1. Expression of egr 1 (zif 268 ) 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: –Egr 1 = zif 268 = zenk = ngfi a = krox 24 = tis 8 –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. Egr 1 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. egr 1 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 egr 1 mRNA 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 Egr 1 levels in confined group not elevated over handled group –Due to habituation to chamber? Exposure to cat more stressful than confinement chamber

19. Results - Amygdala Egr 1 analyzed only in dorsolateral division of the lateral nucleus of amygdala (dlLA) No significant difference found between groups Appears cat exposure did not induce egr 1 mRNA expression compared to controls

20. Fig. 4. Mean ± S.E.M. egr 1 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 expression following 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