calcium - the beginning glial calcium and sodium...

Glial calcium and sodium signalling

How calcium signalling was discovered

Principles of calcium signalling

Calcium signalling in neuroglia

Voltage- and ligand-gated channelsEndoplasmic reticulum calcium storeStore-operated calcium entryCalcium signals in neuronal-glialinteractionsGlial calcium waves

Comcept of Na+ signalling

1

Professor Hamphry Davy(1778-1829)

The scientific story of calcium began in1808, when Sir Humphry Davy was able toshow that lime (which had hitherto beenconsidered to be an inseparable element)was actually a combination of metal andoxygen (hence Calcium - from Latin Calxfor chalk).Sir Humphry tried to purify calcium byexposing a mixture of lime and mercuricacid to electric current; he succeeded inobtaining an amalgam of calcium, butfurther separation of mercury was sodifficult that even Davy himself was notcertain of whether he had obtained puremetallic calcium. In fact, he nevermanaged to isolate this new metal in a pureform, and metallic calcium remained alaboratory curiosity for another 50 years,until Henry Moissan obtained 99% purecalcium by electrolysing calcium iodide.

Calcium - the beginning 2

Calcium signalling: The BeginningThe creator of calcium signalling

Sydney Ringer found that Ca2+ is crucially important for

- survival of fish Ringer S. (1883) The influence of saline media on fishes. J. Physiol. Lond., 4, vi-viii.

- the contraction of the heart and skeletal muscle Ringer S. (1883) A further contribution regarding the influence of different constituents of the blood on the contractions of the heart. J. Physiol. Lond., 4, 29-43.Ringer S. (1886) Further experiments regarding the influence of small quantituies of lime, potassium and other salts on musclular tissue. J. Physiol. Lond., 7, 291-308. Ringer S, Buxton LW. (1887) Concerning the action of calcium, potassium and sodium salts upon the eel's heart and upon skeletal muscles of the frog. J. Physiol. Lond., 8, 15 - 19.

- fertilisation of eggs and development of the tadpoleRinger S, Sainsbury H. (1894) The action of potassium, sodium and calcium salts on Tubifex rivulorum. J. Physiol. Lond., 16, 1-9

Several years later Locke and Overton found that Ca2+ is critical for impulse transmission between nerve and muscle.

Locke FS. (1894) Notiz uber den Einfluss, physiologisher Kochsalzlosung auf die Eregbarkeit von Muscel and Nerve. Zentralbl. Physiol., 8, 166-167.Overton E. (1904) Beitrage zur allgemeinen Muskel- und Nerven physiologie. III. Mittheilung. Studien uber die Wirkung der Alkali- und Erdkali-salze auf Skeletalmuskeln und Nerven. Pflugers Arch., 105, 176-290

3 Calcium chloride added to distilled water sustains life muchlonger than either corresponding quantities of sodium orpotassium salts. For instance, with 30 cc of 1 per cent.solution of calcium chloride to the 1000 c.c. of distilled water,six fish died on average in 47 hours; whilst nine werestill alive on the 12th day.

Ringer, S., 1883,The influence of saline media on fishes,J. Physiol. Lond. 4, vi-viii.

4

Calcium signalling – the idea is born

Lewis Victor Heilbrunn1892-1959 "The sensitivity of protoplasm and its response to

stimulation are believed to be due to a sensitivityto free calcium ion and it is believed that thefreeing of calcium and the reaction of this calciumwith the protoplasm inside the cell is the mostbasic of all protoplasmic reactions.“

An Outline of General Physiology (1943 – 1952)

2. Typically (and perhaps always) the outer part of the protoplasm consistsof a rigid cortex.…7. When a cell is exposed to stimuli, such as heat, cold, mechanical impact,electric shock, ultraviolet radiation, etc., the cortex is liquefied andcalcium is released from the cortex into the cell interior.

The Dynamics of Living Protoplasm (1956)

5 Calcium signalling - Early discoveries

1947: Heilbrunn and Wiercinski observed rapid and strong contractions after directly injecting minute amounts of Ca2+ into muscle fibres Heilbrunn LV, Wiercinsky FJ. (1947) Action of various cations on muscle protoplasm. J. Cell. Comp. Physiol., 19, 15 - 32.

1948 - 1954: Schwarzenbach and Ackerman synthesized EDTA Schwarzenbach, v. G. & Ackermann, H. (1954) Helv. Chim. Acta, 30, 1798 - 1804.

1954: Bozler found that the removal of Ca2+ by EDTA relaxed muscle fibers.Bozler E. (1954) Relaxation in extracted muscle fibers. J. Gen. Physiol., 38, 149 -159.

1959: Anne-Marie Weber discovered that Ca2+ ions after binding to myofibrils activate actomyosinWeber A. (1959) On the role of calcium in the activity of adenosine 5'-triphosphate hydrolysis by actomyosin. J. Biol. Chem., 234, 2764 - 2769.

6

Calcium signalling - Early discoveries

In the 1950s, Setsuro Ebashi, working under the guidance of ProfessorKumagai, found that in glycerol-extracted muscle, ATP could inducecontraction, which was not followed by relaxation, but subsequent addition of amuscle extract induced relaxation. Ebashi and his colleagues called this extractrelaxing factor [1]. This relaxing factor turned out to have characteristics incommon with a Mg2+-activated ATPase described earlier by Wayne Kielley andOtto Meyerhof [2]. In Fritz Lipmann’s laboratory, at the Rockefeller Institute inNew York, Ebashi was later able to demonstrate that the relaxing effect is dueto Ca2+ uptake into sarcoplasmic reticulum vesicles mediated by a Ca2+, Mg2+-activated ATPase [3]; similar results were obtained independently byHasselbach and Makinose [4].

This finding, although originally related specifically to muscle relaxation, is fundamental to ourunderstanding of Ca2+ signalling generally, since it introduced for the first time the concept of anintracellular membrane-bounded Ca2+ store. In 1968, Setsuro Ebashi and Makoto Endo [5] outlined thetheory of muscle contraction as we know it now. They wrote “Ca ion discharged from the sarcoplasmicreticulum under the influence of action potential affects troponin and releases the actin filament from itsdepressed state, resulting in contraction. The sarcoplasmic reticulum then removes Ca ion from troponin atthe expense of ATP and induces relaxation” [5].

Kumagai H, Ebashi S, Takeda F. (1955) Essential relaxing factor in muscle other than myokinase and creatine phosphokinase. Nature, 176, 166.Kielley WW, Meyerhof O. (1948) Studies on adenosintriphosphatase of muscle. II. A new magnesium-activated adenosinetriphosphatase. J. Biol. Chem., 176, 591-601.Ebashi S, Lipmann F. (1962) Adenosine triphophate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. J. Cell. Biol., 14, 389 - 400.Hasselbach W, Makinose M. (1962) ATP and active transport. Biochem. Biophys. Res. Commun., 7, 132-6.Ebashi S, Endo M. (1968) Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol., 18, 123-83.

Setsuro Ebashi

7 Patch-clamp and fluorescent Ca2+ indicators

Calcium probesPatch-ClampErwin Neher Bert Sakmann Roger Tsien

Neher E, Sakmann B. (1976) Single-channelcurrents recorded from membrane of denervatedfrog muscle fibres. Nature, 260, 799-802.Hamill OP, Marty A, Neher E, Sakmann B, SigworthFJ. (1981) Improved patch-clamp techniques forhigh-resolution current recording from cells andcell-free membrane patches. Pflugers Arch, 391,85-100.

Tsien RY. (1980) New calcium indicators andbuffers with high selectivity against magnesium andprotons: design, synthesis, and properties ofprototype structures. Biochemistry, 19, 2396-2404.Tsien RY. (1981) A non-disruptive technique forloading calcium buffers and indicators into cells.Nature, 290, 527-528.Grynkiewicz G, Poenie M, Tsien RY. (1985) A newgeneration of Ca2+ indicators with greatly improvedfluorescence properties. J Biol Chem, 260, 3440-3450.

8

Diversity and versatility of calcium probes

G

Voltage- and ligand-gated calcium channels

Metabotropic receptor

Ca2+

Ca2+

SERCA

InsP3

RyR InsP R3

PMCA

ATPADPH+

Ca2+

Na+

NCE

Leak channel(s)

Cytosolic Ca probes2+

ER Ca probes2+

Mitochoindrial Ca probes2+

9

ERcytosol

Mag-Fura-2

Fluo

-3

Fluo-3

cytosol Fura-2

Fura

-2

20 s

1.0

3.0

F/F

0[C

a](

M)

2+L

m

Caf Caf

1

2

3

4

5

170

80

1 2 3 200

2200

70

500

[Ca

](M

)2+

Lm

F488

(a.u

.)

Combination of patch- voltage-clamp with Ca2+ indicators

Kano M, Garaschuk O, Verkhratsky A & Konnerth A (1995)Journal of Physiology, London., 487, 1- 16

Solovyova N & Verkhratsky A (2002): J.Neurosci Meth, 21: 622-620

10

Calcium as universal signalling molecule

Fluctuations of extracellular [Ca2+]

From: Ward DT (2004)In: Cell Calcium special issue on “Calcium-sensing receptor: physiology, pathology and pharmacological modulation”Ed by D. Riccardi

Calcium as a hormone Calcium as multi-level intracellular messenger

Imm

edia

te e

ffect

sm

secc

onds

- se

cond

sD

ealy

ed e

ffect

sse

cond

s - m

oths

From: Verkhratsky A & Toescu EC (1998) Integrative aspects of calcium signalling, Plenum Press

11 Calcium signalling in neurones and neuroglia: general principles

Nedergaard, M., Rodriguez, JJ & Verkhratsky, A (2010): Cell Calcium v. 47, p. 140-149.

12

Calcium signalling provides the substrate for glial excitability

13 Glial calcium signalling: mechanisms of generation

1. Voltage-gated calcium channels

Glial cells are non-excitable cells in physiological sense (i.e. theycannot generate action potentials. Nonetheless glial cells express aseveral types of voltage-gated ion channels including voltage-gatedcalcium channels.

Voltage-gated Ca2+ channels are generally present in immature glialcells or in glial precursors and their expression is down-regulatedduring development.

14

-45

-35

-25

-15

-5

5

-40

20 40

-700

I m (p

A)

Vm(mV)

HP -75 mVHP -40 mV

250 pA100 ms

40

Calcium currents in glial cells

Immature astrocyte Oligodendrocyte precursor

Akopian, G, Kressin, K, Derouiche, A & Steinhauser, C, (1996): Glia, v. 17, p. 181-194.

Verkhratsky, AN, Trotter, J & Kettenmann, H, (1990): Neurosci Lett, v. 112, p. 194-198.

15

soma (a)

processes (b)

25 s

50 mM K+ 50 mM K+ + 50 μM Ni2+

2.2

F/F 0

2.2

F/F 0

B

1.0

1.0

F/F

0

50 s

A

Low-voltage-activated calcium channels are preferentiallylocalized in the tips of glial precursor cells processes

Kirischuk, S, Scherer, J, Moller, T, Verkhratsky, A & Kettenmann, H, (1995): Glia, v. 13, p. 1-12.

16

Glial calcium signalling: mechanisms of generation

2. Ligand-gated calcium channels

All types of neuroglial cells express ligand-gated ion channels,generally referred to as ionotropic receptors. The most abundant areglutamate receptors (of AMPA, Kainate and NMDA types) andpurinoceptors (or P2X receptors). Many of these channels arepermeable to Ca2+ and can generate cytosolic Ca2+ signals.

17

B C

kainate kainate

30 s0.25

30 s0.25

F 340/380F 340/380

kainate kainate

kainate

30 s100 pA 50 pA

30 s

Ca2+ -free

kainate + CNQXkainate

kainate + CNQX

A

F340/380

control kainate

Purkinje celllayer

Bergmannglial cell

patch pipette loadedwith fura-2

Kainate induces massive calcium influx mediated by AMPA receptors into Bergmann glial cell in cerebellar slice preparation

Verkhratsky, A, Orkand, RK & Kettenmann, H, (1998) : Physiol Rev, v. 78, 99-141.

18


3. Endoplasmic reticulum provides a substrate for glial excitability

Ca2+ release following stimulation of metabotropic receptors and production ofInsP3 forms cytosolic Ca2+ signals, Ca2+ oscillations and propagating Ca2+

waves, which travel along single glial cell and between glial cells, thus allowingintegration within glial networks.

19

Ca2+

Na+

InsP3

InsP R3

InsP R3

RyR- ?

VGCCSOCC

SERCA

Ca - BP2+

ER

ATP

ADP

mGluR1

α1AR

α2AR

P2Y

MGluR5

P2U

P2T

A1

GABAB M1 M2 OXPAFR AT1ETB

5-HT2A

5-HT2C

V1

H1

NK1

B2ETA

Glutamate

Serotonin

Histamine

Substance P

Adrenaline

ATP

Adenosine

GABA Ach Endothelin VasopressinBradikinin Angiotensin IIPAFOxytocin

?

AMPA/KA

NMDA-?

P2X/2Z

GABAA

GLT

Na+

Na+

Ca2+

depolarization

Neurotransmitter receptors and InsP3-mediated Ca2+ signallingin glial cells

20

Visualisation of endoplasmic reticulum by fluorescent thapsigargin in cultured astrocytes

Verkhratsky, Solovyeva & Toescu (2002): In: Glia in synaptic transmission, Ed. by Volterra, Haydon & Magistretti, OUP, p. 91 - 109.

0

2

4

6

8

10

100 150 200 250 300

Num

ber o

f cel

ls

[Ca ] ( M)2+L μ

[Ca

] (

M)

2+Lμ

Ast

rocy

tes

DR

G

Hip

poca

mpa

l

Pur

kinj

e

Neurones

100

200

300

400

500

600

0

[Ca

] (

M)

2+Lu

μ

500

10

Resting Ca2+ concentration within the ER lumen in astroglia


21

M M

InsP

3R

1

InsP

3R

2

InsP

3R

3

100

300

600

astroglia oligodendroglia

InsP

3R

1

InsP

3R

2

InsP

3R

3

Expression of InsP3 receptors in glial cells

Kirchhoff & Verkhratsky, unpublished

22

A

B

0

200

400

600

800

1000

1200

1400

[Ca

] (n

M)

2+i

20 s

KCl90 mM 3 s

ATP/Ca-free100 M 5 sμ

ATP/Ca-free100 M 5 sμ

KCl90 mM 30 s

neuroneastrocyte

1 2

1 2

90 mM KCl 100 M ATP/Ca2+-freeμ

Calcium signalling in neurones and glia: Extracellular vs. intracellular pathway


23

[Ca2+

] i (nM

)

250

65 5 min

ATP

Ca2+-free

80

200

[Ca2+

] i (nM

)

A B

500 nM thapsigargin

30 s

5 min

ATP ATP ATPATPATP

30 s

[Ca2+

] i (nM

)

50

180

ATP

CControl

50 s

ATP

Heparin

25 s

D

>3

F/F

01

ATP triggers calcium release from InsP3-sensitive stores in Bergmann glial cells in cerebellar slices

Kirischuk, S, Moller, T, Voitenko, N, Kettenmann, H & Verkhratsky, A, (1995): J Neurosci, v. 15, p.7861-7871.

24

800 1000

X Axis Title

170

200 160

170

50

40 40

50

50 s

ATP, 100 Mμ ATP, 100 Mμ

InsP , 10 M3 μ

InsP , 10 M3 μ

ATP, 100 Mμ

ATP, 100 Mμ

[Ca

] (

M)

2+Lμ

[Ca

] (

M)

2+Lμ

[Ca

] (

M)

2+Lμ

[Ca

] (

M)

2+Lμ

TG, 5 Mμ

TG, 5 Mμ TG, 5 Mμ

100 s

InsP3 triggers Ca2+ efflux from astroglial endoplasmic reticulum

Solovyova & Verkhratsky, unpublished

25

NA

A

A10 mμ caf caf

InsP3

InsP3

TG

50 100 150

X Axis Title

caf

ATP

ATP

caf caf

650

70

[Ca

] (n

M)

2+i

20 s

180

50

[Ca

] (

M)

2+Lμ

A B

Astrocytes express ryanodine receptors, yet their function remains enigmatic


26

F (a

.u.)

600

700

800

900

1000

[Ca

] (n

M)

2+i

[Ca ] ~ 200 M2+L μ

100

200

300

400

500

600

700

ATP

Ca -free2+

0

Depletion of ER store triggers store-operated Ca2+ entry that is necessary for ER store refilling

ER [Ca2+]

Cytoplasmic Ca2+


27

[Ca2+

] i (nM

)

60

200

100 nM ET-3

Ca-free

200

50

50 s

[Ca2+

] i (nM

)

50 s

50 s

1 μM NA

Ca-free

ATP + NA

Ca-free

[Ca2+

] i (nM

)

60

200

Store-operated Ca2+ is universally present in all types of glia: Store-operated Ca2+ entry in Bergmann glial cells in situ

in cerebellar slice

Tuschick, Kirischuk, Kirchhoff, Liefeldt, Paul, Verkhratsky & Kettenmann, (1997): Cell Calcium, v. 21, p. 409-419.

28

[Ca2+

] i (nM

)

460

5 min

100 μM ATP

0 Ca

100

75 s

[Ca2+

] i (nM

)

60

400

250 s

A

B

0 Ca 0 Ca 0 Ca

100 μM ATP

100 μM ATP

Store-operated Ca2+ is universally present in all types of glia: Activation of purinoceptors triggers both Ca2+ release and store-operated Ca2+ entry in human glioma cells

Hartmann & Verkhratsky (1998): J Physiol, v. 513, p. 411-424.

29

80

450

[Ca2+

] i (nM

)

A

10 μM ATP 100 μM ATP 300 μM ATP

0 Ca 0 Ca 0 Ca

150 s

Δ[Ca2+]cap

[ATP] (μM)

120

80

40

0

1 10 100

Δ[C

a2+] ca

p (n

M)

B(98)

(81)

(12)(0)(0)

0

0.2

0.4

0.6

0.8

1.0

1.2

0.01 0.1 1 10 100[ATP] (μM)

Δ[C

a2+] i/Δ

[Ca2+

] i max

C

Ca2+ release

Ca2+ entry

KH 1.5

KH 7

Dissociation between activation of Ca2+ release and store-operated Ca2+ entry in human glioblastoma cells


30

G

InsP R3

Store-operated channel

Metabotropic receptor

InsP3

SERCA

ATP

ADPCa 2+

Ca release compartment

2+

Store-operated Ca2+ entry is controlled by specific portion of the ER possibly in the very vicinity of the plasmalemma


31

1500

microglia

600

100

1200

430

oligodendrocytes astrocytes

trp 6 5/4 3 2 1 6 5/4 3 2 1 6 5/4 3 2 1

Expression of TRP channels in glial cells

Kirchhoff & Verkhratsky, unpublished

32 Glial calcium signalling: mechanisms of generation

4. Glial cells generate calcium signals in response to neuronal activity

Release of neurotrasnmittters from presynaptic terminals activatesmetabotropic receptors in astroglial perysinaptic processes, that trigger Ca2+

signals. Similarly release of glutamate and ATP from electrically active axonsaxons trigger receptor/InsP3-mediated Ca2+ signals in oligodendroglia.

33

PCL

PF

Pia

BG

STIM

200 mμ

a b

c

12

10 mμ

1 22 s

d

2 s

1.6

1.0

F/F0

4 mμ

0

255

Parallel fibers stimulation triggers calcium signalsin Bergmann glial cell microdomains

Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999): Nat Neurosci, v. 2 p. 139-143.

34

a

4 mμ

Control

TTX

Washout

2 s

1.0

1.5

F/F0

b

Control

Cd2+

2 s

1.0

1.35

F/F0

4 mμ

Inhibition of neuronal excitability abolishes Ca2+ signals in Bergmann glia


35

Synaptically evoked Ca2+ signalling occurs in the microdomains represented by elementary structures which envelope groups of synapses

head

stalk


36

50 s

1000 μM Glu 1000 μM Glu 1000 μM Glu

5 min 10 min

90

250

[Ca2+

] i (nM

)

A

90

230

1000 μM Glu1000 μM Glu 1000 μM Glu

5 min 10 min

[Ca2+

] i (nM

)

B 500 nM thapsigargin

500 nM thapsigargin

50 s

Ca2+-free

Glutamate induces Ca2+ release from thapsigargin-sensitive store

Kirischuk, Kirchhoff, Matyash, Kettenmann & Verkhratsky (1999): Neuroscience, v. 92, p. 1051-1059.

37

1000 μM glutamate

25 s

90

250

[Ca2+

] i (nM

)

A

1000 μM glutamate

25 s

90

250

[Ca2+

] i (nM

)

B

1000 μM glutamate

1000 μM glutamate

Control

Heparin

Heparine inhibits glutamate-induced Ca2+ signalling


38

mGluR1 mGluR5

N-term 7 TMD C-term N-term 7 TMD C-term

# 36 # b11

mGluR1

500

200M

mGluR5b

mGluR5a

b

a

b

a

c

15 ms

3 nA

A

C

B

Br Ø M 453647 42 b143 Br b11b10 b5

Vh = -70 mV

S1 AS2 AS1

S1 S2 AS1

Expression of mGluR 5 in Bergmann glia


39


5. Stimulation of glial cells triggers propagating Ca2+ waves that spread throughglial syncytia

Mechanisms of propagating Ca2+ waves in astroglia are complex and involvediffusion of InsP3 through gap junctions and Ca2+ dependent release oftransmitters (most probably ATP).

40 Propagating calcium waves in astroglial confluent cultures were discoveredby Ann Cornell-Bell and her colleagues in 1990 and are seen in many preparations

in vitro and in situ

Astroglial calcium waves in retinaNewman & Zahs (1997) Science, v.275, p. 844-847

Calcium waves in cultured astrocytesCharles (1998) Glia, v.24, p. 39-49

41

Mechanisms of glial Ca2+ wave propagation

InsP3 diffusion through gap junctions; release of transmitters or combination of both

InsP3

InsP3

InsP3

InsP3

InsP3

InsP3

InsP3

InsP3

InsP3

InsP3Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

ATP

ATP

ATP

ATP

ATP

Gap junction

Metabotropic (P2Y)receptor

ER Ca release2+

Vesicular release of neurotransmitter (ATP)

A

B

C

Verkhratsky & Butt (2007): Glial Neurobiology, A TextbookWiley & Sons

42

Sodium signalling in astroglia

43

Astroglial receptors and transporters regulate Ca2+ and Na+ fluxes 44 Stimulation of ionotropic glutamate receptors induces [Na+] elevation

Kirischuk, Kettenmann & Verkhratsky (2007): Pflugers Arch, 454, 245-252.

45

Application of glutamate triggers inward current and sodium influx

5

5

20

25

[Na

] (m

M)

+i

[Na

] (m

M)

+i

200 pA

200 pA

1000 M glutamateμ 1000 M glutamateμ

100 M CNQXμ Na -free (NMDG)+

100 M kainateμ

100 M kainateμ

100 M CNQXμ

30 s

30 s


46 Stimulation of parallel fibers triggers inward current and [Na+]i elevation


47

Sodium as a signalling molecule in astroglia?

Kirischuk, Parpura & Verkhratsky (2012) TINS, 35, 497-506

48

Conclusions

Stimulation of neuroglial cells with neurotransmitters and neuromodulatorstriggers cytosolic ionic (Ca2+and Na+) signals.

Glial Ca2+ signals are predominantly generated by Ca2+ release from theendoplasmic reticulum though opening of InsP3-gated Ca2+ channels (InsP3receptors).Endoplasmic reticulum membrane represents an excitable mediathat allows generation of propagating calcium waves, which integrateastroglial syncytia.Neuroglial Ca2+ signals can be instrumental for integrativeprocesses in neuronal-glial networks.

Glial Na+ signals are generated by Na+ entry through ionotropic receptors,TRP channels and transporetrs. Cytosolic Na+ concentration regulatemultiple homeostatic pathways.

49

calcium - the beginning glial calcium and sodium...

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