functions in each of these categories. In both groups, the most common R/I function shape was monotonic and saturating, with just under half the units (49.1%) in the acute contra group and just over half the units (57%) in the acute ipsi group. No significant differences in the distribution of R/I function shapes were found between these groups (chi 2 = 6.96, df = 3, not significant).


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 5. Proportion of each type of rate-intensity (R/I) function described in text, in each of (A) acute contra and acute ipsi groups, (B) acute ipsi and 3 neonatal ipsi groups, and (C) acute contra and P5 contra groups.

The distribution of peak discharges for units in the acute ipsi and acute contra groups is shown in Fig. 6A. The peak discharge rate was calculated from 20 repetitions of the BF stimulus after the discharges occurring during a control, nonstimulus period were subtracted. Units with nonsaturating and other R/I functions were not included in this analysis. Peak discharges were higher in the acute contra group, with more values >100 spikes per 20 stimuli than in the acute ipsi group, and this difference was significant (chi 2 = 6.78, df = 1, P < 0.01).


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 6. Distribution of single-unit peak discharges for (A) acute contra and acute ipsi groups, (B) acute ipsi and 3 neonatal ipsi groups, and (C) acute contra and P5 contra groups for 20 repetitions of 310-ms tone burst at BF.

DYNAMIC RANGES. The R/I functions described above were used to calculate the unit dynamic ranges, defined as the stimulus intensity range, in dB, over which the evoked discharge rate increased from 10 to 90% of maximum. All saturating monotonic, saturating nonmonotonic, and other units were included in this analysis. Units with nonsaturating R/I functions were included in the analysis only if the dynamic range over the range of intensities investigated was >= 30 dB (this placed such R/I functions in the final group of the analysis, which included all dynamic ranges of >= 30 dB). The dynamic range of units classified as others was calculated over the widest range of intensities for which the R/I function contained no fluctuations in discharge level >10% of the maximum.

The distribution of dynamic ranges in the two groups is shown in Fig. 7A. Units in the acute contra group tended to have wider dynamic ranges than those in the acute ipsi group, and this tendency was confirmed statistically (chi 2 = 17.12, df = 3, P < 0.001). Specifically, the proportions of units with dynamic ranges between 20 and 30 dB (chi 2 = 10.86, df = 1, P < 0.001) and >30 dB (chi 2 = 5.37, df = 1, P < 0.05) were significantly higher in the acute contra group than in the acute ipsi group.


View larger version (26K):
[in this window]
[in a new window]
 
FIG. 7. Distribution of single-unit dynamic ranges for (A) acute contra and acute ipsi groups, (B) acute ipsi and 3 neonatal ipsi groups, and (C) acute contra and P5 contra groups. See text for details.

SPONTANEOUS DISCHARGE RATES. Single-unit spontaneous discharge rates were calculated as described in the METHODS section. Only units for which R/I functions were obtained were included in this analysis. The distribution of spontaneous rates in the acute contra, and acute and chronic ipsi groups is shown in Fig. 8A. The majority of units in each group (acute contra, 71.7%; acute ipsi, 86.9%; chronic ipsi, 65.5%) had spontaneous discharge rates <5 spikes/s and, for the acute groups, most of these were <1 spike/s. Very few units in any group had spontaneous discharge rates >20 spikes/s. No significant differences were found in the distribution of spontaneous discharge rates between the acute groups (chi 2 = 2.88, df = 2, not significant).


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 8. Distribution of single-unit spontaneous discharge rates for (A) acute contra and acute and chronic ipsi groups, (B) acute ipsi and 3 neonatal ipsi groups, and (C) acute contra and P5 contra groups.

Spontaneous discharge rates were also examined for a relatively small number of units (n = 29) in the chronic ipsi group. Spontaneous discharge rates in this group were significantly higher (chi 2 = 2.88, df = 2, P < 0.05) than in the acute ipsi group, with more units having spontaneous rates >5 spikes/s.

Effect of neonatal cochlear removal on IC responses ipsilateral to the intact ear

In this section we compare ipsilaterally evoked single-unit responses from the P5, 25, and 40 ipsi groups with those of the acute ipsi group. Recording loci data are also compared with those of the normal ipsi group, and recording loci, thresholds, and discharge patterns are compared with those of the chronic ipsi group.

RECORDING LOCI. The proportion of loci at which acoustic stimulation of the intact ear produced excitatory activity in the ipsilateral IC is shown in Fig. 1B. All of the neonatally ablated ipsi groups showed a considerably greater proportion of loci with excitatory activity than was found in the normal ipsi group (Fig. 1A; see above). In addition, a significantly greater proportion of loci in the P5 ipsi group responded with excitation compared with the acute ipsi group (chi 2 = 12.45, df = 1, P < 0.01) but not the chronic ipsi group. The proportion of excited loci gradually declined with advancing age at the time of the removal, and none of the other intergroup differences were significant.

DISCHARGE PATTERNS. Figure 3B shows the proportion of onset and sustained units (see Fig. 2) in each of the acute, chronic, P5, P25, and P40 ipsi groups. Although the proportion of units with sustained discharge patterns was higher in the P5 ipsi group than in the other ipsi groups, this difference was not significant.

SINGLE-UNIT THRESHOLDS. Single-unit thresholds at BF for each of the ipsi groups are shown in Fig. 4B. As in the adult groups (Fig. 4A), there was a wide distribution of BFs, and thresholds for all groups followed the shape of the ferret audiogram. Comparisons of thresholds between groups, with the use of the analysis of covariance described above (Table 1), showed no significant difference between any of the groups, except the P25 ipsi group, which (at P < 0.05) had higher thresholds than the acute ipsi and P5 ipsi groups. Note that all ipsi group thresholds were higher than those for all contra groups. Thus neonatal cochlear removal did not lead to an increase in sensitivity of IC neurons relative to cochlear removal in adulthood.

R/I FUNCTIONS AFTER NEONATAL COCHLEAR REMOVAL. Single-unit R/I functions were classified into one of the four categories (monotonic saturating, nonsaturating, nonmonotonic, and others) defined above. The distribution of R/I functions was similar for the acute and neonatal ipsi groups (Fig. 5B), and no significant difference was found (chi 2 = 12.85, df = 9, not significant).

The effect of unilateral cochlear removal on ipsilaterally evoked peak discharge rates was compared for the acute ipsi and neonatal ipsi groups (Fig. 6B). No significant difference was found between the groups (chi 2 = 20.02, df = 9).

DYNAMIC RANGES AFTER NEONATAL COCHLEAR REMOVAL. R/I functions were used to calculate the dynamic ranges of single units in each of the acute and neonatal ipsi groups (Fig. 7B). The R/I profile criteria used for inclusion in this analysis were as outlined in the first section of RESULTS. Overall, significant differences were observed (chi 2 = 23.94, df = 9, P < 0.005). A greater proportion of units in the acute ipsi group showed dynamic ranges <10 dB compared with all of the neonatal ipsi groups (chi 2 = 17.45, df = 1, < 0.001). The proportion of units with dynamic ranges in each band was also related to the age at which cochlear removal was perfomed. Both the P5 ipsi (chi 2 = 3.94, df = 1, P < 0.05) and P25 ipsi (chi 2 = 5.46, df = 1, P < 0.05) groups, but not the P40 ipsi group, had a higher proportion of units with dynamic ranges >30 dB compared with the acute ipsi group.

SPONTANEOUS DISCHARGE RATES AFTER NEONATAL COCHLEAR REMOVAL. The spontaneous discharge rates of single units for which R/I functions were obtained in the acute and neonatal ipsi groups were calculated and divided into the five categories described in the first section of RESULTS (Fig. 8B). Significant differences were found between the groups (chi 2 = 67.36, df = 12, P < 0.0001). Whereas nearly two-thirds of units in the acute ipsi group had spontaneous rates <1 spike/s, only about one-fifth of units in each of the neonatal ipsi groups had such low rates. In contrast, spontaneous rates in the top two categories (10-20 and >20 spikes/s) accounted for just 3% of units in the acute ipsi group, but >30% of units in the P5, 25, and 40 ipsi groups. Statistically, a greater proportion of units in the neonatal ipsi groups had spontaneous rates in the three highest ranges (5-10 spikes/s, chi 2 = 12.17, df = 1, P < 0.001; 10-20 spikes/s, chi 2 = 13.62, df = 1, P < 0.001; >20 spikes/s, chi 2 = 13.56, df = 1, P < 0.001) compared with the acute ipsi group. In addition, a higher proportion of units had spontaneous rates <1 spike/s in the acute ipsi group than in the neonatal ipsi groups (chi 2 = 22.42, df = 1, P < 0.001). No significant differences were observed in the distribution of spontaneous discharge rates between the P5, 25, and 40 ipsi groups. Finally, all the neonatal ipsi groups had significantly higher spontaneous discharge rates compared with the chronic ipsi group (chi 2 = 4.86, df = 3, P < 0.01), with more units showing spontaneous rates >5 spikes/s.

Thus the effect of unilateral cochlear removal in infancy was to increase the level of spontaneous activity compared with animals that received either acute or chronic cochlear removal in adulthood.

Effect of P5 cochlear removal on IC responses contralateral to the intact ear

In general, IC responses contralateral to the intact ear were little changed by cochlear removal on P5. The proportion of loci at which excitatory activity was recorded in the P5 contra group was almost identical to that of the acute contra group (Fig. 1B). The proportion of units in the P5 contra group that responded to BF tones with a sustained discharge was also similar to that found in the acute contra group (Fig. 3C). Unit thresholds (Fig. 4C) did not differ significantly (Table 1) between the P5 contra group and either the acute contra or normal contra groups. R/I function shapes of the acute and P5 contra groups are shown in Fig. 5C. Despite the lower proportion of monotonic saturating to nonmonotonic saturating units in the P5 contra group, the overall difference between these groups was not significant (chi 2 = 2.32, df = 3, not significant). In addition, the peak discharge rates of units in the two contra groups (Fig. 6C) were not significantly different from each other (chi 2 = 3.72, df = 6, not significant). The dynamic ranges of units in the P5 contra group were significantly broader than those in the acute contra group (Fig. 7C), most notably showing a higher proportion of units with ranges >30 dB (chi 2 = 26.60, df = 1,P  < 0.001). Finally, no significant differences in spontaneous discharge rates were found between the acute and P5 contra groups (Fig. 8C; chi 2 = 3.15, df = 2, not significant).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Cochlear removal in adults

A main finding of this study was that cochlear removal in adulthood produced a rapid and dramatic increase in the responsiveness of the IC on the side of the intact ear to stimulation of that ear. The number of loci at which excitatory activity could be recorded increased from one-third in intact animals to over two-thirds in animals that had one cochlea removed on the day recording commenced. In addition, the yield of ipsilaterally excited single units increased dramatically after acute cochlear removal. These results provide evidence for rapid changes in the function of mature central auditory system neurons that parallels the evoked potential results of Popelar et al. (1994). In that study, adult guinea pigs treated unilaterally with ototoxic drugs showed, within 1-7 days, a decrease in the threshold and latency, and an increase in the amplitude of evoked potentials recorded from large (0.3-0.5 mm) electrodes, implanted in the IC and AI, in response to high-frequency acoustic stimulation of the ipsilateral, untreated ear. Popelar et al. (1994) found a progressive increase in ipsilateral excitation during the 7-day study period. We found a significant increase in excitation from the acutely deafened ferrets, tested on the day of cochlear removal, to the chronically deafened ferrets, tested >= 3 mo after the removal.

By contrast with these results, cochlear removal produced relatively little change in the excitability of neurons in the IC contralateral to the intact ear. In the study of Popelar et al. (1994), little or no change was found in the amplitude of evoked responses in the contralateral IC and, in our study, no change in the overall distribution of excitation or in the threshold of single units was found. However, a significant decrease in the proportion of sustained units was observed, suggesting a possible increase in the level of long-lasting or late inhibition on this side of the brain.

Despite the increased ipsilateral responsiveness produced by cochlear removal in adulthood, excitation in the IC contralateral to the intact ear remained stronger than that in the ipsilateral IC. For example, most IC units in binaurally intact animals responded to contralateral stimulation with a sustained discharge. After acute cochlear removal, most units still responded to contralateral stimulation with a sustained response, whereas those responding to ipsilateral stimulation tended to do so only at onset. Thus the commonly reported excitatory dominance of the contralateral ear in the higher auditory pathway of binaurally intact animals (cat IC: Kuwada et al. 1984; Merzenich and Reid 1974; gerbil IC: Kitzes 1984; Semple and Kitzes 1985; cat AI: Reale et al. 1987) was maintained after unilateral cochlear removal, but at a reduced level.

In summary, it would appear that there is a dynamic interaction between the level of excitation on each side of the higher auditory system and the input from the two ears. Removal of input from the dominant, excitatory, contralateral ear produces a markedly increased level of excitation in response to stimulation of the remaining ear, whereas removal of input from the normally weaker, and more inhibitory, ipsilateral ear produces a much smaller decrease in the strong response to contralateral stimulation.

Cochlear removal in infancy

The second main finding of this study was that cochlear removal in the neonatal period further increased IC responsiveness to ipsilateral stimulation, and that this increase was dependent on the age at which the surgery was performed. After unilateral removal on P5, the proportion of excited recording loci was higher and unit dynamic ranges were wider than in acutely lesioned adults. These findings are consistent with those of Kitzes and Semple (1985), who reported that, after cochlear removal in P0-2 gerbils, responses of units in the IC on the side of the intact ear were generally stronger than those in adult gerbils raised with both cochleas intact. In the ferret, unilateral cochlear removal on P25 or 40 produced changes in IC responsiveness broadly similar to those produced by cochlear removal on P5, but to a progressively lesser degree. Thus the proportion of excitatory loci decreased progressively with age at removal, with only the P5 group differing significantly from the acute ipsi group. In the comparison of unit dynamic ranges, the P5 and P25 groups, but not the P40 group, had more units with broader ranges than did the acute group. On the other hand, cochlear removal on P25 or 40 still produced a greater proportion of IC units with higher spontaneous rates, compared with acutely or chronically lesioned adult animals. Thus the results from the later-operated animals were intermediate between those of the P5- and adult-operated groups. However, this tendency did not vary uniformly between the indexes examined, suggesting that there are at least two developmental sensitive periods in addition to the acute effects of cochlear removal in adulthood.

Two further aspects of the infant data are worthy of comment. First, at no age of cochlear removal were the changes as great as those reported by Kitzes and Semple (1985) in the gerbil. In that study, unit thresholds, peak discharge rates, and the proportion of sustained responses to ipsilateral stimulation all changed to levels normally found for contralateral stimulation. The smaller changes found after cochlear removal in neonatal ferrets may reflect intrinsic species differences or a relative maturity of the neonatal ferret auditory system compared with that of the gerbil. The second point is that we found differences between the neonatal groups and the chronic ipsi group. The most striking of these was the large increase, relative to either of the adult ipsi groups, in the level of spontaneous activity of units in the IC ipsilateral to the intact ear after cochlear removal in all the neonatal groups. This increase was additional to that found to occur after long survival after cochlear removal in adulthood. Thus, although some effects of cochlear removal were found, to about equal degrees, in all three neonatal groups, those effects still had a developmental dependence.

Changes in the contralateral IC after P5 cochlear removal

Most of the alterations in response properties of units in the IC ipsilateral to the intact ear were not found in the IC contralateral to that ear. Previous studies of the IC have examined the effects of unilateral deafening in infancy on the physiology of only the nucleus ipsilateral to the intact ear (Kitzes 1984; Kitzes and Semple 1985; Moore and Kitzes 1986). However, Reale et al. (1987) did examine the effects of unilateral cochlear removal in the cat on unit responses in the contralateral AI. They found that the proportion of excitatory loci was similar (>90%) to that recorded in animals raised with both cochleas intact. Unfortunately, Reale et al. (1987) did not examine single-unit dynamic ranges. The finding in the present study that dynamic ranges of units in the P5 contra group were wider than those in the adult contra group shows that at least one form of induced change occurred in the IC contralateral to the intact ear. Moreover, it may be noted that the direction of this change, associated with a presumed increase in responsiveness, was the opposite to the presumed decrease found in the contralateral IC after cochlear removal in adulthood.

Mechanisms of change after cochlear removal

The different results obtained after cochlear removal in infancy and adulthood, and after survival for varying periods, suggest multiple mechanisms of change. There are at least three general mechanisms by which changes in physiological responses in the IC might be produced; 1) unmasking of existing synapses, 2) formation of new synapses without axogenesis, and 3) axonal sprouting.

The elimination of input from one ear may have "unmasked" ipsilateral excitation through disinhibition or increases in the gain of existing synapses. Unmasking is generally considered to occur almost immediately after a peripheral input modification. For example, in the somatosensory cortex (e.g., Clarey et al. 1996), there is an immediate (within minutes) expansion in the receptive fields of neurons, normally representing a lesioned digit, to include areas of the body adjacent to that digit. Unfortunately, although we know that the excitatory changes in the acute ipsi group reported here occurred within a matter of hours, we do not know whether the changes were immediate. Similarly, Popelar et al. (1994) found changes in high-frequency evoked IC response amplitudes within 24 h of a cochlear lesion, but did not report shorter-term results. We have recently completed a study of single-unit responses in the ferret AI after cochlear removal (Moore et al. 1995a, 1997). Units having a narrow range of BFs (8-12 kHz), recorded from one ferret during 50 h after cochlear removal, showed a gradual reduction in threshold (at BF) rather than an abrupt decrease, as unmasking would seem to predict. Although preliminary, and derived from a different level of the auditory system, this result suggests that even the most rapidchanges reported here may not be due to an immediate release from inhibition. On the other hand, a longer-term change in synaptic gain (e.g., through an increase in receptor expression) would be consistent with both the AI and IC data.

The possibility of synaptogenesis, with or without axon sprouting, is suggested by some of the changes in brain stem morphology and connectivity found after cochlear removal, and outlined in the INTRODUCTION. In altricial mammals, unilateral removal in the early postnatal period (1st 2-3 wk in ferrets) (see Moore 1991) produces profound neuron loss in the CN on the operated side, and increases the extent of the projection from the CN on the side opposite the cochlear removal, both to the SOC bilaterally and to the IC on the intact side. Several response changes (excitatory loci, unit thresholds, and dynamic ranges) found in the present study were qualitatively or quantitatively more marked after cochlear removal during this early period. The freeing up of postsynaptic sites in the IC after the removal of the contralateral cochlea, and the death of a large number of CN neurons, may induce the invasion of new, ipsilaterally derived axons on to the vacant sites, thus increasing the influence of ipsilateral excitation in the IC. Later cochlear removal, or ear plugging, still produces a gradual increase in the projection from the CN to the IC on the normally hearing side, but there is no neuron loss in the CN and no clear change in the projection from the CN to either SOC. The changes in ipsilaterally evoked responses after P25 and P40 removals were somewhat smaller than at P5 and are therefore temporally consistent with the more minor changes in connections in the auditory brain stem that have been observed after removals at these ages (Moore 1994).

A general increase in the excitatory synaptic influence of the intact ear does not account for all the results presented here. For instance, despite the increased dynamic ranges of neurons in the experimental groups, peak discharge rates were no higher than in the control group. One possibility is that an increase occurs in both excitatory and inhibitory inputs deriving from the intact ear. The influence of increased inhibitory inputs, possibly from the dorsal nucleus of the lateral lemniscus (Shneiderman et al. 1988), might be to reduce the rate at which the discharge level increases as stimulus level is increased. Thus dynamic ranges could be extended without an increase in peak discharge level. A combination of new, low-threshold excitatory and high-threshold inhibitory inputs might also account for the general increase in excitability, evidenced by the increase in excited recording loci and spontaneous discharge rates in the neonatally ablated groups compared with both binaurally intact and adult-ablated controls.

A number of recent reports suggested that hearing loss leads to a reduction of inhibition in the IC (Caspary et al. 1995; Miller et al. 1997; Popelar et al. 1994). In particular, the level of gamma -aminobutyric acid decreases within a period of days after deafening. Although it is not yet clear how quickly this occurs, a decrease in local inhibition may explain at least some of the longer-term increase in excitation reported here.

In contrast to the physiological changes that occurred in the IC ipsilateral to the intact ear, responses in the IC contralateral to that ear were relatively unchanged by unilateral cochlear removal. In the normal ferret, the number of CN neurons projecting to the contralateral IC outnumbers that projecting to the ipsilateral IC by ~50 to 1 (Moore 1988). Thus unilateral cochlear removal in the neonatal period, resulting in degeneration of the CN on the operated side, would produce only a minor, excitatory1 deafferentation of the IC contralateral to the intact ear, in contrast to the much larger deafferentation of the ipsilateral IC discussed above. This could create synaptic space for de novo projections from the CN on the intact side, and may contribute to the observed increase in the contralaterally evoked dynamic ranges of single IC neurons. Surprisingly, a retrograde tracing study (Nordeen et al. 1983) actually reported a decrease in the size of the CN-to-IC projection on the side of a neonatal cochlear removal, but further anatomic studies of that pathway (Moore 1994; Moore and Kitzes 1985) have not found any changes. An alternative possibility is that changes may have occurred in the projections to the contralateral IC that are routed through the SOC or the dorsal nucleus of the lateral lemniscus.

It is possible, finally, that the strength and sensitivity of contralaterally evoked responses in normal animals are already close to maximum, and that is why little effect of cochlear removal was found. In this study, 90% of the recording loci in the IC contralateral to the stimulated ear of normal animals responded to tonal stimulation with excitatory responses, and the mean threshold of units was close to the ferret's behavioral threshold (Kelly et al. 1986). The limits to increased responsiveness imposed by the availability of additional, unresponsive IC neurons, and by the mechanical and electrical properties of the auditory periphery, would presumably prevent the expression of such widespread changes as those found in the normally less responsive IC.

    ACKNOWLEDGEMENTS

  We thank D. Fleming and staff for animal care, K. Laman for histological assistance, M. Cortina-Borja for statistical help, A. King and J. Schnupp for critical comments on earlier drafts of the paper, and two anonymous reviewers for helpful criticisms and comments.

  This work was supported by a Wellcome Prize Studentship to D. McAlpine, a C. J. Martin Traveling Fellowship of the Australian National Health and Medical Research Council to R. L. Martin, a Canadian Natural Sciences and Engineering Research Council Studentship and an Oxford McDonnell-Pew Cognitive Neuroscience Studentship to J. E. Mossop, and a Medical Research Council Programme Grant to D. R. Moore.

    FOOTNOTES

   Present address of R. L. Martin: Air Operations Division, Maritime Research Laboratory, Fisherman's Bend, Victoria 3207, Australia. 1   There is also likely to be a more marked, inhibitory deafferentation of the IC on the side of the cochlear removal, produced by neuron loss in the lateral superior olive on that side (Moore 1992; Moore et al. 1995b).

  Present address and address for reprint requests: D. McAlpine, MRC Institute of Hearing Research, University Park, University of Nottingham, Nottingham NG7 2RD, UK.

  Received 24 January 1997; accepted in final form 11 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society