TO A TEST CHAMBER: A 2-DEOXYGLUCOSE STUDY. Jesper MOGENSEN, Gregg WILLIAMS and Ivan DIVAC. Institute of Neurophysiology, Panum Institute, ...
ACTA NEUROBIOL. EXP. 1983, 43: 283-290
ACTIVITY OF THE AUDITORY SYSTEM IN RATS HABITUATED TO A TEST CHAMBER: A 2-DEOXYGLUCOSE STUDY Jesper MOGENSEN, Gregg WILLIAMS and Ivan DIVAC Institute of Neurophysiology, Panum Institute, University of Copenhagen, School' of Medicine, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark
Key words: auditory system, 2-deoxyglucose, habituation, stress, rat.
Abstract. In a number of species, quantitative 2-deoxyglucose method" revealed that the auditory system is much more active than any other neural system. We tested the hypothesis that stressful experimental conditions may be responsible for this hyperactivity. Three rats received transcatheteral injections of [14C] 2-deoxyglucose while exposed to. a wide band noise in a sound-shielded chamber in which they had previously spent 17 habituatio~n sessions. In autonadiograms of these animals the auditory systlem appeared as intensely labelled (active) a s in control animals. We conclude that the high activity of the auditory system in the rat does not seem to result from presumably stressful "normal laboratory conditions". INTRODUCTION
Ability to detect and interpret auditory signals is essential for survival of both predators and 'pray. Even hibernating animals react to, and can be awaken by sounds. ~ u r & hibernation the most active part. ~f the nervous system, as revealed by the 2-deoxyglucose technique(2-DG), is the auditory system (11). The same is true for nonhibernators, e.g., normal rats of different strains and either sex: Sprague-Dawley females (18), Wistar males (4) and a pigmented strain with better vision and therefore presumably less dependence on audition (1). The generality of this phenomenon is even wider: absolute values published for t h e
normal rat (22), rabbit (14) and monkey (10) show a remarkable constancy. In each species, 2-DG consumption in formations involved in auditory functions is 1.34-2.02 times higher than the consumption in the corresponding visual regions. In the tectum this ratio seems to be larger than at thalamic and cortical levels (Table I). TABLE I Consumption of glucose in some auditory and visual formations (pmol/lOOg/min) Formation
1
Species I
Monkeyto Auditory cortex Visual cortex (Ratio Medial geniculate Lateral geniculate (Ratio Inferior colliculus Superior colliculus (Ratio
59
1
65 39 1.67 103 55 1.87
1 i
'1
Ratz2
,
1.41 126 92 1.37 198 99 2.00 -
1
::: 1 '
,
Rabbit14 102.3 76.4 1.34) 108.2 80.7 1.34) 125.5 62.1 2.02) -
As a rule, for the quantitative 2-DG technique, the animals are brought to the laboratory from their home quarters, they are handled, anesthetized, catheterized, injected, restrained and left for about 40 min i n the new environment until they are sacrificed. These treatments are stressful (e.g., 2,3). Results of some studies may be interpreted as suggesting that stress enhances the activity of the auditory system; this effect is in accord with presumably increased significance of auditory signals in potentially dangerous situations. Thus, in swimming rats the activity of the auditory system was higher than in controls (20). Similarly, the activity in the auditory cortex and the medial geniculate was invariably higher in monkeys performing shock-avoidance responses to light stimuli than in the controls (19). The degree of restrain, however, seems to have no influence on the activity of the auditory system (13). It seems interesting to know whether the activity of the auditory system is indeed influenced by the stressful situation or remains relatively high even in animals habituated to the experimental environment. We approached this question by the present experiment in which the 2-DG was injected into rats which were well habituated to their environment land to the circumstances of the treatment. If the high activity of the auditory system was significantly influenced by stress, i.e., by a central mechanism, our habituated rats should show less of this hyperactivity. We opted (a) not to draw blood for the quantitative 2-DG estimation in fear of introducing a new factor of stress, and (b) to have
a high substressful level of background noise (6) to which the animals should habituate, since a decrease of auditory stimulation reduces the activity of the auditory system (15, 21). METHODS
Subjects. Seven male Wistar albino rats were used. The rats weighed 180.0 g at the beginning of the experiment, lived in separate cages, and received water ad libitum and supplement of rat chow once a day, after t h e habituation session. Four animals underwent habituation and catheterization; three of them received 2-DG under habituation conditions and the fot~rthreceived Diazepam prior to 2-DG and spent the test time under conditions to which it had not been habituated. The remaining three rats were treated under "normal laboratory conditions" similar to those in other studies (18, 22). Surgery. The four habituation rats received polyethylen catheters into the right jugular vein 11-12 h y s before the test day and sacrifice. The catheters were flushed with 1010 heparin in saline (0.1 ml) about every second day. On habituation days the catheters were flushed while the animal was in the chamber. The outside of the catheters was painted with dissolved quinine for protection against biting. Procedure. Four days before the beginning of the experiment the habituation animals were transferred to the same room where the test box was located. During the first ten days of the experiment one rat at a time was transferred into the habituation box for 30 min. The box, 28X15.5X28 cm, was placed in a sound-shielded chamber which contained also a source of a masking noise. The noise level was 80 d B (measured by audiometer CASTLE type CS 141 B) inside the box. The rats were given ad libitum mlashed cookies in the box. On the eleventh day of the experiment the rats received intravenous catheters via the right jugular vein and were left in their home cages for 4-5 days to recover. After the recovery period, the rats went through another period of seven days of habituation similar to the preoperative treatment except for flushing of the catheters. For the flushing, the catheters received an extension which led outside the box and the chamber. The hole in the chamber through which the extension passed was stopped with foam rubber. The extension tubing and the syringe were filled with the solution to be injected. A three-way stopcock lallowed flushing of the extension and the catheter. The heparin-saline as well as 2-DG were injected five minutes after the start of the session. After the habituation period three rats were given food, noise and the injection
as usual, and at the customary time, except that the heparin-saline solution was replaced with 120 pCi/kg [14C] 2-DG (Amersham, specific activity 300 pCi/mmol) warmed to the room temperature, followed b y 0.2 ml saline flush through the three-way stopcook. The fourth animaL was pretreated with 10 mg/kg Diazepam 45 min before the 2-DG injection. This animal received 2-DG and the saline flush as soon as i t was placed in the box and then spent the entire test period, lasting 25 min, with the noise switched off and the front door of the soundshielding chamber open allo~wingthe animal to see and hear the activity in the room. The three iistressed" control rats were brought from animal quarters to the laboratory on the day of sacrifice. They received 2-DG through a tail vein while fully conscious, and spent the 40 min postinjection period until sacrifice in the laboratory. Histology and autoradiography. The rats were sacrificed by stunning and decapitation. The brain ,was rapidly taken out, frozen in powdered dry ice, and cut in a cryostat. Every 25th section 10 pm thick was taken on a coverslip and rapidly dried on a 'hot plate. The entire series of sections was placed on Kodak SB-5 film and exposed for 3 wks. RESULTS
The autoradiograms from all animals showed an enhanced activation of the components of the auditory system: the cochlear nuclei, the superior olive, the colliculus inferior, medial geniculate and the auditory cortex (Fig. 1). The degree of activation of the auditory system, judged for each section, seemed to be the same in all animals and looked very much like the illustrations of Schwartz and Sharp (18). DISCUSSION
Comparison of the autoradiograms in the present experiment clearly shows that the auditory system is more strongly labeled than the surrounding gray matter also in rats which are habituated t o the experimental situation. Since our main group should be considered well habituated to the time, place and treatment associated with 2-DG application, we may conclode that the hyperactivity of the auditory system is not caused by acute stress. This suggestion is supported by the normal activity of the auditory system in the rat which was pretreated with Diazepam and by its relative hyperactivity in rats under some anesthetics (7, 9, 22). It is still possible, however, that stress influences the level
'
Fig. 1. Autoradiograms of critical sections to show relative activity levels of the major components of the auditory system in differently treated animals. In each section the activity of the superior olive (S.O.), colliculus inferior (C.I.), medial geniculate (M.G.) and auditory cortex (AUD. CTX.) stands out a s compared to the surrounding gray in the same section. The three levels a r e comparable to Figs. 11A, 14A and 15A respectively of the 2-DG atlas for the normal rat brain by Schwartz and Sharp (18).
of activity of the auditory system to a degree which is below the threshold of the presently used technique and that we may be dealing with different kinds of stress which do not affect the auditory system in the same way. Only parametric studies could produce a decisive answer. The auditory system shows a relatively high activity of succinate dehydrogenase (5), presumably reflecting a chronically elevated energy metabolism in the system. Thus, both enzyme histochemistry and the 2-DG technique suggest that the auditory system of animals, including "olfactory-dominated" rats and "visually-dominated" monkeys, is more active than most other cerebral systems. Why this appears not to be the case in normal humans - in whom studies of regional cortical blood flow do not indicate excessive activity in the auditory cortex (12, 16), and why the brain of schizophrenics shows such relative hyperactivity (8) are tantalizing riddles. One way to approach this riddle is to look for the mechanisms which account for the relatively high activity of the auditory system in animals. One element necessary for the "normal" level of activity of the auditory system is the auditory stimulation itself: a decrease of the intensity of this stimulation reduces the activity of the auditory system (15, 21). This element alme, however, cannot account for the relative hyperactivity of the auditory system because the visual system under "normal" stimulation nevertheless consumes less glucose. The reason for the different levels of glucose consumption must lie ei'ther in different neurobiological properties of the neurons in the two systems or in the presence of a central regulatory mechanism which keeps glucose consumption of the auditory system at a relatively high level. Some evidence suggests that central mechanisms, specifically the adrenergic activation, may influence the activity of the auditory system (17). This study was supported by a grant from t h e Christian X Fund. T h e study w a s kindly helped by Bente Sonne who made the catheters and instructed us in t h e technique of their implantation, Morten Maller who lent us the cryostat, and t h e Department of Radiology of Rigshospitalet where our films were developed. R. Gunilla E. Oberg gave valuable comments on a n earlier draft of t h e paper, Margit Lavgreen helped us with typing, and Flemming Riis with photography.
1. BATIPPS, M., MIYAOKA, M., SHINOHARA, M., SOKOLOFF, L. and KENNEDY, C. 1981. Comparative rates of local cerebral glucose utilization In the visual system of conscious albino and pigmented rats. Neurology 31: 58-62. 2. BHATTACHARYA, S. K. 1982. Stress by restraining elevates brain prostaglandins in the rat. Neurosci. Lett. 33: 165-168.
3. CARLSSON, C., HA'GERDAL, M., KAASIK, A. E. a n d SIESJC), B. K. 1977.. A catecholamine-mediated increase in cerebral oxygen uptake during immobilisation stress in rats. Brain Res. 119: 223-231. 4 . DIVAC, I. a n d DIEMER, N. H. 1980. Prefrontal system in the r a t visualized by means of labeled deoxyglucose - f u r t h e r evidence for functional heterogeneity of the neostriatum. J. Comp. Neurol. 190: 1-13. 5. FRIEDE, R. L. 1966. Topographic Brain Chemistry. Academic Press, N e w York, pp. 22-29. 6. GAMBLE, M. R. 1982. Sound a n d its significance for laboratory animals. Biol. Rev. 57: 395421. 7. HERKENHAM, M. 1981. Anesthetics a n d t h e habenulo-interpeduncular system: selective sparing of metabolic activity. Brain Res. 210: 461-466. 8. INGVAR, D. H. 1976. Functional landscapes of t h e dominant hemisphere. Brain Res. 107: 181-197. 9. INGVAR, M. and SIESJC), B. K. 1982. Effect of nitrous oxide on local cerebral glucose utilization in rats. J. Cereb. Blood Flow Metabol. 2: 481486. 10. KENNEDY, C., SAKURADA, O., SHINOHARA, M., JEHLE, J. a n d SOKOLOFF, L. 1978. Local cerebral glucose utilization in t h e normal conscious macaque monkey. Ann. Neurol. 4: 293-301. 11. KILDUFF, T. S., SHARP, F. R. and HELLER, H. C. 1982. [14C]2-deoxyglucose uptake in ground squirrel brain during hibernation. J. Neurosci. 2: 143157. 12. KNOPMAN, D. S., RUBENS, A. B., KLAS'SEN, A. C., MEYER, M. W. a n d NICCUM, N. 1980. Regional cerebral blood flow patterns during v e r b a l a n d nonverbal auditory activation. Brain Lang. 9: 93-112. 13. OHATA, M., FREDERICKS, W. R., SUNDARAM, U. and RAPOPORT, S. I1981. Effects of immobilization stress on regional cerebral blood flow i n the conscious rat. J. Cereb. Blood Flow Metab. 1: 187-194. 14. PASSERO, S., CARLI, G. a n d BAT TI IS TIN,^, N. 1981. Depression of cerebral glucose utilization during animal hypnosis in the rabbit. Neurosci. Lett, 21: 345-349. 15. PLUM, F., G J D D E , A. a n d SAMSON, F. E. 1976. Neuroanatomical functional mapping by the radioactive 2-deoxy-D-glucose method. Neurosci. Res. Progr. Bull. 14: 457-518. 16. PROHOVNIK, I., HAKANSSON, K. and RISBERG, J. 1980. Observations o n the functional significance of regional cerebral blood flow in "resting" normal subjects. Neuropsychologia 18: 203-217. 17. SAVAKI, H. E., KADEKARO, M., MoCULLOCH, J. and SOKOLOFF, L. 1982. The central noradrenergic system in the r a t : metabolic mapping with a-adrenergic blocking agents. Brain Res. 234: 65-79. 18. SCHWARTZ, W. J., and SHARP, F. R. 1978. Autoradiographic maps of regional brain glucose consumption in resting, awake rats using [14C]2-deoxyglucose. J. Comp. Neurol. 177: 335-360. 19. SCHWARTZMAN, R. J., GREENBERG, J., REVICH, M., KLOSE, K. J. a n d ALEXANDER, G. M. 1981. Functional metabolic mapping of a conditionecf motor task i n primates utilizing 2-[14C]deoxyglucose. Exp. Neurol. 72: 153-163. 20. SHARP, F. R. 1976. Relative cerebral glucose uptake of neuronal p e r i k a r y a
and neuropil determined with 2-deoxyglucose in resting and swimmi~lg rat. Brain Res. 110: 127-139. 21. SHARP, F. R. and EVANS, K. 1982. Regional [I4C] 2-deoxyglucose uptake during vibrissae movements evoked by rat motor cortex stimulation. J. Comp. Neurol. 208: 255-287. 22. SOKOLOFF, L.: RHIVICH, M., KENNEDY, C., Des ROSIERS, M. H., PATLAK, C. S., PETTIGREW, K. D., SAKURADA, 0 . and SHINOHARA, M. 1977. T h e [14C]deoxyglucose method for t h e measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28: 897-9816. Accepted 14 April 1983 C;regg Williams, 2641 So. Ivy. St., Denver, Colorado 80222, USA
