Radley, J. J. et al. J. Neurosci. 2006;26:12967-12976

Regional Differentiation of the Medial Prefrontal Cortex in Regulating Adaptive

Responses to Acute Emotional Stress

Radley, Arias, SawchenkoSalk Institute, La Jolla CaliforniaJournal of Neuroscience 2006

Sawchenko?

• mPFC:– Processing cognitive and emotional info– Modulation attention states– Regulating stress response

• Influence on HPA axis• Autonomic (sympathoadrenal)

Intro

• HPA (hypothalamic-pituitary-adrenal) axis– Paraventricular nucleus (PVN or PVH)

• CRF neurosecretory neurons – ACTH release from anterior Pituitary– Corticosterone from adrenal cortex

• Projections to CNS cell groups involved in autonomic– Sympathetic preganglionic neurons (ACh release)– Stimulates adrenaline and noradrenalin from adrenal medulla

Intro.

Regional specialization?

• mPFC lesion = inhibitory control of HPA – Lesions mainly dorsal; prelimbic (PL) (as opposed

to infralimbic IL)– Large lesions may alter basal, but not stress

induced HPA response in rats– IL may preferentially modulate autonomic– Suggestions that PL and IL differ in both nature of

influence and underlying circuitry

Schematic of mPFC regions

• Current study compares:– Dorsal vs. ventral (mPFCd vs mPFCv) lesions– Acute restraint stress

– Hormonal (B & ACTH)– Histochemical (fos, CRF mRNA)

Rats and Drugs

• Adult male s.dawley 275-325g, indvidually housed, free access to food and H2o

• Bilateral excitotoxic lesions (ibotenic acid) into either v or d mPFC, with a sham surgery group

• 14 day recovery; 30 min restraint stress in plastic tubes, returned to cage for 2 hours

• All animals were terminated within the same time period of the day

Lesions

• Amanita muscaria and Amanita pantherina

• 10 mg/ml ibotenic acid in saline (60-90 nL per side were injected)

• Extent of lesion reconstructed from Nissel stained slides

Labeling of structures

• To measure extent of stress induced Fos immunoreactivity– Fast blue crystals into rostral ventrolateral

medulla to be transported by axons of passage to provide maximal labeling of PVH pre-autonomic cell groups projecting to both dorsal vagal complex and preganglionic neurons in spinal cord

– Fast blue fluid in T1-T2 level of spinal cord – Tracer injected during lesion surgery – Dual IR for fos and fast blue

CRF mRNA

• In situ hybridization • Probe built for CRF identification • Allows visualization of mRNA in very small

amounts, localized to individual cells (as little as 10-20 mRNA copies per cell)

Hormone assays

• In-dwelling jugular catheters implanted 2d before stress exposure (12 d following surgery)– Blood samples collected before stress and again

immediately following stress and 30, 60 and 90 mins after

– B and ACTH measured using RIA kit

Lesion placementAcd = dorsal anterior cingulate

Figure 2. Rostrocaudal extent of mPFCd lesion placements

Figure 2. Rostrocaudal extent of mPFCv lesion placements

Lesion effect on PVH activation following stress

Dp= dorsal parvicellularMpd= hypophysiotropic Mpv= medial parvicellularPm= posterior magnoceullular

Mpd is richest in CRF cell bodies

Stress = sig increased Fos in mpd, dp and mpvFigure 3. sham control vs sham stress

Dorsal lesion = increase Fos in Mpd Ventral lesion attenuates Mpd increase, while increasing Fos in dp and mpv

Ventral lesion is similar to sham lesion, both higher than controls

Lesion effects on PVH activational responses to acute restraint stress• mPFCd lesions

– Overt enhancement

• mPFCv – Mild attenuation– Tendency toward increased response in

preautonomic cell groups (dorsal, Vmedial)

• Of stress induced Fos-IR in the PVH mpd region

What the fos?

• Effects of mPFC lesions on restraint-induced CRF mRNA expression– Directly related to HPA activity

• Stress increases mpd mPFC CRF mRNA 46% compared to unstressed controls

• mPFCd lesion enhanced this effect by 34% • mPFCv lesion n.s. diff compared to sham

stress and no stress groups

Figure 4. Effects of mPFC lesions on acute restraint-induced CRF mRNA expression in the PVH

Figure 4. Effects of mPFC lesions on acute restraint-induced CRF mRNA expression in the PVH

What about blood?

• HPA secretory response to 30 min restraint• ACTH response:

– SHAM- stress = sig increase in ACTH– mPFCv lesion = no increase compared to basal– mPFCd lesion = sig increase compared to basal,

sham and mPFCv lesion

Figure 5. Effects of mPFC lesions on ACTH response to acute restraint

697 +- 164 pg/mL

267 +- 84 pg/mL

CORT

• Similar basal levels of cort among groups• Similar elevation following stress (671-792 pg)• BUT…..• mPFCd lesion remains elevated from baseline

for 90 mins post stress• mPFCv lesion returns to baseline faster than

sham and d lesion groups

Figure 5. Effects of mPFC lesions on CORT response to acute restraint

Whats the deal with mPFCv

• mPFCv involvment in stress-induced HPA output is subtle compared to mPFCd – mPFCv may preferentially modulate autonomic

outputs – Tracer injections into 2 locations

• Rostral ventrolateral medulla• Upper thoracic T1-T2

– Stress, then look for double labeling

Figure 6. Acute restraint stress increases activation of preautonomic PVH after mPFCv lesions

VLM

Stress groupBrown = fast blueBlack = fos

Figure 6. Acute restraint stress increases activation of preautonomic PVH after mPFCv lesions

Dual labelling indicates stress-sensitive preautonomic neurons, and they are localized primarily in dorsal, ventral and lateral parvicellular

Also injected tracer into T1-T2 thoracic spinal cord to more specifically label PVH outputs relevant to sympathetic control. Stress + mPFCv lesion in this groupled to 63% more dbl labeled neurons in PVH

Discussion: talk freely amongst yourselves

• Support regional differentiation of mPFC within its capacity to modulate stress-related PVH outputs

• Stress induced HPA activity was greater following mPFCd lesion, suppressed w/ mPFCv

• mPFCv lesion selectively enhanced stress-induced recruitment of PVH pre-autonomic neurons

• Limitations and difficulties comparing studies– Lesion placement, extent, stressor, duration,

methods used to characterize HPA response, etc

• mPFCv or IL: projects to brainstem cell groups involved in central autonomic control, including NTS (primary central terminus of inputs carried by the vagus and glossopharyngeal nerves)

• PVH innervates both NTS and motor nuclei of vagus and glossopharyngeal nerves

Fight or flight…

• This innervation pattern puts IL in a position to modulate concurrently both cardiac and adrenal medullary activity– “command neurons” for the F or F response

Peri-PVH

• Diencephalic mPFC projections distribute near the PVH, but not within the nucleus proper– Peri-PVH regions are rich in GABA interneurons

• Inhibitory control over HPA response

– mPFC projections are primarily excitatory (glu) • while its effects on HPA is inhibitory

Figure 7. Separate pathways from mPFC may differentially regulate PVH responses to stress

• mPFCd inhibitory mPFCv facilitates HPA• d/v involvment in autonomic output does not

adhere to this scheme

• pPVT (posterior para-vent nuc of thalamus) – Implicated in habituation and facilitation

• Hab: repeated exposure to same stressor = response• Fac: exaggerated response to novel challenge

– pPVT-mPFC connection targets IL preferentially• Implications for chronic stress adaptation

Post Traumatic Stress Disorder

• PTSD patients: functional impairment and shrinkage of mPFC– Correlated with dendritic atrophy and synapse

loss following chronic emotional stress in rodents

• PTSD: HPA axis dysregulation and consistent increases in cardiovascular reactivity

• In the future• Finer grained analysis of mPFC should foster

clarification of functional circuits underlying stress adaptation, and their involvement in affective disorders

This is the end.