Tonic GABAA receptormediated signalling and epilepsy

1
Tonic GABAA receptor-mediated signalling and
epilepsy
Mt Tremblant, Feb 2005
Alexey Semyanov Matthew C. Walker Annalisa Scimemi

• Dimitri M Kullmann
• Institute of Neurology, UCL

2
GABAA receptor-mediated signalling
3
Tonic GABAA receptor-mediated inhibition and
epilepsy

• Where does it occur?
• Which receptors underlie tonic inhibition?
• Does it play a role in epilepsy?

Species
4
Tonic GABAA receptor-mediated inhibition in
cerebellar granule cells
bicuculline
Brickley et al (1996)
5
Extrasynaptic ?6 and ? subunits mediate tonic
inhibition in cerebellar granule cells
Nusser et al (1998)
Brickley et al (2001)
Stell et al (2003)
6
What about the forebrain?
Dentate gyrus granule cells
bicuculline gt 100 mM
Nusser Mody (2002)
Stell et al (2003)
7
Hippocampus
Guinea pig or rat slices Blockers AMPA/Kainate N
BQX NMDA APV GABAB CGP52342 High Cl-
pipette solution GABAA currents are inward when
cell clamped at -70 mV
8
Tonic GABAA receptor-mediated inhibition in the
hippocampus
Cell-type differences in tonic inhibition
hippocampal pyramidal cells
hippocampal interneurons
picrotoxin 100 mM
picrotoxin 100 mM
100 pA 0.5 s
Semyanov et al (2003)
9
Selective reduction in tonic inhibition of
interneurons enhances inhibition of pyramidal
neurons
Interneurons V-clamp INs C-clamp Pyr.
cells V-clamp
Picrotoxin
Semyanov et al (2003)
10
Tonic GABAA receptor-mediated inhibition of
interneurons a possible homeostatic mechanism?
GABAo
? GABAo ? Tonic inhibition of
interneurons ? Interneuron
excitability (NB blocking tonic inhibition
might be anti-epileptic?)
11
Tonic inhibition in the human brain
PC
Interneuron
Picrotoxin (100 ?M)
Picrotoxin (100 ?M)
100 pA
1 s
Tonic IGABA IN 21 6 pA (n 5)
Tonic IGABA PC 41 14 pA (n 3)
Scimemi et al (unpublished)
12
Tonic GABAA receptor-mediated inhibition and
epilepsy

• Which receptors underlie tonic inhibition in the
  hippocampus?

13
Molecular mechanisms GABAA receptors
GABA
BDZ
a1-6 b1-3 g1-3 ? d e p
a
b
g/d/e
a
b
GABA
14
High-affinity GABAA receptors

• ?4-6 and ? (therefore not ?)
• Predicts high sensitivity to
• neurosteroids
• ethanol
• Zn2
• low sensitivity to benzodiazepines and zolpidem

15
Tonic GABAA receptor-mediated inhibition in
interneurons
Low picrotoxin relatively selectively blocks
the tonic conductance
Low SR95531 blocks IPSCs but not the tonic
conductance
Semyanov et al, 2003
16
Tonic GABAA receptor-mediated inhibition in
interneurons
Zolpidem enhances the tonic conductance in
interneurons
Semyanov et al, 2003
17
Blocking GABA uptake uncovers a pharmacologically
similar tonic conductance in pyramidal neurons
In NO711
Semyanov et al (2003)
18
Hippocampal pyramidal cells not ?
Stell et al (2003)
19
?5 extrasynaptic in pyramidal cells?
20
Tonic GABAA current in pyramidal neurons
evidence for ?5
Caraiscos et al (2004)
21
?5 receptors deleterious effect on cognition?
Enhanced learning and memory and altered
GABAergic synaptic transmission in mice lacking
the alpha 5 subunit of the GABAA receptor
Collinson et al J Neurosci. 2002 225572-80.
An orally bioavailable, functionally selective
inverse agonist at the benzodiazepine site of
GABAA alpha5 receptors with cognition enhancing
properties.Chambers et al J Med Chem. 2004
475829-32
22
Tonic GABAA receptor-mediated inhibition
(Hippocampal pyramidal cells)
?5 ?2/3
?
Also SR-sensitive receptors at higher GABA?
23
Under baseline conditions, tonic IGABA is not
mediated by a1-3 or a5 subunit-containing
receptors
However, Caraiscos et al (2003) and others either
supplemented GABA or incubated in vigabatrin to
elevate GABAo
a5 subunit-containing GABAA receptors mediate 40
of tonic IGABA
24
1 ?M GABA is sufficient to evoke an
L655,708-sensitive current
Cultured hippocampal neurons
Suggests that the resting GABAo in a slice is
lt1 ?M
25
Tonic GABAA receptor-mediated inhibition and
epilepsy

• What happens in epilepsy?

26
Down-regulation of ?5 in experimental epilepsy
Control
Rice et al (1996)
Epileptic
Houser et al (2003)
27
Methods
8 week-old rats
?5 down-regulated (G. Sperk Innsbruck)
28
Tonic IGABA is not decreased in spite of ? ?5
29
The cell-type specificity of tonic IGABA is
maintained
30
No major changes in mIPSCs or sIPSCs
Miniature action potential- independent IPSCs
Spontaneous IPSCs
31
Blocking GABA transporters increases tonic IGABA
in PCs (but not in interneurons) both in control
and in epileptic rats
32
Tonic IGABA has a higher sensitivity to ? GABAo
in epilepsy
33
Tonic IGABA in 5 ?M GABA is no longer mediated by
a5 subunits
34
The late component of eIPSCs increases in Ep PCs
35
(No Transcript)
36
Epileptogenesis
? ?5
GABA uptake unchanged
tonic IGABA unchanged in low GABAo, ? in high
GABAo
Another subunit takes over
Questions Is this protective? Can it contribute
to drug resistance? Does it contribute to
cognitive deterioration accompanying
long-standing temporal lobe epilepsy?
37
Acknowledgements
Annalisa Scimemi Alexey Semyanov ? Riken Brain
Science Institute, Japan Matthew C. Walker Joost
Heeroma Kirill Volynski Günther Sperk (Innsbruck
Medical University, Austria)
38
Conclusions
- Tonic inhibition can be detected in the
hippocampus proper of adult rats
- It shows a similar cell-type specificity to
that previously described in guinea pigs
- It is maintained in the epileptic hippocampus,
despite a down-regulation of the a5 subunit
- Tonic inhibition has multiple components,
depending on GABAo
Under baseline conditions, ?-subunit containing
GABAA receptors mediate the tonic current in
control and epileptic PCs
In control PCs, the a5 subunit mediates 40 of
the tonic current only when the extracellular
GABA concentration is increased to 5 ?M
In epileptic PCs, tonic inhibition might be
mainly mediated by ?-subunit containing GABAA
receptors
- The sensitivity to GABA increases during
epilepsy
- Functional relevance dampen hyperexcitable
networks and curtail seizures?
Tonic IGABA?
39
Extracellular solution (mM)
NaCl 119 KCl 9 MgSO4 1.3 NaH2PO4 1 CaCl2 2 Gl
ucose 22 pH 7.4 296 mOsm
Intracellular solution (mM)
CsCl 120 CsOH Hepes 10 CsOH EGTA 2 NaCl 8 MgCl2
0.2 MgATP 2 Na3GTP 0.3 QX314 5 pH 7.2 290 mOsm
40
The PPR of eIPSCs does not change during epilepsy
Normalized IPSC amplitude
41

• ?1 ?Zn2-sensitivity ?ZOL-sensitivity marker
  of the majority of IN
• ?2 synaptic
• ?4 Zn2-sensitive furosemide-sensitive
• ?5 ZOL-insensitive L-655,708-sensitive
  midazolam-sensitive largely restricted to PC
  ?desensitization at depolarized membrane
  potentials mainly extrasynaptic
• b2/b3 etomidate-sensitive
• ?2 ?Zn2-sensitivity
• ? Zn2-sensitive extrasynaptic sites (?4?6?
  ?4?3? ?6?3? lowEtOH-sensitive)
  THDOC-sensitive(?) ?desensitization ?EC50 GABA
  (0.2 µM for a6ß2d vs 10 µM for a1ß2?2)
• The isoform of the ß-subunit (13) has little
  influence on the GABA-modulatory actions of the
  pregnane steroids.
• BDZ but not barbiturates require a g subunit

42
An alternative way of looking at GABAA receptors
in epilepsy
43
Protocol
Adapted from Miledi et al. 2002
44
GABAA Receptor Subunits
Jones-Davis and Macdonald, 2003
45
Evidence for alteration in GABAA receptor
function in epilepsy
46
Evidence for changes in GABAA receptor subunit
expression in epilepsy
47
GABAA Currents in Injected Oocytes
48
Current Enhancement by Pentobarbital
Frontal
100 mM Pentobarb
30 mM GABA
Hippocampal
49
Current Amplitudes - Control vs. Epileptic
50
Dose-Response - Control vs. Epileptic
51
Zinc sensitivity - Control vs. Epileptic
Epileptic Hippocampus
52
(No Transcript)
53
Selectively reducing GABA release at synapses on
interneurons does not affect the tonic GABAA
receptor-mediated conductance
54
Amplitude and decay time of mIPSCs
55
Amplitude and decay time of sIPSCs
56
Pharmacological sensitivity of tonic IGABA in PCs
Tonic IGABA is mediated by ? subunit-containing
GABAA receptors
57
Pharmacological sensitivity of tonic IGABA in the
presence of GABA (5 mM )
The THDOC sensitivity of tonic IGABA is similar
to that observed at low GABAo
THDOC (10 nM)
Fraction of the PTX sensitive Ihold
?Ihold (pA)
PC Ep PC (3) (5)
PC Ep PC (3) (5)
58
Pharmacological sensitivity of tonic IGABA in the
presence of GABAo 5 mM
The zolpidem sensitivity of tonic IGABA increases
during epilepsy
zolpidem (200 nM)



Fraction of the PTX sensitive Ihold
?Ihold (pA)
PC Ep PC (7) (6)
PC Ep PC (7) (6)