Jeffrey B. Rosen, Robert E. Adamec, Barbara L. Thompson

1. Expression of egr1 (zif268) mRNA in select fear-related brain regions following exposure to a predator Jeffrey B. Rosen, Robert E. Adamec, Barbara L. Thompson

2. Neuroanatomical Circuit Of Fear J.B. Rosen, M.P. Donley / Biological Psychology 73 (2006) 49–60

3. Immediate-Early Genes • Expressed within minutes of an environmental change • Induced rapidly • mRNA expression returns to baseline (typically) within one hour • Includes: • Egr1 = zif268 = zenk = ngfia = krox24 = tis8 • fos, arc, jun, J.B. Rosen et al. / Behavioural Brain Research 162 (2005) 279–288

4. Conditioned vs. Unconditioned Fear Paradigms • Can be derived from and used in animal’s native environment • Unconditioned fear stimuli derived from the animal’s natural history better represent natural conditions and responses • Responses the same, stimuli different • Unconditioned paradigms do not rely on learning and memory

5. Previous Lesion Studies(small lesion/chemical inactivation) • Basal, lateral, or central nuclei of the amygdala • No major effects on unconditioned freezing • Severely disrupt fear conditioned responses

6. c-fos • activated in several amygdala nuclei and periaqueductal gray with fear conditioning and fear learning memory retention tests (some studies did not find this result) • expression not increased in lateral or basal nuclei after exposure to live cat or predator odors, but is increased in periaqueductal gray

7. Egr1 • Increases in response to fear in lateral nucleus of amygdala • Whether due to stress or part of fear conditioning/learning not known

8. Aims • Examine expression of egr-1 after predator/predator odor exposure • Lateral nucleus of amygdala, paraventricular nucleus of hypothalamus, periaqueductal gray and sensory cortex

9. Methods • 30 male Long-Evans rats (60 days old) • Divided into three groups (n=10 each) • Handled • Confined • Cat exposed

10. Test Day • Handled group • handled for one minute • Confined group • confined for 5 minutes • Cat exposed group • placed in chamber with cat for 5 minutes • All behaviors videotaped for later analysis

11. In Situ Hybridization • Four brain sections per brain area (per animal) • Egr-1 areas labeled and density measured • Dorsolateral portion of lateral nucleus of amygdala, paraventricular nucleus of hypothalamus, periaqueductal gray, and visual and somatosensory cortices

12. Results - Behavior • Cat exposed rats spent 114 +/- 15s in defensive immobile posture • Confined rats spent 189 +/- 16s in immobile position

15. Results - PVN • Combined control (handled and confined) vs. cat exposure showed significant difference in egr-1 mRNA levels

16. Fig. 2. Mean ± S.E.M. egr1 mRNA levels in the paraventricular nucleus of the hypothalamus. Expression levels in the cat-exposed group differed from a combined handled and confined group (p < 0.04).

17. Fig. 3. Representative images of egr1mRNA in the paraventricular nucleus of the hypothalamus (PVN), periaqueductal gray (PAG), and visual cortex of handled, confined and cat-exposed rats. V, ventricle; A, aqueduct. The digitized images are reversed to dark field and the contrast increased for demonstration purposes.

18. Discussion - PVN • Egr1levels in confined group not elevated over handled group • Due to habituation to chamber? • Exposure to cat more stressful than confinement chamber

19. Results - Amygdala • Egr1analyzed only in dorsolateral division of the lateral nucleus of amygdala (dlLA) • No significant difference found between groups • Appears cat exposure did not induce egr1 mRNA expression compared to controls

20. Fig. 4. Mean ± S.E.M. egr1 mRNA levels in the dorsolateral division of the lateral nucleus of the amygdala. There were no statistical differences between any of the groups.

21. Discussion - Amygdala • Previous fear conditioning studies show increased egr-1 in LaDL • Cat exposure did not induce egr-1 expression in LaDL • Egr-1 involved in transcriptional processes during fear conditioning, but not unconditioned fear of a predator • Using egr-1 antisense oligodeoxynucleotide and reducing egr-1 levels blocks long term memory of conditioned fear but not unconditioned freezing to predator odor

22. Results - Periacueductal Gray • Analyzed in dorsal and lateral aspects of PAG • Differences between right and left sides of lateral PAG analyzed • No differences in dorsal or lateral PAG • Significant interaction of group by side of the PAG

24. Discussion - PAG • PAG important in both active escape (dorsolateral) and passive immobile responses (ventrolateral) to fear and pain • No change in egr-1 expression in dorsal PAG • Probably not appropriate marker in PAG

26. Results - Visual and Somatosensory Cortex • Cat exposure induced increased egr-1 expression in primary and secondary visual cortices • Shown with 2 outliers removed • Using combined control groups • Egr-1 not expressed in somatosensory cortex

27. Conclusions • Egr-1 and c-fos expressionfollowing predator/predator odor exposure suggest overlapping conditioned/unconditioned fear circuits • Amygdala nuclei in conditioned fear not in unconditioned fear • Lesions to medial nucleus interfere with unconditioned fear • Medial nucleus does not appear to be involved in fear conditioning