Spencer et al 2005 J. Comparative Neurology

BNST Subdivisions

Ac=anterior commAL= anterior lateralAM= anterior medialDL= dorsal lateralIc=internal capsuleJXC=juxtacapsularLV=Lateral ventriclePL=posterior lateralPS=parastrialVL=ventral lateralVM=ventral medial

Hypothalamic Map

Intro

PVN: role in generating adaptive autonomic, behav, & hormonal stress responses

Suppressive inputs are a major target for stress related issues

mPFC: activation generates cardiac depressor responses, inhib influence on sympathetic vasomotor function, role in modulation of PVN….

IntroAnatomical studies fail to show direct projections from mPFC to PVNModulation must involve one or more relay through other regions

Candidate regions: medial region of hypothalamus BNST, Amygdala, paraventricular nucleus of the thalamus (PVT) LS, and brainstem catecholamine groups

All of the candidate regions receive mPFC inputs and have been implicatedin control of the PVN function during stress

Question?Do mPFC lesions that increase PVN responses to a psychological stressor also elicit corresponding changes in activation of any of the candidates?

c-fos activity

Intro

Answer!

mPFC lesion and air puff challenge causes increased c-fos activity in vBNST, no other candidate regions

Further characterize role of BNST in PVN cell response to air puffThink in terms of integration of info from mPFC to PVN in response to stress

Retrograde tracing to determine if BNST directly innervates PVN Direct projections from mPFC to BNST recruited by air puff stress

Examine effects of BNST lesions on recruitment of the PVN cells to air puff stress

Determine specific regions of PVN involved

Methods Adult male Wistar Rats 250-500 g YIKES!!!

Individually housed, 24C 12-12 light cycle, lights on at 6 am

Food and water ad libitum Food intake and body weight recorded throughout

Surgery: Ibotenic acid used for bilateral lesions “Excitotoxicity” Death by glutamate

mPFC lesions covered both prelimbic (PrL) and infralimbic (IL) regions

BNST lesions covered both dorsal and ventral BNST

Vascular cannulae also implanted to allow for easy collection of bloodleft femoral artery cannula, routed through skin up to scapulaeflushed with gentamicin in heparinized saline during recovery

Methods

Retrograde Tracing using the cholera toxin b subunit (CTb) ask me how this works?can be visualized using immunocytochemistry (Ctb-IR)

CTb injected iontophoretically (using electric magic) into:PVN, dBNST, both d and vBNST

6 uA current for 7 seconds on and 7 seconds off, 20 minutes total

Plasma ACTH conc. Determined using one giant Radioimmunoassay

Headshot from an air cannon Series of 27 puffs of 300 kPa pressurized

air aimed at the head of a freely moving animal 5-10cm away

15 minute period Nine blocks, each block with 3 2-second puffs

with 10 seconds between puffs and 1 minute between blocks

2 separate lesions mPFC lesion first BNST lesion

second

Substantial cell loss through all PrL and IL No c-FOS-IR in mPFC of air puff + lesion

animals

Sham Lesion mPFC Lesion

Effects of air puff on sham lesioned animals Air puff = sig in Fos-IR in all regions Post hoc tests on those that sig. interact w/lesion

mPFC both PrL and IL mpPVN CRF cells vBNST

Compared to non-stressed sham lesion control

Similar Fos profile as seen with other psychological stressors (noise, restraint)

Lesion had no effect in non-stressed animals Table 1 sham no stress vs mPFC no stress

Lesion + stress = fos in mpPVN Increase in the number of activated CRF

cells Sig increase in plasma ACTH with stress

No difference between sham or mPFC lesion Lesion + stress = fos in vBNST

Sham+stress = less fos

mPFC lesion + stress = more fos

Right mpPVN

mPFC lesion

Fos in vBNST following mPFC lesion

Sham= less fos

lesion= more fos

http://ngm.nationalgeographic.com/your-shot/top-shots-2008

http://www.sciencemag.org/cgi/content/full/319/5859/101/DC1

BNST lesions vary in how much of the structure they cover

BNST lesion + stress = fos compared to sham lesion + stress group

Some BNST lesion + stress groups still showed high levels of fos and they were excluded from analysis as an incomplete lesion group

BNST sham injection

BNST lesion

No effect of BNST lesion on food intake No effect on body mass

No effect in any region in non stressed groups Table 2 sham no stress vs lesion no stress

Lesion+stress = fos in vBNST,PVN, mPFC, MeA mpPVN showed fos Air puff induces increased ACTH release No sig. attenuation of the ACTH response in the

BNST lesion group, despite the decreased fos expression in the CRF cells of the mpPVN

BNST lesion

N=18: intra-PVN CTb 7d before stress 10 have sig. tracer deposits in mpPVN Of these 10, all showed large numbers of

retrogradely labeled neurons in the vBNST ~14% of these were co-labeled with fos

mpPVN-projecting vBNST cells sensitive to stress

No retrograde labeling in the mPFC

Retrograde labels fromCTb injections into the Mp-PVN

dBNST inj. dBNST + vBNST inj.

mPFC labeling of fos andCtb and co-localizationFrom BNST injections

Labeling only occurred In the v and dBNST group

mPFC lesion enhanced recruitment of mpPVN CRF cells by air puff stress

BNST lesion + stress = decreased fos in all regions studied

mPFC and BNST important in influencing the response to a psychological stress

2 categories of acute, unconditioned stressor Psychological: threat of a disturbance Physical: actual disturbance of tissue integrity

Air cannon = moderate intensity Avoids saturation of stress response Psychological in nature (they think)

<10% total CRF cell activation in mpPVN ~25% of what they see w/ restraint or noise

stress

Air puff (along w/restraint&swim stress) elicited MeA fos activation

Hemorrhage & immune challenge activate CeA

Air puff in BNST activated only ventral portion Immune Challenge activates v and dBNST

Air puff meets criteria of psychological stressor

mPFC lesion increases expression of fos protein by mpPVN CRF cells in response to air cannon Not accompanied by an increase in ACTH Could be due to altered release of other

secretagogues like oxytocin or AVP CRF release could be doing other things

mpPVN CRF also stim. NE synthesis & release in bstem

vBNST = increased fos following airpuff and lesion to mPFC (only candidate region that did)

vBNST gets a direct projection from mPFC, and projects directly to mpPVN Difficult to lesion only vBNST vBNST sends Glu to mpPVN which could be

a direct stimulation following a stressor

Direct projection from mPFC to ipsalateral vBNST (but not dBNST) However, vBNST tracer injections did not

retrogradely label any mPFC neurons…… May be another brain region involved

Psychological stressor= increased mpPVN CRF and vBNST cell recruitment in mPFC lesioned animals

BNST lesion = decreased mpPVN cell response to psychological stressor

mPFC may suppress mpPVN CRF cell response to stress via the a BNST pathway