The C1 cells and acute stress responses

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1 Institut Pasteur, 2014 The C1 cells and acute stress responses Patrice Guyénet University of Virginia SOM

2 Peter Burke Seth Depuy Ruth Stornetta Postdocs G. Boshorishvili Melissa Coates Roy Kanbar Under grads Steve Abbott Lab specialists Ken Viar Gavin West Thanh Nguyen Ben Holloway Funding: NIH HL & HL (PGG) Soc Française d hypertension (RK), AHA (SBA)

3 C1 TH+PNMT What are the C1 neurons? A1 TH only tyrosine TH L-DOPA AADC dopamine DA β-ohase norepinephrine PNMT epinephrine RVLM 1 mm Hillarp, A. Dahlstrom, K Fuxe, 1966: A1 CA group identified Hokfelt, Fuxe, Goldstein,and Johansson, O. 1973: C1 adrenergic group identified IHC by Card and Sved 2006

4 C1 cells and BP control: early concepts Baroreceptors C1 TH+PNMT NTS Glu Glu GABA A1 TH only RostralVLM a.k.a. pressor region a.k.a. RVL (DJ Reis) Glu IHC by Card and Sved 2006

5 Regulation of immune and glucoprivic responses by the C1 cells to PVH C1 TH+PNMT NTS Feeding? A1 TH only 1 mm LPS - IL1- CRF-ACTH-corticosterone (P Sawchenko) Glucoprivic responses (S. Ritter) to adrenal medulla IHC by Card and Sved 2006

release From Card and Sved 2006 (SD rat, PRSx8 lenti)

6 The CNS projections of the C1 cells suggest a much wider role than BP regulation, ACTH and epinephrine release From Card and Sved 2006 (SD rat, PRSx8 lenti) or Abbott et al (DIO ChR2-mCherry, DBH Cre/0 mouse)

7 Aggregate role of the C1 cells: adaptive responses to injury or looming injury? Vigilance & attention Physical stresses: hypoxia/asphyxia hypotension pain, injury, infection hypoglycemia infection LPS/IL-1 Anticipation of injury psychological stress Astrocyte stimulation Breathing, sighing NE neurons H 2 O retention PVH Magno SON SNS BP, CO PVH parvo CRF / ACTH / corticosterone Arousal PSNS Liver / metabolism Heart GI motility Stomach acid SNS Antiinflammation? Metabolism Antiinflammmation? C1 cells

8 Optogenetic evidence that the C1 cells regulate the circulation ChAT ChR2-mCherry TH / C1 cells Targeting ChR2 to the C1 cells with PRSx8-ChR2-mcherry lenti Kanbar & Guyenet Am J Respir Crit Care Med. 2010

9 C1 cell stimulation in awake rats increases sympathetic tone Small BP rise HR slows SNA increases Kanbar & Guyenet Am J Respir Crit Care Med. 2010

10 C1 cells and arousal: the C1 cells as components of the reticular activating system

11 Selective expression of ChR2 by the C1 cells in a TH-Cre rat >95% TH + TH-Cre rat: BAC Ilana Witten & Karl Deisseroth 87% bulbospinal Burke & Guyenet, unpublished

Guyenet, unpublished Rf 100 150 50 Flow EEG EEG (Hz)

12 Photostimulation of the C1 neurons in a sleeping rat produces: BP breathing f Sighing Arousal HR Burke & Guyenet, unpublished Rf Flow EEG EEG (Hz) EMG HR BP ml/s 0.5 mv nrem 0.1 mv 60s

13 Selective C1 cell stimulation produces EEG desynchronization and sighs 100 laser on nrem 20Hz 10Hz 2 Hz nrem * * *** *** 20 Control Time (sec) 0 Control (n=6) 2Hz 10Hz 20Hz (n=5) Burke & Guyenet, unpublished

Pons / midbrain RTN/ VRC NTS C1 neurons pain hypoxia hypotension hypoglycemia

14 C1 cells target arousal-promoting areas such as the LC, the raphe and the orexin system Arousal / vigilance networks Raphe LC PAG lpb Orexin neurons Pons / midbrain RTN/ VRC NTS C1 neurons pain hypoxia hypotension hypoglycemia Breathing Cervical cord SNS Thoracic cord Bochorishvili, Stornetta Guyenet, unpublished

15 The C1 cells activate noradrenergic neurons throughout the brain and body cortex NE neurons olf hippo cereb basal f.brain LC A2 dienceph A5 Hypotension Hypoglycemia Hypoxia Infection Pain Psychological stress C1 A1 SNS spinal cord

16 C1 cells activate LC monosynaptically by releasing glutamate Cre-inducible AAV2* RVLM 4 weeks + DBH Cre/0 mouse * DIO-ef1α-ChR2mCherry or DIO-ef1α-ChR2eYFP Holloway Guyenet J Neurosci. 2013

17 Photostimulation of ChR2-expressing C1 axons activates LC neurons in slices (adult mice) Laser: hη 473 nm, 1 ms, 5 mw C1 axon varicosity Holloway et al., JN 2013

18 Laser 1 ms, 5mW Input from RVLM-CA neurons to LC is monosynaptic and glutamatergic Vclamp LC Control Add 4-AP X laser X Add glur antags X X X Add TTX + TTX + TTX + 4-AP + TTX + 4-AP + CNQX + AP5 Holloway Guyenet J Neurosci 2013

19 The C1 cells express VGLUT2, are potentially glutamatergic VGLUT2 mrna TH-ir PNMT-ir VGLUT2-ir 10 μm Stornetta et al JCN

20 C1 neurons target most perhaps all CNS NE neurons Pain Hypoxia Hypotension Hemorrhage A1 A2 C1 A6 (LC) A7? A5

21 Functional significance LC & other NE neurons α & β-ars on Astrocytes Increased CBF Glycogenolysis Increased K & glutamate uptake, lactate production Neurons increased activity / excitability C1 cell activation Hypotension, hypoglycemia, hypoxia, hemorrhage, infection, etc. SNS activation (Epi/ NE release) Metabolic effects (e.g. glycogenolysis, in creased K uptake In skel muscle, liver glucose release) α & β-ars on heart and VSM Depolarization / increase contractility

22 The C1 cells activate parasympathetic efferents by releasing glutamate Pancreas Stomach + other GI Heart? Spleen/immune? IVLM DMV = dorsal motor nucleus of the vagus nerve

23 Optogenetic activation of the C1 cells in mice: effects on DMV Cre-inducible AAV2* RVLM TH mcherry both Control: DβH Cre/0 cko: DβH Cre/0 ;;VGLUT2 fl/fl * DIO-ef1α-ChR2mCherry Selectivity is virtually perfect in mice >98%

24 C1 projections to DMV in the mouse DMV hypoglossal mcherry ChAT

25 C1 cells excite DMV neurons by releasing glutamate: CRACM evidence hη 473 nm Laser, 5mW, 1 ms ChR2+ axons DMV 10 ms Average of 30 stimuli Depuy et al., JN 2013

26 How many classes of C1 neurons are there? Vigilance & attention enteroceptive stresses: hypoxia/asphyxia hypotension pain, injury, infection hypoglycemia Glia stimulation Anticipation of looming injury psychological stress Breathing, sighing NE neurons SNS BP, CO PVH parvo CRF / ACTH / corticosterone Arousal PSNS Liver / metabolism Heart GI motility Stomach acid Immunosuppression SNS Metabolism immunosuppression C1 cells

Conclusions The C1 cells (total ~2000 in rats; ~800 in mice) are glutamatergic and synthesize numerous additional transmitters, including catecholamines, the function of which remains unclear.

27 Conclusions The C1 cells (total ~2000 in rats; ~800 in mice) are glutamatergic and synthesize numerous additional transmitters, including catecholamines, the function of which remains unclear. Collectively, the C1 cells innervate most if not all subcortical regions suspected to mediate acute stress responses. The C1 cells collectively respond to internal and external danger signals (tissue injury, pain, hypoxia, hypotension, hypoglycemia, hemorrhage, bacterial infection). The C1 cells elicit (facilitate? contribute to?) a repertoire of neuroendocrine and autonomic responses designed to help the organism survive these threats. These responses include glucose production, corticosteroid release, maintenance of blood pressure and respiration, immune system modulation (via corticosterone and, possibly, cholinergic antiinflammation), fluid conservation and increased vigilance. These responses are mediated via the sympathetic and parasympathetic nervous systems, the HP axis and arousal-promoting pathways such as the locus coeruleus & the orexinergic neurons. There are probably several subsets of C1 neurons that are each capable of triggering distinct yet overlapping sets of physiological responses.

29 Evidence for extensive network of axonals collaterals from single C1 cells PAG PBN LC DVC SC Evidence of C1 cell collateralization based on antidromic mapping and retrograde tracer evidence e.g. Haselton & Guyenet Janssen Loewy 1995 Science

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