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Unit-activity in the central amygdalar nucleus of rats in response to immobilization—stress
Bwirr Re.trrrw/~ Bd/cfit~,
Vol.
10, pp. 833-837.
1983. d Ankho
International. Printed in the
U.S.A
Unit-Activity in the Central Amygdalar Nucleus of Rats in Response to Immobilization-Stress’ PETER St. Francis Xavier i.fnivtmity,
G. HENKE
Antigonish,
Received
5 January
Nova Scotia, Canada B2G ICCl 1983
FIENKE, P. G. Wtl~r-a~,t~~,it~ in t/w wnfral u~z~~~ala~ ntrclc~ztsof rut.~ in re.yxm.w to imnrohilitation-,rtrc>ss. BRAIN RES BULL tO(6) 833-837, 1983.-Multiple-unit actrvity in the central nucleus of the amygdala of rats was measured during immobilization and during presentations ofa white-noise stimulus that had been paired with the restraint treatment. Results showed that some units responded with an increase, others with a decrease in firing rate during immobilization. Several units also showed “conditioned” rate changes to the auditory stimulus. A behavioral assessment of the presumed conditioned aversive properties of the stimulus showed that the rats which had experienced the pairing of restraint and the noise stimulus escaped faster when presented with the stimulus than the unpaired controls. It is suggested that the centrai amygdala might be part of a system in which stressful inputs influence autonomic functions. Central amygdalar nucleus
Unit-activity
immobilization
PHYSICAL restraint is one of the most commonly used methods to induce stress-pathology, e.g., in the stomach. The mechanisms by which immobilization produces such gastric pathology are unclear, but recently it has been suggested that telencephahc limbic structures might be importantly involved [ 14,201. For example, bilateral lesions of the centromedial amygdala attenuated the effects of restraining the animal on the development of such pathology. On the other hand, damage to posterolateral regions of the amygdala increased the severity of the pathological changes in the stomach [16,17]. Against the background of these data, one troublesome aspect of the immobilization technique is the findings that indicate augmented corticosterone release during restraint; even after surgically separating the basal hypothalamus from the rest of the brain [Il. 121. In fact, the suggestion has been made that these particular findings compromise the usefulness of this method as an experimental paradigm to investistress factors (one assumes it to be gate “psychological” contrasted with “physical” stress). Although it is felt that this conceptual distinction is primarily a philosophical issue [20], nevertheless, it was judged to be important to determine whether or not inputs related to immobilization experiences reach the amygdala, modify the neural activity in this area, and also whether they might be experienced as being “aversive” by the animal. An additional objective was to study the possibility that changes in amygdala unit-activity could be “conditioned” to an external stimulus that had been paired with the immobilization treatment. METHOD Male Wistar rats, approximately
ISupported by grant NSERC A8617.
150 days old, were used.
Stress
Multiple-unit activity was recorded with stainless-steel electrodes (Medwire Corp.), 76 pm diameter, Teflon insulated, and cut with scissors to the appropriate length. The electrodes were positioned stereotactically, using a hydrolic microdrive (David Kopf), through a guide-tube attachment which was used to penetrate the dura. The animals were anesthetized with a mixture of chloralose (0.1 g/kg) and urethan (1.0 g/kg), and the electrodes were advanced to within 0.5 mm of the target coordinates: AP 6.4 (5.4), H -2.0 (-1.5). L 3.8 (4.4) 191. The electrode was lowered until “spikes” (3:1 signal to noise; >I00 pV) appeared on the oscilloscope (Tektronix 5 113). The unit-activity was recorded differentially through a battery-powered, high-impedance preamplifier/amplifier system with low-frequency cut-off (Grass Instruments). its output was stored on FM-tape (Tandberg FM-recorder). Four probes were lowered into either the right or left side of the brain. The indifferent electrode was a 250 pm wire which was placed l-2 mm away from the recording probes. The differential recording between probes and indifferent electrode virtually eliminated the EMG-interference, sometimes generated from the facial muscles. The electrodes were connected to a multi-wire cable through MS-343 connectors (Plastic Products Co.). The connector was anchored to the skull with stainless steel screws and dental acrylic. The low-noise microdot cables and one open-ended movement detector (hearing-aid) wire were then connected via a commutator and counter-balanced arm (BRWLVE) to the amplification system, oscilloscope. and FM-recorder. Units were tested prior to the restraint treatment by placing the rat into a Plexiglas box (40~30x42 cm), two days after surgery. The box was inside a shielded, sound-
HI;NKt
x34
attenuating cubicle, equipped with a one-way mirror (BRSILVE). The rat was kept in the Plexiglas box for I hr, and during the last 30 min a white-noise stimulus (75 dB, re 20 pN/m2) was also presented. Only two units responded to the introduction of the white-noise, and they had returned to baseline firing rates by the end of the session. The signals from the movement detector were fed into a Schmitt-trigger which was set to discriminate the periods during which the rat showed no obvious body movements. Only during these periods was neuronat activity recorded from the central amygdala. Mean spontaneous unit-activity during these recording periods was used as the index of the unrestrained-baseline-activity. Immediately thereafter, the rat was immobilized in a Plexiglas restraining cage (Fisher Scientific Co.) for 2 hr. Rats in the so-called “paired-condition” also received presentations of the white-noise stimulus during restraint. The other rats did not experience the pairing of restraint treatment and the white-noise stimulus. Following the 2 hr restraining session, the rats were returned to their homecages for 2 hr. After this “rest” period, the rat was placed again into the 40~30x42 cm Plexiglas box, and 10 min later the white-noise stimulus came on (30 min) for “conditioning” tests. A behavioral assessment of the presumed “aversive” properties of the white-noise stimulus was carried out during the next two days. Escape responses to the stimulus were measured in the “paired” and “unp~red-co~ition” rats. A Modular Testing Unit (Lafayette, Model 85000) was the start box of the one-way escape apparatus. An automatic guillotine-door (Lafayette, Model 85013) separated the Modular Testing Unit from a 38x 18x 17 cm compartment, which had been constructed from plywood and was painted black. During testing, the rat was placed into the start box, 10 set later the guillotine-door was raised, the white-noise came on, and an electric stop-clock started (Lafayette, Model 58007). When the animal entered the black compartment the door was lowered and the clock was stopped. Following the escape response, the rat was removed from the apparatus and then was placed into a holding cage. After an intertrial interval of 60 set the next trial began. Each rat was tested for 15 trials/day. If the animal did not escape from the stimulus within 60 set the trial was terminated, and a latency score of 60 set was recorded. Following testing, the animals were sacrificed with an overdose of sodium pentobarbital, perfused through the heart with physiological saline and 10% formaline, and then the brains were removed. After embedding in parafIln, sections were cut at 9 p, and every third section was stained with thionin. RESULTS
Sixteen units were lost during the study, reducing the number of active units to eighty-four. As a measure of variation in neural activity, the mean frequency and the standard deviation of activity counts during successive 100 msec bins of the unrestrained baseline period were obtained. Standard scores, based on the baseline distribution of activity measures and the mean unit-activity during the restraining session, were then computed. This procedure provides a statistical assessment of changes in unit-activity during immobilization, relative to the baseline [5]. This analysis showed that during immobilization, 31% of the units responded with a statistically significant increase, relative to baselines.
TABLE
1
MEAN BASELINE ACTIVITY OF FACfLITATORY (I?) AND SUPPRESSED(S) UNITS IN THE CENTRAL AMYGDALAR NUCLEUS
Treatment
Restraint + Noise Restraint Only
Restraint + Noise Restraint Only
whereas, 23% showed significantly decreased firing- rates (zscores> -+1.96, p
AMYGDALA
AND STRESS
835
UP
11
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.2 * i=
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2 0 Iz 9.1 3 9.2
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MIN FIG. 2. Mean activity counts of facilitatory unit during the first and second 15 min-periods of the conditioning test. Broken lines represent unrestrained baseline activity (B) and mean counts during body restraint (R). Location of the electrode is indicated on the left. Brain map is reproduced from KZinig and Ktippel 1281.
.
73 -.4
P
I UP
-R
FIG. I. Mean unit-ratios of facilitated and suppressed units in the central amygdalar nucleus during the immobilization treatment. Some units were paired (P) with the noise stimulus, others were unpaired (UP) controls.
mobilization. Figure 3 atso indicates that this conditioned suppressed unit was Iocated in the dorsolateral quadrant of the central amygdalar nucleus. Although the central nucleus can be divided into subregions on the basis of cytoarchitectural, and histochemical characteristics [6, 10,35,38,43,44], no clear pattern of the distribution of suppressed units could be found. On the other hand, the majority of facilitated units (17 of 26 units) were located in the ventral portion of the central nucleus. The behavioral measurements of the escape latencies of those animals which had shown significant unit-changes during the immobilization treatment are shown in Table 2. The behavior of the eleven “paired-rats” and nine “unpaired rats” indicates that the presentation of the white-noise during the restraint session, subsequently, facilitated escape responses from that stimulus. Paired animals escaped with significantly b~O.05) shorter latencies than the unpaired rats (Table 2). DISCUSSION
The present findings indicate that the immobilization of rats initiated changes in some units in the central amygdala. Several units responded with increases in unit-rates, others were suppressed by the stress stimulus. Apparently, this change in unit-activity can also be conditioned to an auditory stimulus that had been associated with the restraint-
l-15
16-30 MIN
FIG. 3. Mean activity counts of suppressed unit during first and second 15 min-periods of the conditioning test. Broken lines represent baseline (B) and mean restraint-induced activity (R). Location of the electrode is indicated on the left. Brain map is reproduced from Kiinig and Klippel [28].
treatment. However, the data atso showed that not all of these so-called restraint-units could be conditioned in this fashion, suggesting that some of the units only respond to the immobilization-stress itself. The plasticity of the conditionable units may, possibly, reflect learning-related processes in the central amygdala under stressful conditions. This suggestion is in line with the behavioral measurements obtained in the escape apparatus. Apparently, the white-noise stimulus had acquired aversive properties, presumably by being associated with the stress-treatment. It seems that restraining the animal is experienced as aversive, and based on the gastric pathology literature [ 14,201, probably stressful. This stress-input, the present results indicate, modified the neural activity of the central amygdala {and, possibly, other brain structures, as well). Recent findings also indicate that the unit-activity in the central amygdalar nucleus of rabbits
HEN lil..
836
TABLE 2 MEAN ESCAPE LATENCIES (SEC) FROM THE NOISE STIMULUS PAIRED AND UNPAIRED RATS
Group
Day I
Day 2
Paired
6.9*
IX..5
Unpaired
24.3
IN
21.7
*p<0.05
seemingly correlated with the development of conditioned changes in the heart-rate in response to electric shock stimulation [I]. Taken together, the data suggest a more generalized responsiveness of central amygdalar neurons to aversive (stressful) inputs. In fact, the proposal has been made that this area of the limbic system may be part of a so-called “ancillary” pain system, mediating the presumed affective-motivational components of aversive experiences [20]. The results of lesions in this area also suggest a more generalized effect on aversive inputs, e.g., frustration due to nonreward and aversive body handling 113, 15,211. In general, lesions in the amygdala reduce the animal’s reaction to various noxious inputs, i.e., damage in this area reduces shock-motivated avoidance behavior, shock-motivated fighting, and gastric pathology following restraint treatment or electric shock stimulation [13, 16, 17, 19, 321. On the other hand, electrical stimulation of the centromedial amygdala has been reported to induce pathological effects in the stomachs of rats and cats [17, 26, 401. Aversive inputs may reach the central amygdala via the thalamus or neocortex or, directly, from nociceptive fibers associated with the substantia nigra and locus coeruleus. Recordings of unit activity in these areas have indicated that noxious inputs reach these structures, which then may be relayed to the central nucleus of the amygdala [3,4,7, 8, 29. 3 I]. Studies have also shown that this area of the amygdala is particularly rich in enkephalins and Substance P-positive cells, 12, 6, 23, 35, 41, 42, 441. In addition, it has been found that micro-injections of morphine into the amygdala reduced the animal’s reactions to noxious inputs [36,37]. It is possible
that the suppression of neural activity found with sonic urut~ reflects the presumed inhibitory functions associated with enkephalins [22]. It is interesting to note that the suppressed units, in the present study, were found in widespread arcas of the central nucleus. and it has been reported that cnkephalins are fairly evenly distributed throughout this nucleus [44]. On the other hand, the facilitatory units were located mostly in the ventral areas. Recent data have shown that the cell bodies of output neurons tcl lower brainstem autonomic control areas were concentrated in the medial region. including areas from which facilitation was recorded from several units in the present study [3X]. Immuno~yto~hemi~al studies also suggest that Substance-P terminals, usually assumed to have exitatory effects. are highly concentrated in the ventrolateral section of the central nucleus [44]. Anatomical studies also suggest that afferent information from the stomach might reach the amygdala. Fibers from the solitary tract nucleus project to the central amygdala 133.341. and the solitary tract nucleus is also the recipient of afferent fibers from the stomach [30]. Conversely, direct projections from the central amygdalar nucleus to the dorsal motor nucleus of the vagus also exist (24. 25, 33. 381. These data suggest that the central amygdala is, therefore, in a position to both monitor visceral functions and to influence visceraf activities. A model of psychosomatic disorders by Schwartz [39] has proposed that negative feedback (“biofeedback’.) participate in the CNS-regulation (or disreguIation) of stress-induced somatic effects. The model assumes that feedback information is used to maintain stability (homeostasis) of the brain-gut regulatory systems. It seems possible that the amygdala might be involved in autonomic and behavioral regulatory actions (e.g.. flight or fight) when this brain-gut regulatory system is under stress. Prolonged interference (e.g., restraint) with behavioral adjustments may. as a consequence, impair the effectiveness of negative feedback to regulate the stability of the brain-gut mechanisms; in turn, producing somatic pathology. In summary, the data indicate that the unit-activity in the central amygdaia can be altered by stressful inputs. Stimuiation of this area also modifies gastric and cardiovascular functions, sometimes to the point of pathology [17. 26, 27, 401. Taken together, it seems that the central amygdala is part of a system in which stressful inputs influence au-
tonomic functions.
REFERENCES 1. Applegate, C. D., R. C. Frysinger, B. S. Kapp and M. Gallagher. Multiple unit activity recorded from amygdala central nucleus during Pavlovian heart rate conditioning in rabbit. Bruilz Ras 238: 457-462, 1982. 2. Atweh, S. F. and M. J. Kuhar. Autor~iog~phic localization of opiate receptors in rat brain. II. TeIencepbaion. Brain Res 134: 393-405, 1977. 3. Barasi, S. Responses of substantia nigra neurons to noxions stimulation. Brain Res 197: 121-130, 1979. 4. Barasi, S. and S. Pay. Electrophysiological investigation of the connection between the substantia nigra and the amygdala in rat. Neurosci Lett 17: 265-269, 1980. 5. Berger, T. W. and R. F. Thompson. Neuronal plasticity in the limbic system during classical conditioning of the rabbit nictating membrane response. I. The hippocampus. Brain Rcs 145: 323-346, 1978.
6. Cassel, M. D., N. J. Mankovich, T. S. Gray and T. H. Williams. Computer-assisted image analysis of the distribution of peptidergic terminals in the central nucleus of the amygdala: A prelimin~y study. Peptides 3: 283-290, 1982. 7. Cederbaum, J. M. and G. K. Aghajanian. Noradrenergic neurons of the locus coeruleus: Inhibition by epinephrine and activation by the a-antagonist piperoxane. Brciin Rcs ! 12: 4 l3419, 1976. 8. Cederbaum, J. M. and G. K. Aghajanian. Afferent projections to the rat locus coeruleus as determined by a retrograde tracing technique. J Camp Neurnl 178: I-16, 1978. 9. de Groot, J. The rat forebrain in stereotaxic coordinates. Vcrh K Ned Akad Wet 52: l-40, 1959. 10. Fallon, J. H. Histochemical characterization of dopaminergic nonadrenergic and serotonergic projections to the amygdala. In: The Amygdaloid Complex. edited by Y. Ben-Ari. Amsterdam: ElsevierlNorth-Holland, 1981.
AMYGDALA
AND
STRESS
11. Feldman, S. The integration of neural and endocrine factors regulating hypothalamic activity. In: Brain-Pituitary-Adrrnal Intrrrclationships. edited by A. Brodish and E. S. Redgate. Base]: S. Karger, 1973. 12. Feldman, S., N. Conforti, 1. Chowers and J. M. Davidson. Pituitary-adrenal activation in rats with medial basal hypothalamic islands. Acta Endoc~rinol (Copmh) 63: 405-414, 1970. 13. Goddard, G. V. Functions of the amygdala. Psycho/ Bull 62: 89-109, 1964. 14. Henke, P. G. The hypothalamus-amygdala axis and experimental gastric ulcers. Neurosri Biobrhav Rev 3: 75-82, 1979. 15. Henke, P. G. Limbic lesions and the energizing, aversive, and inhibitory effects of non-reward in rats. Con J Psycho/ 33: 133140, 1979. 16. Henke, P. G. The amygdala and restraint ulcers in rats. J Camp Physiol Psycho/ 94: 3 13-323, 1980. 17. Henke, P. G. The centromedial amygdala and gastric pathology in rats. PhyAiol Behav 25: 107-I 12, 1980. 18. Henke, P. G. Facilitation and inhibition of gastric pathology after lesions in the amygdala of rats. Physiol Bchuv 25: 575-579, 1980. 19. Henke, P. G. Attenuation of shock-induced ulcers after lesions in the medial amygdala. Physiol Behuv 27: 143-146, 1981. 20. Henke, P. G. The telencephalic limbic system and experimental gastric pathology: A review. Neurosci Biohehuv Rev 6: 381-390, 1982. 21. Henke, P. G. and D. Maxwell. Lesions in the amygdala and the frustration effect. Physiol Bchuv 10: 647-650, 1973. 22. Hokfelt, T., 0. Johansson, A. Ljungdahl, J. M. Lundberg and M. Schultzberg. Peptidergic neurons. Nuture 284: 515-521, 1980. 23. Hokfelt, T., A. Ljungdahl, L. Terenius, R. Elde and G. Nillson. Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalins and Substance P. Proc Nut1 Acud Sc,i USA 74: 3081-3085. 1977. 24. Hopkins, D. A. Amygdalotegmental projections in the rat, cat and rhesus monkey. Neurosc~i L&t 1: 263-270, 1975. D. A. and G. Holstege. Amygdaloid projections to the 25. Hopkins, mesencephalon, pons and medulla oblongata in the cat. Exp Bruin Rrs 32: 529-547, 1978. 26. Innes, D. L. and M. F. Tansy. Gastric mucosal ulceration associated with electrochemical stimulation of the limbic system. Bruin RPS Bull 5: Suppl I, 33-36, 1980. M. D. Underwood, C. L. McNall 27 Kapp, B. S., M. Gallagher, and D. Whitehorn. Cardiovascular responses elicited by electrical stimulation of the amygdala central nucleus in the rabbit. Bruin Rrs 234: 251-262. 1982. 28. Konig, J. F. R. and R. A. Klippel. T/X, Rut Bruin. Baltimore: Williams and Wilkins, 1963. 29. Korf. J., B. S. Bunney and G. K. Aghajanian. Noradrenergic neurons: Morphine inhibition of spontaneous activity. Eur J Phurmuc,ol 25: 165-169, 1974.
837
30. Leslie, R. A., D. G. Gwyn and D. A. Hopkins. The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Bruin Rcs Bull 8: 37-43, 1982. 31. Mehler, W. R. Subcortical afferent connections of the amygdala in the monkey. J Camp Neural 190: 733-762, 1980. 32. Miczek, K. A.. T. Brykczynski and S. P. Grossman. Differential effects of lesions in the amygdala, periamygdaloid cortex. and stria terminalis on aggressive behaviors in rats. ./ ( ,,trtp Physiol Ps_vchol 87: 760-77 I. 1974. 33. Nauta, W. J. H. and V. B. Domesick. Neural associations ofthe limbic system. In: The Neural Busis of’Bchuvior. edited by A. L. Beckman. New York: Snectrum Publications. 1982. 34. Ricardo, J. A. and E. T. Koh. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Bruin Ras 153: l-26, 1978. 35. Roberts, G. W.. J. M. Polak and T. J. Crow. The peptidergic circuitry of the amygdaloid complex. In: Thr Amygdtrloicl Complex. edited by Y. Ben-Ari. Amsterdam: Elsevier/NorthHolland, 1981. 36. Rodgers, R. J. Influence of intra-amygdaloid opiate injections on shock threshold, tail-flick latencies and open field behaviour in rats. Bruin Rcs 153: 21 l-216, 1978. 37. Rodgers. R. J. Goioid neutides. brain and behaviour: A brief review. Ir J Mrd sc,i 14?:‘57-62. 1978. 38. Schwaber, J. S., B. S. Kapp and G. Higgins. The origin and extent of direct amygdala projections to the region of the dorsal motor nucleus of the vagus and the nucleus of the solitary tract. Neurosc,i Lctt 20: 15-20, 1980. 39. Schwartz, G. E. Psychosomatic disorders and biofeedback: A psychobiological model of disregulation. In: P.ryc.hoprrtho/o~:y: Expcrimc,ntul Modds. edited by J. D. Maser and M. E. P. Seligman. San Francisco: W. H. Freeman, 1977. 40. Sen, R. N. and B. K. Anand. Effect of electrical stimulation of the limbic system of brain (“visceral brain”) on gastric secretory activity and ulceration. Indiun J Mctl Rcs 45: 515-S21. 1957. 41. Uhl, G. R., M. J. Kuhar and S. H. Snyder. Enkephalincontaining pathway: Amygdaloid efferents in the stria terminalis. Bruin Rcs 149: 223-228, 1978. 42. Uhl, G. R., M. J. Kuhar, R. R. Goodman and S. H. Snyder. Histochemical localization of the enkephalins. In: Endorphins in Mrntul Hculth Rcswrch. edited bv E. Usdin, W. E. Bunney and N. S. Kline. London: MacMillan, 1979. 43 Wakefield, C. and E. Hall. Some observations on the ultrastructure of the central amygdaloid nucleus in the cat. (‘e/I Ti,s.~rrc Rrs 1.51: 489-498, 1974. 44. Williams, T. H.. M. D. Cassell, N. J. Mankovich. H. K. Huang and T. S. Gray. The distribution of neuropeptides in the central nucleus of the amygdala: A quantitative approach using image analysis. In: The Amy&u/ok/ Complcr. edited by Y. Ben-Ari. Amsterdam: Elsevier/North-Holland. 1981.
Vol.
10, pp. 833-837.
1983. d Ankho
International. Printed in the
U.S.A
Unit-Activity in the Central Amygdalar Nucleus of Rats in Response to Immobilization-Stress’ PETER St. Francis Xavier i.fnivtmity,
G. HENKE
Antigonish,
Received
5 January
Nova Scotia, Canada B2G ICCl 1983
FIENKE, P. G. Wtl~r-a~,t~~,it~ in t/w wnfral u~z~~~ala~ ntrclc~ztsof rut.~ in re.yxm.w to imnrohilitation-,rtrc>ss. BRAIN RES BULL tO(6) 833-837, 1983.-Multiple-unit actrvity in the central nucleus of the amygdala of rats was measured during immobilization and during presentations ofa white-noise stimulus that had been paired with the restraint treatment. Results showed that some units responded with an increase, others with a decrease in firing rate during immobilization. Several units also showed “conditioned” rate changes to the auditory stimulus. A behavioral assessment of the presumed conditioned aversive properties of the stimulus showed that the rats which had experienced the pairing of restraint and the noise stimulus escaped faster when presented with the stimulus than the unpaired controls. It is suggested that the centrai amygdala might be part of a system in which stressful inputs influence autonomic functions. Central amygdalar nucleus
Unit-activity
immobilization
PHYSICAL restraint is one of the most commonly used methods to induce stress-pathology, e.g., in the stomach. The mechanisms by which immobilization produces such gastric pathology are unclear, but recently it has been suggested that telencephahc limbic structures might be importantly involved [ 14,201. For example, bilateral lesions of the centromedial amygdala attenuated the effects of restraining the animal on the development of such pathology. On the other hand, damage to posterolateral regions of the amygdala increased the severity of the pathological changes in the stomach [16,17]. Against the background of these data, one troublesome aspect of the immobilization technique is the findings that indicate augmented corticosterone release during restraint; even after surgically separating the basal hypothalamus from the rest of the brain [Il. 121. In fact, the suggestion has been made that these particular findings compromise the usefulness of this method as an experimental paradigm to investistress factors (one assumes it to be gate “psychological” contrasted with “physical” stress). Although it is felt that this conceptual distinction is primarily a philosophical issue [20], nevertheless, it was judged to be important to determine whether or not inputs related to immobilization experiences reach the amygdala, modify the neural activity in this area, and also whether they might be experienced as being “aversive” by the animal. An additional objective was to study the possibility that changes in amygdala unit-activity could be “conditioned” to an external stimulus that had been paired with the immobilization treatment. METHOD Male Wistar rats, approximately
ISupported by grant NSERC A8617.
150 days old, were used.
Stress
Multiple-unit activity was recorded with stainless-steel electrodes (Medwire Corp.), 76 pm diameter, Teflon insulated, and cut with scissors to the appropriate length. The electrodes were positioned stereotactically, using a hydrolic microdrive (David Kopf), through a guide-tube attachment which was used to penetrate the dura. The animals were anesthetized with a mixture of chloralose (0.1 g/kg) and urethan (1.0 g/kg), and the electrodes were advanced to within 0.5 mm of the target coordinates: AP 6.4 (5.4), H -2.0 (-1.5). L 3.8 (4.4) 191. The electrode was lowered until “spikes” (3:1 signal to noise; >I00 pV) appeared on the oscilloscope (Tektronix 5 113). The unit-activity was recorded differentially through a battery-powered, high-impedance preamplifier/amplifier system with low-frequency cut-off (Grass Instruments). its output was stored on FM-tape (Tandberg FM-recorder). Four probes were lowered into either the right or left side of the brain. The indifferent electrode was a 250 pm wire which was placed l-2 mm away from the recording probes. The differential recording between probes and indifferent electrode virtually eliminated the EMG-interference, sometimes generated from the facial muscles. The electrodes were connected to a multi-wire cable through MS-343 connectors (Plastic Products Co.). The connector was anchored to the skull with stainless steel screws and dental acrylic. The low-noise microdot cables and one open-ended movement detector (hearing-aid) wire were then connected via a commutator and counter-balanced arm (BRWLVE) to the amplification system, oscilloscope. and FM-recorder. Units were tested prior to the restraint treatment by placing the rat into a Plexiglas box (40~30x42 cm), two days after surgery. The box was inside a shielded, sound-
HI;NKt
x34
attenuating cubicle, equipped with a one-way mirror (BRSILVE). The rat was kept in the Plexiglas box for I hr, and during the last 30 min a white-noise stimulus (75 dB, re 20 pN/m2) was also presented. Only two units responded to the introduction of the white-noise, and they had returned to baseline firing rates by the end of the session. The signals from the movement detector were fed into a Schmitt-trigger which was set to discriminate the periods during which the rat showed no obvious body movements. Only during these periods was neuronat activity recorded from the central amygdala. Mean spontaneous unit-activity during these recording periods was used as the index of the unrestrained-baseline-activity. Immediately thereafter, the rat was immobilized in a Plexiglas restraining cage (Fisher Scientific Co.) for 2 hr. Rats in the so-called “paired-condition” also received presentations of the white-noise stimulus during restraint. The other rats did not experience the pairing of restraint treatment and the white-noise stimulus. Following the 2 hr restraining session, the rats were returned to their homecages for 2 hr. After this “rest” period, the rat was placed again into the 40~30x42 cm Plexiglas box, and 10 min later the white-noise stimulus came on (30 min) for “conditioning” tests. A behavioral assessment of the presumed “aversive” properties of the white-noise stimulus was carried out during the next two days. Escape responses to the stimulus were measured in the “paired” and “unp~red-co~ition” rats. A Modular Testing Unit (Lafayette, Model 85000) was the start box of the one-way escape apparatus. An automatic guillotine-door (Lafayette, Model 85013) separated the Modular Testing Unit from a 38x 18x 17 cm compartment, which had been constructed from plywood and was painted black. During testing, the rat was placed into the start box, 10 set later the guillotine-door was raised, the white-noise came on, and an electric stop-clock started (Lafayette, Model 58007). When the animal entered the black compartment the door was lowered and the clock was stopped. Following the escape response, the rat was removed from the apparatus and then was placed into a holding cage. After an intertrial interval of 60 set the next trial began. Each rat was tested for 15 trials/day. If the animal did not escape from the stimulus within 60 set the trial was terminated, and a latency score of 60 set was recorded. Following testing, the animals were sacrificed with an overdose of sodium pentobarbital, perfused through the heart with physiological saline and 10% formaline, and then the brains were removed. After embedding in parafIln, sections were cut at 9 p, and every third section was stained with thionin. RESULTS
Sixteen units were lost during the study, reducing the number of active units to eighty-four. As a measure of variation in neural activity, the mean frequency and the standard deviation of activity counts during successive 100 msec bins of the unrestrained baseline period were obtained. Standard scores, based on the baseline distribution of activity measures and the mean unit-activity during the restraining session, were then computed. This procedure provides a statistical assessment of changes in unit-activity during immobilization, relative to the baseline [5]. This analysis showed that during immobilization, 31% of the units responded with a statistically significant increase, relative to baselines.
TABLE
1
MEAN BASELINE ACTIVITY OF FACfLITATORY (I?) AND SUPPRESSED(S) UNITS IN THE CENTRAL AMYGDALAR NUCLEUS
Treatment
Restraint + Noise Restraint Only
Restraint + Noise Restraint Only
whereas, 23% showed significantly decreased firing- rates (zscores> -+1.96, p
AMYGDALA
AND STRESS
835
UP
11
.4 ‘3
. //
.2 * i=
R
/ /
:
.l
i 1-15
2 0 Iz 9.1 3 9.2
B
-_-l._16 -30
MIN FIG. 2. Mean activity counts of facilitatory unit during the first and second 15 min-periods of the conditioning test. Broken lines represent unrestrained baseline activity (B) and mean counts during body restraint (R). Location of the electrode is indicated on the left. Brain map is reproduced from KZinig and Ktippel 1281.
.
73 -.4
P
I UP
-R
FIG. I. Mean unit-ratios of facilitated and suppressed units in the central amygdalar nucleus during the immobilization treatment. Some units were paired (P) with the noise stimulus, others were unpaired (UP) controls.
mobilization. Figure 3 atso indicates that this conditioned suppressed unit was Iocated in the dorsolateral quadrant of the central amygdalar nucleus. Although the central nucleus can be divided into subregions on the basis of cytoarchitectural, and histochemical characteristics [6, 10,35,38,43,44], no clear pattern of the distribution of suppressed units could be found. On the other hand, the majority of facilitated units (17 of 26 units) were located in the ventral portion of the central nucleus. The behavioral measurements of the escape latencies of those animals which had shown significant unit-changes during the immobilization treatment are shown in Table 2. The behavior of the eleven “paired-rats” and nine “unpaired rats” indicates that the presentation of the white-noise during the restraint session, subsequently, facilitated escape responses from that stimulus. Paired animals escaped with significantly b~O.05) shorter latencies than the unpaired rats (Table 2). DISCUSSION
The present findings indicate that the immobilization of rats initiated changes in some units in the central amygdala. Several units responded with increases in unit-rates, others were suppressed by the stress stimulus. Apparently, this change in unit-activity can also be conditioned to an auditory stimulus that had been associated with the restraint-
l-15
16-30 MIN
FIG. 3. Mean activity counts of suppressed unit during first and second 15 min-periods of the conditioning test. Broken lines represent baseline (B) and mean restraint-induced activity (R). Location of the electrode is indicated on the left. Brain map is reproduced from Kiinig and Klippel [28].
treatment. However, the data atso showed that not all of these so-called restraint-units could be conditioned in this fashion, suggesting that some of the units only respond to the immobilization-stress itself. The plasticity of the conditionable units may, possibly, reflect learning-related processes in the central amygdala under stressful conditions. This suggestion is in line with the behavioral measurements obtained in the escape apparatus. Apparently, the white-noise stimulus had acquired aversive properties, presumably by being associated with the stress-treatment. It seems that restraining the animal is experienced as aversive, and based on the gastric pathology literature [ 14,201, probably stressful. This stress-input, the present results indicate, modified the neural activity of the central amygdala {and, possibly, other brain structures, as well). Recent findings also indicate that the unit-activity in the central amygdalar nucleus of rabbits
HEN lil..
836
TABLE 2 MEAN ESCAPE LATENCIES (SEC) FROM THE NOISE STIMULUS PAIRED AND UNPAIRED RATS
Group
Day I
Day 2
Paired
6.9*
IX..5
Unpaired
24.3
IN
21.7
*p<0.05
seemingly correlated with the development of conditioned changes in the heart-rate in response to electric shock stimulation [I]. Taken together, the data suggest a more generalized responsiveness of central amygdalar neurons to aversive (stressful) inputs. In fact, the proposal has been made that this area of the limbic system may be part of a so-called “ancillary” pain system, mediating the presumed affective-motivational components of aversive experiences [20]. The results of lesions in this area also suggest a more generalized effect on aversive inputs, e.g., frustration due to nonreward and aversive body handling 113, 15,211. In general, lesions in the amygdala reduce the animal’s reaction to various noxious inputs, i.e., damage in this area reduces shock-motivated avoidance behavior, shock-motivated fighting, and gastric pathology following restraint treatment or electric shock stimulation [13, 16, 17, 19, 321. On the other hand, electrical stimulation of the centromedial amygdala has been reported to induce pathological effects in the stomachs of rats and cats [17, 26, 401. Aversive inputs may reach the central amygdala via the thalamus or neocortex or, directly, from nociceptive fibers associated with the substantia nigra and locus coeruleus. Recordings of unit activity in these areas have indicated that noxious inputs reach these structures, which then may be relayed to the central nucleus of the amygdala [3,4,7, 8, 29. 3 I]. Studies have also shown that this area of the amygdala is particularly rich in enkephalins and Substance P-positive cells, 12, 6, 23, 35, 41, 42, 441. In addition, it has been found that micro-injections of morphine into the amygdala reduced the animal’s reactions to noxious inputs [36,37]. It is possible
that the suppression of neural activity found with sonic urut~ reflects the presumed inhibitory functions associated with enkephalins [22]. It is interesting to note that the suppressed units, in the present study, were found in widespread arcas of the central nucleus. and it has been reported that cnkephalins are fairly evenly distributed throughout this nucleus [44]. On the other hand, the facilitatory units were located mostly in the ventral areas. Recent data have shown that the cell bodies of output neurons tcl lower brainstem autonomic control areas were concentrated in the medial region. including areas from which facilitation was recorded from several units in the present study [3X]. Immuno~yto~hemi~al studies also suggest that Substance-P terminals, usually assumed to have exitatory effects. are highly concentrated in the ventrolateral section of the central nucleus [44]. Anatomical studies also suggest that afferent information from the stomach might reach the amygdala. Fibers from the solitary tract nucleus project to the central amygdala 133.341. and the solitary tract nucleus is also the recipient of afferent fibers from the stomach [30]. Conversely, direct projections from the central amygdalar nucleus to the dorsal motor nucleus of the vagus also exist (24. 25, 33. 381. These data suggest that the central amygdala is, therefore, in a position to both monitor visceral functions and to influence visceraf activities. A model of psychosomatic disorders by Schwartz [39] has proposed that negative feedback (“biofeedback’.) participate in the CNS-regulation (or disreguIation) of stress-induced somatic effects. The model assumes that feedback information is used to maintain stability (homeostasis) of the brain-gut regulatory systems. It seems possible that the amygdala might be involved in autonomic and behavioral regulatory actions (e.g.. flight or fight) when this brain-gut regulatory system is under stress. Prolonged interference (e.g., restraint) with behavioral adjustments may. as a consequence, impair the effectiveness of negative feedback to regulate the stability of the brain-gut mechanisms; in turn, producing somatic pathology. In summary, the data indicate that the unit-activity in the central amygdaia can be altered by stressful inputs. Stimuiation of this area also modifies gastric and cardiovascular functions, sometimes to the point of pathology [17. 26, 27, 401. Taken together, it seems that the central amygdala is part of a system in which stressful inputs influence au-
tonomic functions.
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