CEREBRAL VENOUS POTASSIUM EFFLUX DURING SPREADING DEPRESSION

CEREBRAL VENOUS POTASSIUM EFFLUX DURING SPREADING DEPRESSION JESSICA SEIDEL Ph.D. FIELDS INSTITUTE: Workshop on CSD and Related Neurological Phenomena JULY 7-11, 2014

OUTLINE 1. Introduction Hypothesis and Methods 2. Results Cortical and Venous K + Recordings Models of Ischemia Venous Thrombosis dmcao Role of Astrocytes in Vascular K + Clearance Role of K + Channels in Venous K + Clearance 3. Future directions

HYPOTHESIS Clearance of extracellular K + post-sd is done by astrocytic siphoning into the vasculature.

ASTROCYTE CLEARANCE OF [K + ] e K + Na + K + NKCC1 K + Glu ADP ATP ATP K ir ADP Na + GLT-1 K + SN-1 Gln Glu

SIGNALLING BETWEEN ASTROCYTES AND VASCULATURE NKCC1 Modified from: Dunn KM. et.al., Circulation Journal (2010) 74: 608-616.

OBJECTIVES 1. Measure [K + ] e in cerebral venous blood during SD under normoxic and ischemic conditions. 2. Determine the contribution of active K + uptake by astrocytes to the clearance of [K + ] e post-sd.

METHODS ANIMALS: Mice (C57BL/6, male, 20-30 g) SURGICAL PROCEDURE: Arterial Cannulation NO Mechanical Ventilation Arterial BP, ph, po 2 and pco 2 were monitored. n Weight (g) ph pco2 (mmhg) po2 (mmhg) BP (mmhg) 93 25 ± 2 7.35 ± 0.03 37 ± 4 108 ±14 82 ± 10 SD INDUCTION: Topical 300 mm KCl Application

METHODS CORTICAL RECORDINGS: K + -sensitive and electrodes were ~300 μm deep, equidistant to the KCl stimulation site. K + KCl VENOUS RECORDINGS: Made from pial veins ~20 μm in diameter. electrode was ~300 μm deep in the cortex, adjacent to the K + electrode. K + KCl For all studies: *p<.05, **p<.01 and ***p<.001

OUTLINE 1. Introduction Hypothesis and Methods 2. Results Cortical and Venous K + Recordings Models of Ischemia Venous Thrombosis dmcao Role of Astrocytes in Vascular K + Clearance Role of K + Channels in Venous K + Clearance 3. Future directions

[K + ] c (mv) [K + ] v (mv) POTASSIUM RECORDINGS DURING SD CORTEX VEIN K + K + KCl KCl [K + ] e [K + ] e 100 10 mv 60 s 60 s 20 mv 100 10 mv 60 s 20 mv 60 s 80 80 60 60 40 40 20 20 0 0 0 50 100 150 0 50 100 150 Time (s) Time (s) n=14 n=11

Amplitude VENOUS RECORDINGS DURING SD K + Electrode t 1/2 KCl *** ** Cortex: n=14 and Vein: n=11

CONFIRM VENOUS RECORDINGS K + Electrode 30 mm KCl with Francesco Blasi

VENOUS THROMBOSIS VENOUS THROMBOSIS: Thrombin (2500 i.u./ml) was injected into a pial vein (~1-2 mm from midline) using a glass micropipette. Clot was allowed to stabilize for 10 min prior to inducing SD. Thrombin LDP K + KCl with Francesco Blasi n=6

VENOUS THROMBOSIS (Cortical K + Recordings) Thrombin LDP K + KCl PRE POST 20 mv 60 s Resting [K + ] e was increased by 0.72±0.50 mm post-thrombin injection. with Francesco Blasi n=6

VENOUS THROMBOSIS (Averaged Data) Thrombin LDP K + KCl * * * 50% of all SD recorded post-thrombin injection were spontaneous. with Francesco Blasi n=6

VENOUS THROMBOSIS (Laser Speckle Flowmetry) Thrombin KCl with Francesco Blasi and Daniel Von Bornstädt n=5

VENOUS CLEARANCE OF K + DURING FOCAL ISCHEMIA K + FOCAL ISCHEMIA: K + and electrodes were placed in the cortex prior to placing the clip on the dmca. Spontaneous peri-infarct depolarizations (PIDs) were monitored for 2H. Average increase in baseline [K + ] e after the 1 st PID was 8.4±7.1 mm. with Daniel Von Bornstädt

CHANGES IN TISSUE K + FOLLOWING ISCHEMIA Dorsal Ischemic Edge Ischemic Core Ventral Ischemic Edge Yushmanov VE et.al., Brain Res. (2013) 1527: 199-208

VENOUS CLEARANCE OF K + DURING FOCAL ISCHEMIA K + *** 20 mv with Daniel Von Bornstädt 60 s n=7

ASTROCYTE CLEARANCE OF [K + ] e K + Na + K + NKCC1 K + Glu ADP ATP ATP K ir ADP Na + GLT-1 K + SN-1 Gln Glu

SELECTIVE INHIBITION OF ASTROCYTE OXIDATIVE METABOLISM (FAc: 10 mm, 90 min) K + Electrode CORTEX FAc KCl FAc Vehicle Aceytl-CoA FAc citrate synthase Citrate FC VEIN 2 min 20 mv TCA Cycle aconitase Isocitrate FAc Vehicle 20 mv 2 min

SELECTIVE INHIBITION OF ASTROCYTE OXIDATIVE METABOLISM (FAc: 10 mm, 90 min) K + Electrode FAc KCl *** ** p =.07 Cortex: n=14 and Vein: n=10

K + SIGNALLING BETWEEN ASTROCYTES AND VASCULATURE NKCC1 Slo -/- OR Paxilline (10 μm) NS-1619 (10 μm) Ba 2+ (100 μm) Modified from: Dunn KM. et.al., Circulation Journal (2010) 74: 608-616.

BaCl 2 : K ir ANTAGONIST (100 μm, 30 min) * K + Electrode BaCl 2 KCl ** Cortex: n=6 and Vein: n=4

PAXILLINE: BK CHANNEL ANTAGONIST (0.5-20 μm, 30 min) * K + Electrode Paxilline KCl * * * Cortex: n=18 and Vein: n=13

BK KNOCKOUT MOUSE (Slo -/- ) Targeted mutation of the pore-forming subunit (encoded by the mslo1 gene) by homologous recombination in embryonic stem cells. FVB/NJ background 40% Slo -/- mice die at ~2.2 months of unknown causes, but surviving mutants have normal life spans (10 mo). Significantly reduced ability to produce offspring: Slo -/- male bred with WT female. Pervasive phenotype is moderate ataxia (shorter stride, worse performance on rotarod and hanging wire). Motor learning NOT impaired. Southern Blot Western Blot Modified from: Meredith AL. et.al., J of Biological Chem (2004) 279 (35): 36746-36752.

BK KNOCKOUT MOUSE (Slo -/- ) 2 K + Electrode 2 mm KCl VEIN CORTEX Cortex: n=2 and Vein: n=2

NS-1619: BK CHANNEL AGONIST (10 μm, 30 min) K + Electrode NS-1619 KCl Cortex: n=7 and Vein: n=5

OUTLINE 1. Introduction Hypothesis and Methods 2. Results Cortical and Venous K + Recordings Models of Ischemia Venous Thrombosis dmcao Role of Astrocytes in Vascular K + Clearance Role of K + Channels in Venous K + Clearance 3. Future directions

FUTURE DIRECTIONS 1. Can we determine at what level K + is entering the vasculature? Use in vivo TPM to image vascular increases in K +. Asante Potassium Green (APG2) Good photostability Better K + selectivity (over Na + ) than previous indicators Rapid loss of dye in vivo (i.a. injection) 2. Are there differences in K + clearance in different regions of the brain during focal ischemia/stroke? Simultaneously with LSF Determine if there are differences in K + clearance in penumbral vs. non-ischemic tissue.

AKNOWLEDGEMENTS Primary Investigator Cenk Ayata The Lab Yahya Atalay Mustafa Balkaya Francesco Blasi* Shih-Pin Chen Ali Daneshmand Katharina Eikermann-Haerter Fanny Herisson Qin Tao Daniel Von Bornstädt* Ying Wei Nilufer Yalcin Esther Yu Yi Zheng Funding AHA Postdoctoral Fellowship Award, Seidel (PI) MGH Internal Sundry: Heitman Foundation, Ayata (PI) NINDS: 2P50NS051343-06, Ayata (PI Project 2) Leducq Foundation: Transatlantic Centers of Excellence Award 2012D000293, Ayata (PI MGH)

K + SIGNALLING BETWEEN ASTROCYTES AND VASCULATURE NKCC1 Furosemide (5 mm) IbTx (150 nm) OR Paxilline (10 μm) NS-1619 (10 μm) Ba 2+ (100 μm)

K + Decay Duration (t 1/2, s) Shift Duration (s) K + Amplitude (mm) Shift Amplitude (mv) FUROSEMIDE: NKCC1 ANTAGONIST (5 mm, 30 min) CORTEX (n=5) VEIN (n=5) 80 60 30 20 ** 40 20 10 0 Vehicle Furosemide 0 Vehicle Furosemide 120 80 100 80 60 40 20 60 40 20 0 Vehicle Furosemide 0 Vehicle Furosemide

CLINICAL MANIFESTATION OF CADASIL Chabriat H. et.al., Lancet Neurol. (2009) 8: 643-53.

SD SUSCEPTIBILITY INCREASED IN CADASIL MUTANTS Eikermann-Haerter, et.al., Annal. Neurol. (2011) 69: 413-418.

CSD speed (mm/min) Electrical threshold ( C) CSD frequency (#/h) SD SUSCEPTIBILITY INCREASED IN CADASIL MUTANTS 1000 15 Male Female * * 10 100 5 * 0 WT TgNotch3 R90C 10 5 4 * * 3 2 1 1 WT TgNotch3 R90C 0 WT TgNotch3 R90C Eikermann-Haerter, et.al., Annal. Neurol. (2011) 69: 413-418.

INCREASED PID FREQUENCY IN CADASIL MUTANTS TgNotch3 R90C WT Group TgR90C PID Freq (/h) 4.4 ± 0.7 Cum PID Dur (sec) 375 ± 90 WT 2.7 ± 0.3 146 ± 31 Lee et.al., unpublished data.

INCREASED PID FREQUENCY IN NOTCH3 -/- Group Notch 3 -/- WT PID Freq (/h) 6.0 ± 2.5 2.9 ± 2.5 LSF 10-12 week-old, Male Mean ± SEM; p<0.05; n=8-10 each Arboleda-Velasquez et.al., PNAS. (2008) 105: 4856-61.