The consequences of uncontrollable stress are sensitive to duration of

prior wheel running

Benjamin N. Greenwood, Teresa E. Foley, Dan Burhans, Steven F. Maier, Monika Fleshner

• Chronic exposure to uncontrollable stressors (UcS)– behavioral depression– learned helplessness

(LdH)

LdH

• LdH– psychological condition in which a human being/animal

has learned to believe that a situation is helpless– tail shock

• impaired escape performance• exaggerated fear conditioning

• Prevented/reversed by – Antidepressant/anxiolytic drugs– Physical activity

• 6+ weeks of voluntary access to running wheels

Prior works

• Activation of 5-HT neurons in the DRN is both necessary and sufficient to produce LH behaviors.

• dorsal raphé nucleus (DRN) and serotonin (5-HT) play a role in – stress-related affective disorders (depression/anxiety)– pharmacological action of antidepressant and

anxiolytic drugs– the modulation of the behavioral effects of

uncontrollable stressors

Stress

Controllable• Increase in 5-HT activity in

the DRN – involved in protection against

LH provided by wheel running– 5-HT neurons in the DRN can

be hyper-activated and sensitized

uncontrollable• greater c-Fos induction in the

DRN 5-HT neurons – exaggerated 5-HT efflux

(during stress and later behavioral testing)• within the DRN• in DRN projection sites

• manipulations that decrease 5-HT neural activity in the DRN during uncontrollable stress eliminate LH behaviors

c-Fos• … is a cellular proto-oncogene belonging to the immediate

early gene family of transcription factors.

• …transcription is upregulated in response to many extracellular signals, e.g. growth factors.

• …phosphorylation by MAPK, PKA, PKC or cdc2 alters the activity and stability of c-Fos.

• …dimerises with Jun to form the AP-1 transcription factor, – which upregulates transcription of a diverse range of genes

involved in everything from proliferation and differentiation to defense against invasion and cell damage.

corticotropin-releasing hormone (CRH)

• Release is influenced by – stress– cortisol – sleep/wake cycle

• Acts as a NT and a hormone– CRHRs in the …

• Central nucleus of the amygdala(CeA) • Bed nucleus of the stria terminalis (BNST)

5-HT & SSRIs• citalopram

– (Celexa, Cipramil, Emocal, Sepram, Seropram)

• escitalopram oxalate – (Lexapro, Cipralex, Esertia)

• fluoxetine – (Prozac, Fontex, Seromex, Seronil,

Sarafem, Fluctin (EUR)) • fluvoxamine maleate

– (Luvox, Faverin) • paroxetine

– (Paxil, Seroxat, Aropax, Deroxat, Rexetin, Xetanor, Paroxat)

• sertraline – (Zoloft, Lustral, Serlain)

CRH

• …is critical for mediation of behavioral consequences of UcS

• …has excitatory influences on 5-HT neurons located in the caudal DRN (dorsal raphé nucleus)– Caudal DRN CRH-R2 ant. injection prevent LdH

– No effect if injected rostral

• LdH behaviors are dependent upon hyperactivity of DRN 5-HT neurons during exposure to uncontrollable stress.– 6 weeks of wheel running prevents LdH and attenuates

uncontrollable stress-induced activity of DRN 5-HT neurons.

• Possible reasons:– 5-HT1A (autoreceptor)

• Increases in 5-HT1A inhibition can result in increased DRN 5-HT activity

– Other regions• Regions afferent to DRN could play their own game

• Physically active and sedentary rats have equal corticosterone responses during exposure to uncontrollable tail shock

• c-Fos is not globally attenuated in all stress reactive brain regions

Candidates regions

• Intra-DRN microinjections of CRH-R2/EAA antagonists prevent symptoms of UcS

• CRH and NMDA agonists can influence DRN 5-HT neurons– Afferent regions containing

• CRH• EAAs (excitatory amino acids) [eg. Glutamate or aspartate]

BNST• Activation of BNST

– produces neuroendocrine and behavioral responses that resemble those produced by stress

– inactivation of the BNST decreases neuroendocrine and behavioral responses to stress

• Lesion:– eliminate typical

behavioral conseq. of UcS

BLA & CeA• Basolateral amygdala (BLA):

– Info about environmental signals are associated w/ aversive unconditioned stimuli in the BLA

• CeA– stimulation produces

behavioral, cardiovascular and neuroendocrine components of fear

• Lesions– reduce or eliminate fear

LHb

• Electrical stimulation of the LHb excites DRN neurons

• Lesions– eliminate stress-induced

increases in 5-HT in the DRN.

– eliminate typical behavioral conseq. of UcS

• Anxiogenic and fear responses elicited by BNST and AMG stimulation are similar to stimulation of the DRN

• Take them out!

Goals

• effects of 3 weeks and 6 weeks of wheel running on LdH behaviors and uncontrollable stress-induced – c-Fos in the BNST, AMG and LHb– 5-HT & c-Fos in the DRN

Animals

• Adult, male Fischer F344 rats – 12:12 h light/dark cycle

(lights on 6am–6pm)– individually housed with

attached running wheel– 2 weeks acclimatization

(locked wheels)

Activity

2 groups

• No stress (home cage)• Stressed (tail shock)– Plexiglas tube + 100, 5 s tail shocks (1.5 mA) on a

1-min variable-interval schedule. • (8 am – 10 am (light)

– Returned to home cage

human equivalent

conditioned fear/shuttle box (24h post stress)

1. Freezing (Every 8s): 4 paws on floor + no movement

2. Foot shock: auto off after 30 s

Immuno

• Rats (n = 8/group) – Terminally sampled 90 min after tail shock (c-Fos

optimum)

• 35 m coronal sections (cryostat) of– DRN– BNST– AMG– LHb

c-Fos labeling

c-Fos/5-HT double labeling (DRN)

c-Fos + 5HT 5-HT

c-Fos

c-Fos immunoreactivity

Activity and body weights

Running Distance/week

Escape

Freezing

5-HT

c-Fos

# double labeled

c-Fos expression in the BNST

c-Fos Expression in the AMG

c-Fos Expression in the LHb

Summary

• Run long!– More sensitive to

duration (less to distance)

– Burghardt (12) • + maze

– 8 weekies yes– 4 weekies no

Summary

• Attenuation of 5-HT activity in the DRN contributes to the prevention of LdH by wheel running.

• BNST has a role in mechanism by which wheel running prevents LdH– But not the AMG or LHb