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

1. Regional Differentiation of the Medial Prefrontal Cortex in Regulating Adaptive Responses to Acute Emotional Stress Radley, Arias, Sawchenko Salk Institute, La Jolla California Journal of Neuroscience 2006 Radley, J. J. et al. J. Neurosci. 2006;26:12967-12976

2. Sawchenko?

3. Intro • mPFC: • Processing cognitive and emotional info • Modulation attention states • Regulating stress response • Influence on HPA axis • Autonomic (sympathoadrenal)

4. 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

5. 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

6. Schematic of mPFC regions

7. Current study compares: • Dorsal vs. ventral (mPFCdvsmPFCv) lesions • Acute restraint stress • Hormonal (B & ACTH) • Histochemical (fos, CRF mRNA)

8. 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

9. 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

10. 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

11. 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)

12. 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

13. Lesion placement Acd = dorsal anterior cingulate

15. Figure 2. Rostrocaudal extent of mPFCd lesion placements

16. Figure 2. Rostrocaudal extent of mPFCv lesion placements

17. Lesion effect on PVH activation following stress Dp= dorsal parvicellular Mpd= hypophysiotropic Mpv= medial parvicellular Pm= posterior magnoceullular Mpd is richest in CRF cell bodies

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

19. 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

20. 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

21. 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

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

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

24. 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

25. Figure 5. Effects of mPFC lesions on ACTH response to acute restraint 697 +- 164 pg/mL 267 +- 84 pg/mL

26. 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

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

28. 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

29. Figure 6. Acute restraint stress increases activation of preautonomic PVH after mPFCv lesions VLM Stress group Brown = fast blue Black = fos

30. 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 group led to 63% more dbl labeled neurons in PVH

31. 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

32. 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

33. 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

34. 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

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

36. 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

37. 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

38. In the future • Finer grained analysis of mPFC should foster clarification of functional circuits underlying stress adaptation, and their involvement in affective disorders

39. This is the end.