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Striato-amygdaloid transition area lesions reduce the duration of tonic immobility in the lizard Podarcis hispanica
Brain Research Bulletin, Vol. 57, Nos. 3/4, pp. 537–541, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter
PII S0361-9230(01)00687-6
Striato-amygdaloid transition area lesions reduce the duration of tonic immobility in the lizard Podarcis hispanica D. C. Davies,1* F. Martı´nez-Garcı´a,2 E. Lanuza3 and A. Novejarque2 1
Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, London, UK; Departments of 2Biologia Animal; and 3Celular, Universitat de Vale`ncia, Burjassot, Spain
ABSTRACT: Neuroanatomical data suggest that the lizard striato-amygdaloid transition area is homologous with the mammalian central amygdala. In order to investigate possible functional similarities, tonic immobility was induced in adult lizards and its duration recorded. Each lizard was then randomly assigned to one of three treatments: (1) bilateral striatoamygdaloid transition area lesions, (2) bilateral dorsal cortex lesions or (3) untreated controls. Three days after trial 1, each lizard was subjected to a second trial and the tonic immobility duration recorded. The mean tonic immobility duration in lizards with striato-amygdaloid transition area lesions was significantly shorter (80.5%; p < 0.0033) in trial 2 than in trial 1. There were no inter-trial differences within dorsal cortex-lesioned lizards or untreated controls. There was a significant treatment effect on tonic immobility duration in trial 2 (p < 0.0001). The mean tonic immobility duration of lizards with striato-amygdaloid transition area lesions was significantly shorter than that of dorsal cortex-lesioned lizards (72.2%; p < 0.01) or untreated controls (78.2%; p < 0.01). There was no significant difference in mean tonic immobility duration between dorsal cortex-lesioned lizards and untreated controls. Tonic immobility is considered to be an anti-predator behaviour that reflects the underlying state of fear. Therefore, the reduced tonic immobility duration in lizards with striato-amygdaloid transition area lesions reflects a reduction of fear. These results provide the first data to indicate a functional similarity between the lizard striato-amygdaloid transition area and the mammalian central amygdala. © 2002 Elsevier Science Inc.
a variety of other tests [15,16,28]. The duration of TI therefore appears to be a reliable measure of an animal’s underlying state of fear [8,14]. There is considerable evidence to suggest that the mammalian amygdala plays a role in fear behaviour [20] and there is also evidence that the amygdala is involved in the control of the TI response [22]. Tonic immobility occurs in a wide range of vertebrate species, including reptiles [32]. A variety of lizards have been demonstrated to exhibit the TI response, including Iguana iguana [31], Anolis carolinensis [12,13,35] and Hemidactylus turcicus [12]. Little is known about the telencephalic structures involved in the TI response in lizards. Neuroanatomical data suggest that the striato-amygdaloid transition area (SAT) of the lizard telencephalon is homologous with the mammalian central amygdala [19,33]. However, there is little functional evidence for a role of the SAT in fear behaviour to support this suggestion. Therefore, as a first step towards elucidating its function, the role of the SAT in TI was investigated by means of a lesion study. MATERIALS AND METHODS Adult lizards (Podarcis hispanica) of either sex ranging from 45–58 mm in snout-cloaca length, were captured in Vale`ncia during the months of February/March and October. They were communally housed in terraria heated by lamps, and food and water were available ad libitum. Throughout the experimental work, the animals were treated according to the European Communities Council Directive of November 24, 1986 (86/609/EEC). Each lizard was removed from its home terrarium and taken to a neighbouring quiet room, where TI was induced by placing the animal on its back on a flat surface and applying pressure to the thorax and pelvis, restraining the limbs (Fig. 1). When the lizard ceased struggling, it was slowly released and the time taken for it to resume an upright posture was recorded. If the lizard was still immobile after 900 s, the trial was terminated and a TI duration of 900 s recorded. The lizard was then randomly assigned to one of three experimental groups: (1) lizards that received bilateral SAT lesions (n ⫽ 9), (2) lizards that received bilateral dorsal cortex (DCx) lesions (n ⫽ 9), and (3) untreated controls (n ⫽ 9), coded and returned to its home terrarium. Soon after the first induction of TI, lizards that were to receive lesions were anaesthetised by intramuscular injection of ketamine
KEY WORDS: Amygdala, Amniote vertebrates, Archistriatum, Dorsal cortex, Fear.
INTRODUCTION Tonic immobility (TI) is a reversible state of motor inhibition that is triggered by repetitive stimulation, pressure on body parts, inversion, and restraint [6]. It is considered to be the last line of defence in a sequence of anti-predator behaviours [32]. The TI response is thought to be of great adaptive value because it can result in a predator losing interest in its prey [5,34,37]. Tonic immobility is considered to be positively related to fear since procedures designed to increase fears increase the length of the response, whereas fear-reducers shorten it [5,14]. Moreover, there is a close association between TI duration and measures of fear in
* Address for correspondence: Dr. D.C. Davies, Department of Anatomy and Developmental Biology, Cranmer Terrace, Tooting, London SW17 0RE, UK. Fax: ⫹44-(0)20-8725-3326; E-mail: [email protected]
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DAVIES ET AL. (3.25 mg/kg; Parke-Davis, El Prat de Llobregat, Spain) and placed in a small animal stereotaxic apparatus. The cranium was then surgically exposed, a hole drilled through it and the dura mater incised. A stainless steel electrode (0.3 mm in diameter) with a lacquer-insulated shaft was then lowered through the leptomeninges into the telencephalon. Electrolytic lesions were then made by passing a radio frequency current of 6 mA through the uninsulated tip (0.25 mm) of the electrode for 30 s. Lesions were placed in both hemispheres. Taking the anterior vertex of the pineal scale as zero, the SAT co-ordinates were 0.2 mm rostral, 0.8 mm lateral to the midline, and 0.9 mm below the surface of the brain. The DCx co-ordinates were similar, except that the lesions were placed 0.2 mm below the surface of the brain. After the lesions were completed, the entry wounds were treated with Veterin-Fenicol (chloramphenicol and lidocaine, Hoechst-Roussel, Barcelona, Spain), closed with Biocatalase (Fournier Labs., Tres Cantos, Madrid, Spain) and the lizards were returned to their home terraria. Three days after the initial induction of TI, each lizard was subjected to a second trial and the duration of TI was recorded. At the end of this trial, the lesioned animals were given a lethal dose of anaesthetic and perfused through the heart with 4% paraformaldehyde in phosphate buffered (0.1 M, pH 7.6) physiological saline. The brains were then removed and stored in similar fixative, before serial coronal sections (40 m) were cut in a cryostat. Every third section was collected onto glass slides, stained with toluidine blue and viewed in a light microscope. The sections were then drawn and the extent of the lesion sites recorded with the aid of a camera lucida drawing attachment. Brains that did not contain appropriate bilateral lesions of the SAT or DCx were excluded from the experiment. The codes were then broken and the behavioural data united with experimental treatment for each lizard. The effect of trial number on the duration of TI for each treatment was investigated by means of paired t-tests. The effect of treatment on TI duration in the second trial was investigated using analysis of covariance adjusting for TI duration in the first trial. Multiple comparisons were then performed between pairs of treatment groups using the Newman-Keul’s Studentized range test. Two-sample t-tests were used to investigate the effects of sex and season on TI duration in the first trial. RESULTS
FIG. 1. The stages of tonic immobility in Podarcis hispanica. (A) The lizard is placed on its back and pressure applied to the thorax and pelvis, restraining the limbs and tail. (B) Once the lizard stops struggling, pressure and restraint is slowly removed. (C) Tonic immobility. (D) The end of tonic immobility is typically signalled by head movement and torsion of the upper trunk, prior to resumption of an upright posture.
Striato-amygdaloid transition area lesions frequently encroached upon the overlying dorsal ventricular ridge and occasionally the DCx. Because some of the DCx lesions also impinged upon the underlying dorsal ventricular ridge, the effect of SAT lesions can be attributed to damage to deeper structures, predominantly the SAT. The largest and smallest SAT and DCx lesions are illustrated in Fig. 2. In lizards that had received SAT lesions, the duration of TI was significantly lower (80.5%) in trial 2 than in trial 1 (t ⫽ 4.12295; p ⬍ 0.0033). There were no inter-trial differences in TI duration in either untreated control or DCx lesion groups (Fig. 3). Analysis of covariance adjusting for TI duration in the first trial, revealed a significant effect of treatment on TI duration in the second trial [F(2,23) ⫽ 15.81; p ⬍ 0.0001]. Breaking down the data, the duration of TI in SAT lizards in trial 2 was significantly shorter than that of untreated control (78.5%, p ⬍ 0.01) and DCx (72.2%, p ⬍ 0.01) lizards (Fig. 3). There was no significant effect of sex or season on the TI duration of lizards in trial 1. The mean TI duration of males (N ⫽ 14) was 529.5 s ⫾ 105.3 SEM and that of females (N ⫽ 13) was 523.5 s ⫾ 107.8 SEM. The mean TI duration in February/March (N ⫽ 15) was 540.1 s ⫾ 104.0 SEM and that in October (N ⫽ 12) was 509.8 s ⫾ 108.5 SEM.
AMYGDALA LESIONS REDUCE TONIC IMMOBILITY
FIG. 2. Reconstructions of striato-amygdaloid transition area (SAT) and dorsal cortex (DCx) lesions. The largest and smallest lesions in each group are indicated by light and dark stippling respectively. The levels of the sections are indicated on the drawing of the lateral view of the brain. Scale bar: 400 m. Abbreviations: ac, anterior commissure; Acc, nucleus accumbens; ADVR, anterior dorsal ventricular ridge; aot, accessory olfactory tract; BNST, bed nucleus of the stria terminalis; DBN, nucleus of the diagonal band of Broca; DCx, dorsal cortex; DLA, dorsolateral amygdaloid nucleus; DMCx, dorsomedial cortex; GP, globus pallidus; LA, lateral amygdala; LCx, lateral cortex; lfb, lateral forebrain bundle; MA, medial amygdala; MCx, medial cortex; mfb, medial forebrain bundle; Naot, nucleus of the accessory olfactory tract; NS, nucleus sphericus; PDVR, posterior dorsal ventricular ridge; PT, pallial thickening; PVA, periventricular anterior nucleus; RC, retrochiasmatic area; S, septum; SAT, striato-amygdaloid transition area; St, striatum; VPA, ventral posterior amygdala.
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DAVIES ET AL. not prevent the TI response. Similarly, transection of the rabbit brain behind the thalamus leave the TI response intact [2]. These results suggest that the principal control centre for the TI response is located in the brainstem or spinal cord. However, telencephalic ablation in chickens results in an increase in the duration of TI [10], suggesting that ‘higher’ centres exert an inhibitory influence on TI. Klemm [18] proposed that the limbic system might potentiate the TI response by inhibition of the neocortex. This view is supported by the result of the current study that bilateral ablation of the SAT in Podarcis reduced the duration of the TI response. However, the neural pathways subserving the interaction between brainstem and suprabulbar centres await elucidation. ACKNOWLEDGEMENTS
This work was supported by the Acciones Integradas Hispano-Brita´ nicas (HB-1997-0211) and the Conselleria de Educacio´ i Cultura Vale`nciana (GV00-161-05). We are grateful to Professor Martin Bland for advice about the statistical analysis.
FIG. 3. A bar diagram showing the effects of striato-amygdaloid transition area (SAT) and dorsal cortex (DCx) lesions given after trial 1, on the duration of tonic immobility in trial 2. Number is given within each bar and bars bearing the same letter are significantly different from each other (p ⱕ 0.01 for all comparisons). Error lines indicate ⫾ SEM.
DISCUSSION Bilateral lesions of the SAT significantly reduced the duration of TI in trial 2 compared to that in the trial 1. This effect was not present in untreated controls or lizards with DCx lesions. Therefore, the reduction in TI duration in SAT-lesioned lizards is unlikely to be due to habituation in the second trial or to non-specific brain damage. The fact that neither sex nor season differences were observed in TI duration, justified the pooling of the data for analysis. Lesions of the mammalian amygdala reduce fear and aggressive behaviour and impair avoidance responses [1,4,11,38]. Lesions of the avian archistriatum also result in a reduction of escape/fear motivated behaviour [25,26,29,30]. The archistriatum is considered to be, at least in part, homologous with the amygdala [3,39]. Similarly, large lesions of the ‘amygdaloid’ region of the posterior dorsal ventricular ridge have also been reported to abolish aggressive behaviour in Sceloporus occidentalis [36] and reduce attack and flight responses in Caiman crocodilus [17]. Because procedures that reduce fear levels also reduce TI duration [7,14] and SAT lesions reduce TI duration, it is reasonable to conclude that the effect of SAT lesions is due to a reduction in fear. Thus, the results of the current study provide the first functional evidence and reinforce the neuroanatomical evidence [19,33] for a homology between the SAT and the amygdala. The results of experiments on mammals suggest that the amygdala is not only involved in the expression of fear, but also in the acquisition of conditioned fear [5]. The domestic chick archistriatum is also involved in learning with an avoidance component [23,24]. There appears to be an interaction between the expression of innate fear and conditioned fear, since after exposure to a fear-conditioning paradigm, both young chickens [9] and adult guinea pigs [21] exhibit an increased duration of TI. However, whether the lizard SAT is involved in fear conditioning in a similar way to the central nucleus of the rat amygdala [5], remains to be investigated. Brain transection studies of frogs [27] have revealed that coronal cuts just posterior to the anterior border of the cerebellum do
REFERENCES 1. Blanchard, D. C.; Blanchard, R. J. Innate and conditioned responses to threat in rats with amygdaloid lesions. J. Comp. Physiol. Psychol. 81:281–290; 1972. 2. Carli, G. Sub-cortical mechanisms of rabbit hypnosis. Arch. Ital. Biol. 109:15–26; 1971. 3. Davies, D. C.; Csillag, A.; Sze´ kely, A. D.; Kabai, P. The efferent connections of the chick archistriatum. A phaseolus lectin anterograde labelling study. J. Comp. Neurol. 389:679 – 693; 1997. 4. Davis, M. The role of the amygdala in fear and anxiety. Annu. Rev. Neurosci. 15:353–375; 1992. 5. Davis, M. The role of the amygdala in conditioned fear. In: Aggleton, J. P., ed. The amygdala: Neurobiological aspects of emotion, memory and mental dysfunction. New York: Wiley-Liss; 1992:255–305. 6. Gilman, T. T.; Marcuse, F. L. Animal hypnosis. Psychol. Bull. 46: 151–165; 1949. 7. Gallup, G. G. Jr. Tonic immobility: The role of fear and predation. Psychol. Rec. 27:41– 61; 1977. 8. Gallup, G. G. Jr. Tonic immobility as a measure of fear in domestic fowl. Anim. Behav. 20:166 –169; 1979. 9. Gallup, G. G. Jr.; Rosen, T. S.; Brown, C. W. Effect of conditioned fear on tonic immobility in domestic chicks. J. Comp. Physiol. Psychol. 78:22–25; 1972. 10. Gentle, M. L.; Jones, R. B.; Maguire, S. Telencephalic removal and tonic immobility in the domestic hen (Gallus domesticus). Behav. Proc. 10:265–271; 1985. 11. Goddard, G. V. Functions of the amygdala. Psychol. Bull. 62:89 –109; 1964. 12. Henning, C. W.; Dunlap, W. P. Circadian rhythms and the effects of lighting on tonic immobility in two species of lizard (Anolis carolinensis and Hemidactylus turcicus). Behav. Biol. 20:523–528; 1977. 13. Henning, C. W.; Dunlap, W. P.; Harston, C. T. Tonic immobility and skin colour in anoles: Effects of serotonin precursors and metabolites. Physiol. Behav. 22:1079 –1088; 1979. 14. Jones, R. B. The tonic immobility reaction of the domestic fowl: A review. Worlds Poultry Sci. J. 42:82–96; 1986. 15. Jones, R. B. The assessment of fear in adult laying hens: Correlational analysis of methods and measures. Br. Poultry Sci. 28:319 –326; 1987. 16. Jones, R. B.; Mills, A. D. Estimation of fear in two lines of the domestic chick: Correlations between various methods. Behav. Proc. 8:243–253; 1983. 17. Keating, E. G.; Korman, L. A.; Horel, J. A. The behavioral effects of stimulating and ablating the reptilian amygdala. Physiol. Behav. 5:55– 59; 1970. 18. Klemm, W. R. Neurophysiologic studies of the immobility reflex (“animal hypnosis”). In: Ehrenpreis, S.; Solnitzky, O. C., eds. Neuroscience research, vol 4. New York: Academic Press; 1971:165–212. 19. Lanuza, E.; Font, C.; Martı´nez-Marcos, A.; Martı´nez-Garcı´a, F. Amygdalo-hypothalamic projections in the lizard Podarcis hispanica:
AMYGDALA LESIONS REDUCE TONIC IMMOBILITY
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PII S0361-9230(01)00687-6
Striato-amygdaloid transition area lesions reduce the duration of tonic immobility in the lizard Podarcis hispanica D. C. Davies,1* F. Martı´nez-Garcı´a,2 E. Lanuza3 and A. Novejarque2 1
Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, London, UK; Departments of 2Biologia Animal; and 3Celular, Universitat de Vale`ncia, Burjassot, Spain
ABSTRACT: Neuroanatomical data suggest that the lizard striato-amygdaloid transition area is homologous with the mammalian central amygdala. In order to investigate possible functional similarities, tonic immobility was induced in adult lizards and its duration recorded. Each lizard was then randomly assigned to one of three treatments: (1) bilateral striatoamygdaloid transition area lesions, (2) bilateral dorsal cortex lesions or (3) untreated controls. Three days after trial 1, each lizard was subjected to a second trial and the tonic immobility duration recorded. The mean tonic immobility duration in lizards with striato-amygdaloid transition area lesions was significantly shorter (80.5%; p < 0.0033) in trial 2 than in trial 1. There were no inter-trial differences within dorsal cortex-lesioned lizards or untreated controls. There was a significant treatment effect on tonic immobility duration in trial 2 (p < 0.0001). The mean tonic immobility duration of lizards with striato-amygdaloid transition area lesions was significantly shorter than that of dorsal cortex-lesioned lizards (72.2%; p < 0.01) or untreated controls (78.2%; p < 0.01). There was no significant difference in mean tonic immobility duration between dorsal cortex-lesioned lizards and untreated controls. Tonic immobility is considered to be an anti-predator behaviour that reflects the underlying state of fear. Therefore, the reduced tonic immobility duration in lizards with striato-amygdaloid transition area lesions reflects a reduction of fear. These results provide the first data to indicate a functional similarity between the lizard striato-amygdaloid transition area and the mammalian central amygdala. © 2002 Elsevier Science Inc.
a variety of other tests [15,16,28]. The duration of TI therefore appears to be a reliable measure of an animal’s underlying state of fear [8,14]. There is considerable evidence to suggest that the mammalian amygdala plays a role in fear behaviour [20] and there is also evidence that the amygdala is involved in the control of the TI response [22]. Tonic immobility occurs in a wide range of vertebrate species, including reptiles [32]. A variety of lizards have been demonstrated to exhibit the TI response, including Iguana iguana [31], Anolis carolinensis [12,13,35] and Hemidactylus turcicus [12]. Little is known about the telencephalic structures involved in the TI response in lizards. Neuroanatomical data suggest that the striato-amygdaloid transition area (SAT) of the lizard telencephalon is homologous with the mammalian central amygdala [19,33]. However, there is little functional evidence for a role of the SAT in fear behaviour to support this suggestion. Therefore, as a first step towards elucidating its function, the role of the SAT in TI was investigated by means of a lesion study. MATERIALS AND METHODS Adult lizards (Podarcis hispanica) of either sex ranging from 45–58 mm in snout-cloaca length, were captured in Vale`ncia during the months of February/March and October. They were communally housed in terraria heated by lamps, and food and water were available ad libitum. Throughout the experimental work, the animals were treated according to the European Communities Council Directive of November 24, 1986 (86/609/EEC). Each lizard was removed from its home terrarium and taken to a neighbouring quiet room, where TI was induced by placing the animal on its back on a flat surface and applying pressure to the thorax and pelvis, restraining the limbs (Fig. 1). When the lizard ceased struggling, it was slowly released and the time taken for it to resume an upright posture was recorded. If the lizard was still immobile after 900 s, the trial was terminated and a TI duration of 900 s recorded. The lizard was then randomly assigned to one of three experimental groups: (1) lizards that received bilateral SAT lesions (n ⫽ 9), (2) lizards that received bilateral dorsal cortex (DCx) lesions (n ⫽ 9), and (3) untreated controls (n ⫽ 9), coded and returned to its home terrarium. Soon after the first induction of TI, lizards that were to receive lesions were anaesthetised by intramuscular injection of ketamine
KEY WORDS: Amygdala, Amniote vertebrates, Archistriatum, Dorsal cortex, Fear.
INTRODUCTION Tonic immobility (TI) is a reversible state of motor inhibition that is triggered by repetitive stimulation, pressure on body parts, inversion, and restraint [6]. It is considered to be the last line of defence in a sequence of anti-predator behaviours [32]. The TI response is thought to be of great adaptive value because it can result in a predator losing interest in its prey [5,34,37]. Tonic immobility is considered to be positively related to fear since procedures designed to increase fears increase the length of the response, whereas fear-reducers shorten it [5,14]. Moreover, there is a close association between TI duration and measures of fear in
* Address for correspondence: Dr. D.C. Davies, Department of Anatomy and Developmental Biology, Cranmer Terrace, Tooting, London SW17 0RE, UK. Fax: ⫹44-(0)20-8725-3326; E-mail: [email protected]
537
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DAVIES ET AL. (3.25 mg/kg; Parke-Davis, El Prat de Llobregat, Spain) and placed in a small animal stereotaxic apparatus. The cranium was then surgically exposed, a hole drilled through it and the dura mater incised. A stainless steel electrode (0.3 mm in diameter) with a lacquer-insulated shaft was then lowered through the leptomeninges into the telencephalon. Electrolytic lesions were then made by passing a radio frequency current of 6 mA through the uninsulated tip (0.25 mm) of the electrode for 30 s. Lesions were placed in both hemispheres. Taking the anterior vertex of the pineal scale as zero, the SAT co-ordinates were 0.2 mm rostral, 0.8 mm lateral to the midline, and 0.9 mm below the surface of the brain. The DCx co-ordinates were similar, except that the lesions were placed 0.2 mm below the surface of the brain. After the lesions were completed, the entry wounds were treated with Veterin-Fenicol (chloramphenicol and lidocaine, Hoechst-Roussel, Barcelona, Spain), closed with Biocatalase (Fournier Labs., Tres Cantos, Madrid, Spain) and the lizards were returned to their home terraria. Three days after the initial induction of TI, each lizard was subjected to a second trial and the duration of TI was recorded. At the end of this trial, the lesioned animals were given a lethal dose of anaesthetic and perfused through the heart with 4% paraformaldehyde in phosphate buffered (0.1 M, pH 7.6) physiological saline. The brains were then removed and stored in similar fixative, before serial coronal sections (40 m) were cut in a cryostat. Every third section was collected onto glass slides, stained with toluidine blue and viewed in a light microscope. The sections were then drawn and the extent of the lesion sites recorded with the aid of a camera lucida drawing attachment. Brains that did not contain appropriate bilateral lesions of the SAT or DCx were excluded from the experiment. The codes were then broken and the behavioural data united with experimental treatment for each lizard. The effect of trial number on the duration of TI for each treatment was investigated by means of paired t-tests. The effect of treatment on TI duration in the second trial was investigated using analysis of covariance adjusting for TI duration in the first trial. Multiple comparisons were then performed between pairs of treatment groups using the Newman-Keul’s Studentized range test. Two-sample t-tests were used to investigate the effects of sex and season on TI duration in the first trial. RESULTS
FIG. 1. The stages of tonic immobility in Podarcis hispanica. (A) The lizard is placed on its back and pressure applied to the thorax and pelvis, restraining the limbs and tail. (B) Once the lizard stops struggling, pressure and restraint is slowly removed. (C) Tonic immobility. (D) The end of tonic immobility is typically signalled by head movement and torsion of the upper trunk, prior to resumption of an upright posture.
Striato-amygdaloid transition area lesions frequently encroached upon the overlying dorsal ventricular ridge and occasionally the DCx. Because some of the DCx lesions also impinged upon the underlying dorsal ventricular ridge, the effect of SAT lesions can be attributed to damage to deeper structures, predominantly the SAT. The largest and smallest SAT and DCx lesions are illustrated in Fig. 2. In lizards that had received SAT lesions, the duration of TI was significantly lower (80.5%) in trial 2 than in trial 1 (t ⫽ 4.12295; p ⬍ 0.0033). There were no inter-trial differences in TI duration in either untreated control or DCx lesion groups (Fig. 3). Analysis of covariance adjusting for TI duration in the first trial, revealed a significant effect of treatment on TI duration in the second trial [F(2,23) ⫽ 15.81; p ⬍ 0.0001]. Breaking down the data, the duration of TI in SAT lizards in trial 2 was significantly shorter than that of untreated control (78.5%, p ⬍ 0.01) and DCx (72.2%, p ⬍ 0.01) lizards (Fig. 3). There was no significant effect of sex or season on the TI duration of lizards in trial 1. The mean TI duration of males (N ⫽ 14) was 529.5 s ⫾ 105.3 SEM and that of females (N ⫽ 13) was 523.5 s ⫾ 107.8 SEM. The mean TI duration in February/March (N ⫽ 15) was 540.1 s ⫾ 104.0 SEM and that in October (N ⫽ 12) was 509.8 s ⫾ 108.5 SEM.
AMYGDALA LESIONS REDUCE TONIC IMMOBILITY
FIG. 2. Reconstructions of striato-amygdaloid transition area (SAT) and dorsal cortex (DCx) lesions. The largest and smallest lesions in each group are indicated by light and dark stippling respectively. The levels of the sections are indicated on the drawing of the lateral view of the brain. Scale bar: 400 m. Abbreviations: ac, anterior commissure; Acc, nucleus accumbens; ADVR, anterior dorsal ventricular ridge; aot, accessory olfactory tract; BNST, bed nucleus of the stria terminalis; DBN, nucleus of the diagonal band of Broca; DCx, dorsal cortex; DLA, dorsolateral amygdaloid nucleus; DMCx, dorsomedial cortex; GP, globus pallidus; LA, lateral amygdala; LCx, lateral cortex; lfb, lateral forebrain bundle; MA, medial amygdala; MCx, medial cortex; mfb, medial forebrain bundle; Naot, nucleus of the accessory olfactory tract; NS, nucleus sphericus; PDVR, posterior dorsal ventricular ridge; PT, pallial thickening; PVA, periventricular anterior nucleus; RC, retrochiasmatic area; S, septum; SAT, striato-amygdaloid transition area; St, striatum; VPA, ventral posterior amygdala.
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DAVIES ET AL. not prevent the TI response. Similarly, transection of the rabbit brain behind the thalamus leave the TI response intact [2]. These results suggest that the principal control centre for the TI response is located in the brainstem or spinal cord. However, telencephalic ablation in chickens results in an increase in the duration of TI [10], suggesting that ‘higher’ centres exert an inhibitory influence on TI. Klemm [18] proposed that the limbic system might potentiate the TI response by inhibition of the neocortex. This view is supported by the result of the current study that bilateral ablation of the SAT in Podarcis reduced the duration of the TI response. However, the neural pathways subserving the interaction between brainstem and suprabulbar centres await elucidation. ACKNOWLEDGEMENTS
This work was supported by the Acciones Integradas Hispano-Brita´ nicas (HB-1997-0211) and the Conselleria de Educacio´ i Cultura Vale`nciana (GV00-161-05). We are grateful to Professor Martin Bland for advice about the statistical analysis.
FIG. 3. A bar diagram showing the effects of striato-amygdaloid transition area (SAT) and dorsal cortex (DCx) lesions given after trial 1, on the duration of tonic immobility in trial 2. Number is given within each bar and bars bearing the same letter are significantly different from each other (p ⱕ 0.01 for all comparisons). Error lines indicate ⫾ SEM.
DISCUSSION Bilateral lesions of the SAT significantly reduced the duration of TI in trial 2 compared to that in the trial 1. This effect was not present in untreated controls or lizards with DCx lesions. Therefore, the reduction in TI duration in SAT-lesioned lizards is unlikely to be due to habituation in the second trial or to non-specific brain damage. The fact that neither sex nor season differences were observed in TI duration, justified the pooling of the data for analysis. Lesions of the mammalian amygdala reduce fear and aggressive behaviour and impair avoidance responses [1,4,11,38]. Lesions of the avian archistriatum also result in a reduction of escape/fear motivated behaviour [25,26,29,30]. The archistriatum is considered to be, at least in part, homologous with the amygdala [3,39]. Similarly, large lesions of the ‘amygdaloid’ region of the posterior dorsal ventricular ridge have also been reported to abolish aggressive behaviour in Sceloporus occidentalis [36] and reduce attack and flight responses in Caiman crocodilus [17]. Because procedures that reduce fear levels also reduce TI duration [7,14] and SAT lesions reduce TI duration, it is reasonable to conclude that the effect of SAT lesions is due to a reduction in fear. Thus, the results of the current study provide the first functional evidence and reinforce the neuroanatomical evidence [19,33] for a homology between the SAT and the amygdala. The results of experiments on mammals suggest that the amygdala is not only involved in the expression of fear, but also in the acquisition of conditioned fear [5]. The domestic chick archistriatum is also involved in learning with an avoidance component [23,24]. There appears to be an interaction between the expression of innate fear and conditioned fear, since after exposure to a fear-conditioning paradigm, both young chickens [9] and adult guinea pigs [21] exhibit an increased duration of TI. However, whether the lizard SAT is involved in fear conditioning in a similar way to the central nucleus of the rat amygdala [5], remains to be investigated. Brain transection studies of frogs [27] have revealed that coronal cuts just posterior to the anterior border of the cerebellum do
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