Camp. Biochem. Physiol. Vol. 102A, No. 3, pp. 513-517, Printed in Great Britain

1992 0

0300-9629/92 65.00 + 0.00 1992 Pergamon Press Ltd

AUDITORY-EVOKED BRAINSTEM RESPONSES IN THE HIBERNATING WOODCHUCK MARMOT MONAX BHARTIKATBAMNA,*CHRY~XXJLA THODI,JEROMEB. SENTuntAt and DAVIDA. METZ Department of Speech and Hearing, Cleveland State University, 1983 East 24th Street, Cleveland, OH 44115, U.S.A.; iDepartment of Biology, Cleveland State University, Cleveland, OH, U.S.A. Telephone (216) 687-3804 (Receiued 28 October 1991)

Abstract-1. This study measured the changes in the auditory-evoked brainstem responses in the woodchuck (Marmota monux) during hibernation and arousal. 2. The auditory brainstem response of the euthermic woodchuck consisted of four waves occurring in a 10msec time window after stimulation. 3. In the hibernating woodchuck, waves I and II could be traced down to the lowest body temperatures. 4. As temperatures increased all the components of the ABR emerged. The latencies of all the waves showed systematic decrease with temperature increments, the effect being cumulative across the time window. 5. These findings reflect activity in the VIIIth cranial nerve and the cochlear nuclei during hibernation and restoration of the functional integrity of the brainstem auditory pathway during arousal.

INTRODUCTION Hibernation is considered to be an active process characterized by highly dynamic and integrated activity of the various levels of the brain as well as the specific and non-specific pathways to the brain. Such integrated activity promotes the sequential inhibition and facilitation required to enter into and arouse from the torpid state (Mihailovic, 1972). The curled up posture with the head buried beneath the tail assumed during hibernation is an indication of controlled motor activity in certain pathways. Measurement of evoked electrical activity in response to olfactory, visual and auditory stimulation (Chatfield et al., 1951; Lyman and Chatfield, 1953, 1955; Mihailovic, 1972) provides further evidence of the functional integrity of ascending pathways during from hibernation. various stages of arousal Strumwasser (1959) also reported a behavioral response to auditory stimulation in the hibernating ground squirrel. The response was characterized by uncurling and orientation of the pinna towards the sound source for 20-30min even after cessation of the sound stimulus and in some cases the animals aroused completely, rewarming to euthermia. Recent studies involving measurement of neural activity along the auditory brainstem pathways, to define the physiological processes responsible for this sensory sensitivity, have supported different physiological processes. Kilduff et al. (1982) measured [‘4C]2-deoxyglucose uptake to determine the magnitude of neural activity at various levels of the brainstem pathways in the hibernating ground squirrel. Ascending from the cochlear nucleus to the inferior colliculus, they showed a minimum decline (38.9%) in the neural activity at the cochlear nucleus, 52.0 and 50.3% reductions at the superior olivary nucleus *To whom all correspondence should be addressed.

and the nucleus of the lateral lemniscus, respectively and the greatest decrease in activity (65.0%) at the inferior colliculus. These findings suggest a sequential decline in metabolic activity along the ascending auditory pathway, the most rostra1 nuclei showing a maximum suppression of activity during hibernation. Hamill et al. (1989) measured auditory brainstem responses (ABR) during hibernation and arousal in the same species. In small animals, since the ABR waves I to V emanate from the VIIIth cranial nerve, the cochlear nucleus, the superior olivary complex, the lateral lemniscus and the inferior colliculus, respectively (Anchor and Starr, 1980a, b; Buchwald and Huang, 1975; Henry, 1979; Huang and Buchwald, 1977; Wada and Starr, 1983), its absence or alteration provides an index of the functional integrity of this portion of the ascending auditory pathway. Since Hamill et al. (1989) observed all the components of the ABR at temperatures as low as 9.8”C, they concluded that all the nuclei within the brainstem auditory pathway were activated simultaneously during the arousal phase. This all or none type of response pattern, however, does not explain the differential sensitivity of hibernators to sensory stimuli compared to the euthermic state. This study measured the changes in the ABR evoked with slow and fast repetition rates to elucidate the details of this response pattern in the hibernating woodchuck.

MATERIALS

AND METHODS

Animal care Four, 2-3-year-old woodchucks (Marmota monux) trapped in the Cleveland area served as experimental subjects. They were housed individually in a temperaturecontrolled environment and received standard laboratory (rabbit and mouse) chow, fresh greens and drinking water. 513

BIURTI KATUMNAet al.

514 Measurement procedures

Table 2. Means

Prior to the induction of hibernation, hearing screening and baseline recordings were obtained from all the woodchucks. These electrophysiological recordings were made with the Bio-logic Traveler (Bio-logic Systems Corp. clinical averager). Three silver wire loops implanted subcutaneously on the vertex, antero-ventral to the stimulus ear and anteroventral to the contralateral ear served as non-inverting, inverting and common electrodes, respectively. The tests (hearing screening and baseline recordings) were performed under sedation with acepromazine (Promace maleate, Aveco Co.) administered intramuscularly (4.0 me/kg body weight). The body temperature of the animal was maintained at 37°C during these recordings. Subsequent measurements were made sequentially during hibernation and during the arousal phase. The data were stored for later analysis. Induction of hibernation

Hibernation was induced by placing the subject in a thermostatically controlled cold room set at lO”C, with a 8L:l6D lightdark cycle and restricting food intake. Tympanic temperatures were measured with a YSI thermistor probe connected to a digital thermometer (sensitivity & 0. IQ, placed within 5 mm of the tympanic membrane. Hibernation was defined as a state characterized by the curled posture, low body temperature (< 30°C) and delayed responsiveness to external stimuli. Measurements were initiated when the animal demonstrated the above characteristics and were stopped when the responses recorded excessive muscle artefact indicating that the animal had aroused from hibernation. After the recordings the animals were fed and removed from the cold room. Stimulation and recording parameters The screening criterion consisted of measurement of a replicable ABR evoked in response to clicks presented via insert earphones at a repetition rate of 21.l/sec and intensity of 75 dB peSPL (peak sound pressure level). Subjects that showed no response in either ear were eliminated from the study. For baseline recordings and subsequent measurements, the stimuli were delivered oia a standard earphone attached to the cage at a distance of 12 in. from the subject’s head. The stimuli consisted of broad-band clicks presented at 90 dB peSPL (measured at the head) and repetition rates of 21.1 and 6l.l/sec. Fifteen hundred responses were averaged in a time window of 10 msec and a band-pass filter of 100-3000 Hz. The time window was extended to 45 msec for ABR measurements made below 20°C. Although this time window did not permit stimulation with the fast repetition rate, it ensured the measurement of components likely to show pronounced delays. The artefact reject was set at 16 p V with a gain of 150,000. Each waveform was replicated to ensure reliability of the response.

and

Click rate Wave I

wave II

Wave

III

of

the

ABR

wave

2l.llsec

61.11sec

Combined

Mean SD Range Mean SD Range

0.42 0.10 0.30-0.63 0.32 0.11 0.09-0.47

Mean SD Rann

0.24 0.12 0.08-0.41

SD Range

0.20 0.11 0.10-0.41

0.35 0.13 0.24-0.66 0.32 0.10 0.1 l-O.45 0.21 0.10 0.08-0.32 0.15 0.05 0.10-0.26

0.38 0.12 0.24-0.66 0.32 0.10 0.09-0.47 0.23 0.11 0.08-0.41 0.18 0.08 0.10-0.42

Mea;;

Wave IV

standard deviations amDlitu&?

ring within a time window of 10 msec. The means and standard deviations of the absolute latencies for waves I through IV measured at the two click rates are shown in Table 1 and those of the absolute amplitudes are shown in Table 2. T-tests for repeated measures yielded no significant differences between the mean latencies or amplitudes obtained for each wave at the slow repetition rate versus the fast repetition rate. Hence, the data for the two click rates were collapsed. The means and standard deviations of the collapsed data for the late&es and amplitudes are also shown in Tables 1 and 2, respectively. The ABR during hibernation At tympanic temperatures of 16°C and lower, auditory-evoked brainstem activity disappeared completely. Neither of the two click rates could elicit a repeatable response. Between tympanic temperatures of approximately 17 and 19”C, the changes were striking. Wave(s) I and/or II emerged and could be

RESULTS

The auditory-evoked euthermic woodchuck Table 1. Means and standard Click rate Wave I

Mean SD

Range WaveII Wave III

Wave IV

Mean SD Range Mean SD Range Mean SD Range

brainstem responses of the consisted of four waves occurdeviations

of the ABR wave latencies

Zl.l/sec

6l.l/sec

Combined

1.24 0.12 1.12-1.40 2.33 0.21 2.08-2.69 3.23 0.19 3.04-3.48 4.36 0.32 4.02-5.06

I .25 0.09 1.16-1.43 2.41 0.19 2.16-2.66 3.32 0.16 3.12-3.56 4.65 0.35 4.24-5.12

I .24 0.10 1.12-1.43 2.37 0.20 2.08-2.69 3.28 0.18 3.04-3.56 4.50 0.36 4.02-5.12

I

t

I

LATENCY(Pme/div) Fig. 1. Auditory-evoked brainstem responses obtained during hibernation showing wave(s) I and/or II compared to those measured during the initial phase of arousal (the last tracing).

Woodchuck auditory-evoked

brainstem response

515

0' 19.0

22.7

26.4

30.1

Temperature +

b

WI

-0.31 (0.03)

A

WI1

-0.35 (0.03)

33.6

37.5

(in ‘C) 0

Will

-0.38 (0.05)

+

WIV

-0.53 (0.07)

fi 0.84 0.86 0.78 0.80 Fig. 3. Latency-temperature relationship and associated parameter estimates, b [slope (standard error)] indicating the cumulative effect of latency change and rz (coefficient of determination) showing good fit of the regression lines for ABR waves I (WI) through IV (WIV). 0

4

8

12

LATENCY (in

16

20

24

ma)

Fig. 2. Serial recordings of auditory-evoked brainstem responses obtained at various temperatures during arousal from hibernation.

measured at both the click repetition rates. The latencies of both the waves were significantly prolonged compared to the baseline recordings obtained at 37°C. The amplitudes of the waves were repeatable, but somewhat lower than the euthermic measurements. Figure 1 depicts these changes in the ABRs obtained at the slow repetition rate. The ABR during the arousal phase The arousal phase was marked by increase in body temperature as well as by the simultaneous emergence of all the ABR waves. Figure 2 shows the ABRs obtained at various temperatures from a woodchuck arousing from hibernation. As temperature increased the latencies of all the waves decreased, the effect being cumulative across the time frame, so that wave I showed the least delay, whereas wave V showed the maximum delay. Regression analyses performed to delineate the rate of latency change per degree Celsius showed this cumulative effect as well. Figure 3 shows the regression lines along with the slopes, the standard errors and the coefficients of determination (adjusted r’s) for each

wave. The standard errors indicate low variability and the adjusted rzs indicate good fit of regression lines for all the waves, i.e. 78% or more variation in the wave latencies can be accounted for by the change in temperature. Although the amplitudes of all the waves were extremely variable, waves I and II showed an overall rising trend with temperature increments, which reached a maximum between 25 and 32°C and declined thereafter (Fig. 4A). The variability of the amplitudes of waves III and IV can be seen vividly in the scatter diagram of the temperature-amplitude relationship shown in Fig. 4B. The morphology of the waveform showed striking changes in the successive measurements made during arousal, the early recordings showing incomplete separation of all the waves compared to the later ones. As tympanic temperatures rose, the waveforms became similar to those seen during the euthermic measurements.

DXSCUSSION The ABRs recorded from the euthermic woodchuck showed that waves I and II were the most robust components. These components could also be traced down to the lowest temperatures in the ABRs measured during hibernation. In small animals, since waves I and II have been shown to originate from the

516

BHARTIKATBAMNA er

al.

0.80

0.60

z

0.60

-

0.50

+

0.40

-

0.30

-

0.20

-

0

g : 2

0 0 i +

0 +

0.10

+

Q

+

+

0

19.0

22.7

26.4

30.1

Temperature +

WI

33.6

37.5

19.0

22.7

WII

30.1

Temperature

(in ‘C) A

26.4

0

Will

33.6

37.5

(in ‘C) +

WIV

Fig. 4. (A) Amplitude-temperature relationship for ABR waves I (WI) and II (WII) showing a maximum between 25 and 32°C. (B) Showing the variability of the amplitudes as a function of temperature of waves III (WIII) and IV (WIV).

VIIIth cranial nerve and the cochlear nuclei, respectively (Anchor and Starr, 1980a, b; Buchwald and Huang, 1975; Henry, 1979; Huang and Buchwald, 1977; Wada and Starr, 1983), their presence at the lowest temperatures may be attributed to persistent activity at these levels of the auditory brainstem pathway. As temperatures increased above 19°C all the components of the ABR emerged. Although waveform morphology changed dramatically with temperature increments, all the components could be identified in successive recordings. These observations indicate that audition in the hibernating woodchuck may be mediated via an alternative ascending auditory pathway. Since the VIIIth cranial nerve carries auditory information from the periphery to the first order neurons, the dominance of the components originating from these structures is not surprising. Once relayed to the first order neurons, further conduction and processing may occur through other ascending routes. Alternatively, variable contribution of higher order neurons during hibernation may be responsible for the absence of rostra1 components of the ABR. Such findings have been documented by Kilduff et al. (1982). These investigators measured cellular uptake of [“C’jZ-deoxyglucose at various levels of the auditory brainstem pathway in the hibernating ground squirrel. The cochlear nuclei showed the highest level of [“Cl2-deoxyglucose uptake (38.9%) reflecting minimal change in neural activity from the euthermic state. Successive higher order nuclei showed decrease in neural activity from approximately 50% at the superior olivary complex and the lateral lemniscus nucleus to 65% at the inferior colliculus, indicating pronounced differences in functional contribution beyond the cochlear nuclei. Thus, differential contribution past the cochlear nuclei may partly explain the

absence of waves III and IV at the lowest temperatures measured. The emergence of all the components during the arousal phase suggests restoration of the functional integrity of the primary brainstem auditory pathway. The findings of latency prolongations of all the waves with temperature decrements, more so of the later waves than the earlier ones, are consistent with the results of previous studies (Hamill et al., 1989; Markand et al., 1984; Rosenblum et al., 1985; Rossi and Britt, 1984, Schom et al., 1977; Stockard et al., 1978). However, as the regression coefficients indicate, the changes in latency are much larger during hibernation than during hypothermia (Hamill et al., 1989). These findings may be related to the differences in the two physiological states. The amplitudes of the robust components I and II of the ABR showed a maximum between 25 and 32”C, declining at either end of this range. Since the woodchucks in this study aroused very rapidly from hibernation with auditory stimulation, these results may be attributed to rapid elevation of their body temperature. Similar results have been documented in experiments involving sudden systemic cooling (Rossi and Britt, 1984, Williston and Jewett, 1982). The variability of the amplitudes of waves III and IV may be attributable to poor wave separation seen in all recordings during arousal. In conclusion, ABR waves I and II could be traced down to the lowest body temperatures in the hibernating woodchuck. As temperatures increased all the components of the ABR emerged. These fhrdings reflect markedly higher levels of activity in the VIIIth cranial nerve and the cochlear nuclei during hibernation and restoration of the functional integrity of the brainstem auditory pathway during arousal.

Woodchuck auditory-ev #oked brainstem response REFERENCES

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Auditory-evoked brainstem responses in the hibernating woodchuck Marmota monax.

1. This study measured the changes in the auditory-evoked brainstem responses in the woodchuck (Marmota monax) during hibernation and arousal. 2. The ...
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