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

Neuroanatomical Circuit Of Fear

J.B. Rosen, M.P. Donley / Biological Psychology 73 (2006) 49–60

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

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

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

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

Egr1

• Increases in response to fear in lateral nucleus of amygdala– Whether due to stress or part of fear

conditioning/learning not known

Aims

• Examine expression of egr-1 after predator/predator odor exposure– Lateral nucleus of amygdala,

paraventricular nucleus of hypothalamus, periaqueductal gray and sensory cortex

Methods

• 30 male Long-Evans rats (60 days old)

• Divided into three groups (n=10 each)– Handled– Confined– Cat exposed

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

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

Results - Behavior

• Cat exposed rats spent 114 +/- 15s in defensive immobile posture

• Confined rats spent 189 +/- 16s in immobile position

Results - PVN

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

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).

Fig. 3. Representative images of egr1 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.

Discussion - PVN

• Egr1 levels in confined group not elevated over handled group– Due to habituation to chamber?

• Exposure to cat more stressful than confinement chamber

Results - Amygdala

• Egr1 analyzed 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

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.

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

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

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

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

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