2. 2
ABSTRACT
Pannexin1 (Panx1) is a large-pore channel present in the post-synaptic site that has been
implicated in an in vitro model of seizure-like burst firing. However, the precise contribution of
Panx1 to seizure activity remains controversial, with many inconsistent findings. Furthermore,
the contribution of Panx1 to the development of ictogenic nervous tissue (‘epileptogenesis’) has
not been investigated. The present experiments pursue three lines of research in an attempt to
further characterize the role of Panx1 in seizure and epileptogenesis.
The contributions of Panx1 to susceptibility to seizure, seizure severity and seizure
duration were studied in vivo using both the pilocarpine model of seizure and status epilepticus,
and the electrical kindling model of seizure and epileptogenesis. Further, the effect of Panx1
block on the process of epileptogenesis itself in the electrical kindling model was examined.
Finally, the role of Panx1 in local excitability and short-term potentiation following
epileptogenesis were studied using acute hippocampal slices obtained from kindled and sham
kindled rats.
A single early central infusion of Panx1 blocker was sufficient to chronically attenuate
epileptogenesis. Furthermore, Panx1 block acutely decreased seizure severity and afterdischarge
threshold in fully kindled animals in the electrical kindling model. Panx1 block was also found
to acutely decrease seizure severity and increase seizure latency, but not seizure duration in the
pilocarpine model.
Finally, Panx1 block was also found to increase paired-pulse facilitation but not
differentially between kindled and non-kindled tissue.
3. 3
Acknowledgements
Supervisor:
Dr. G. Campbell Teskey
Co-Supervisory:
Dr. Roger J. Thompson
Supervisory Committee:
Dr. Quentin J. Pittman
Dr. Michael A. Colicos
Examining Committee:
Dr. Quentin J. Pittman
Dr. Michael A. Colicos
. Dr. Andrea B. Protzner
Members of the Teskey Lab:
Ryan McCarthy
Kathleen Scullion
Andrew Brown
Bonita Ma
Members of the Thompson Lab:
5. 5
TABLE OF CONTENTS
Abstract 2
Acknowledgements 3
Table of Contents 5
List of Abbreviations 6
Chapter 1 – General Introduction 9
Introduction to Seizures and Epilepsy 9
Introduction to Pannexin Channels 11
Pannexin Channels in Seizure and Epileptogenesis 17
Modelling Seizure and Epileptogenesis 20
Kindling: Phenomenon, Technique and Model 22
Mechanisms of Kindling 26
Pilocarpine Model 32
in vitro Modelling – The Acute Slice 33
Hypotheses and Specific Aims 37
Chapter 2 – Empirical Paper 40
Introduction 40
Methods 42
Results 50
Discussion 64
Conclusion 69
Chapter 3 – General Discussion 70
Chapter 4 – References 76
6. 6
List of Abbreviations
µA microamp
10
panx Pannexin1 blocking mimetic peptide
A1R Adenosine-1 Receptor
aCSF Artificial Cerebrospinal Fluid
AD Afterdischarge
ADD Afterdischarge Duration
Ado Adenosine
ADT Afterdischarge Threshold
AMPA 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid
AMPAR 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid Receptor
ANOVA Analysis of Variance
ATP Adenosine Triphosphate
BDNF Brain Derived Neurotrophic Factor
CA1 cornu ammonis 1
CA3 cornu ammonis 3
cDNA Complementary DNA
CNS Central Nervous System
DG Dentate Gyrus
EEG electroencephalogram
EP Evoked Potential
fEPSP field Excitatory Postsynaptic Potential
GABA Gamma-amino-butyric Acid
GABAR Gamma-amino-butyric Acid Receptor
GFAP Glial Fibrillary Acidic Protein
7. 7
HIV Human Immunodeficiency Virus
I2nd Secondary inward current
IP3 Inositol Triphosphate
ISH in situ Hybridization
KA Kainic Acid
KIP Kindling-Induced Potentiation
KS Kolmogorov-Smirnov
LTD Long-Term Depression
LTP Long-Term Potentiation
M1R Muscarinic Receptor 1
mV millivolt
MW Mann-Whitney
NMDA N-methyl-D-aspartate
NMDAR N-methyl-D-aspartate Receptor
OGD Oxygen Glucose Deprivation
P2XN Purinergic Receptor P2X type N
Panx Pannexin
Panx1 Pannexin1
PBS Phosphate Buffered Saline
PDS Paroxysmal Depolarizing Shift
PFA Paraformaldehyde
pop. Spike population spike
PPR Paired pulse ratio
PS Population Spike
PSD95 Post-Synaptic Density protein 95
8. 8
S.C. Schaffer Collaterals
SE status epilepticus
SEM Standard Error of the Mean
SO stratum oriens
SP/s.p. stratum pyramidale
SR/s.r. stratum radiatum
SS Seizure Severity
STP Short-Term Plasticity
TAT Transactivator of Transcription
trkB tyrosine receptor kinase B
UTP Uridine-5’-triphosphate
vHPC Ventral Hippocampus
9. 9
CHAPTER 1 – GENERAL INTRODUCTION
Introduction to Seizures and Epilepsy
A Brief History of Seizures and Epilepsy
Epilepsy is a neurological disorder affecting the central nervous system, characterized by
abnormal electrical activity that may result in a behavioural seizure (Engel and Pedley, 2008).
The first known reference to epilepsy is in a Babylonian medical text dating to the first
millennium BCE (Wilson and Reynolds, 1990). This text is a part of a larger medical diagnostic
document known as ‘All Diseases’, and attempts to differentiate between types of seizure and
their respective causative demons of possession. Interestingly, the text makes note of many types
of seizure caused by demons which specifically possess children, the infirm, and those with
unresolved sins. This folkloric association was prescient not only of the tendency for epilepsy to
occur predominantly in vulnerable populations (Engel and Pedley, 2008), but of the stigma
against people living with epilepsy (which continues to be a global problem into the 21st
century;
de Boer, 2010).
Epidemiology
In 2001, there were an estimated 175000 Canadians living with epilepsy (Tellez-Zentino
et al, 2004) and a total global prevalence of around 50 million people (Engel and Pedley, 2008).
Cases of epilepsy tend occur with a greater frequency in the developing world (Kotsopoulos et
al, 2002; Bell and Sander 2001). Although the cause of this increased incidence has not been
determined, it is possible that increased exposure to parasitic infection might be associated with
comorbid seizure disorders (Bell and Sander 2001). And though a diagnosis of epilepsy may
occur at any age, the incidence of epilepsy is highest in children under the age of 12 and second
10. 10
highest in seniors over the age of 60 (Kotsopoulos et al., 2002). Together, these two age groups
account for 50% of all emerging diagnoses of epilepsy (Bell and Sander 2001).
Despite the enormous burden epilepsy places on global health, it remains that the
majority of seizures occur in people who do not have epilepsy. An estimated 4% of Americans
will have a single unprovoked seizure before the age of 80 with only 30-40% of those people
going on to have a second unprovoked seizure, obtaining a diagnosis of epilepsy. Seizures are
not benign events (Herman, 2004). It is therefore evident that research into the mechanisms of
seizure and its translation into clinical medicine extend beyond an application to the treatment of
epilepsy alone.
Pathophysiology of Seizures and Epilepsy
A behavioural or clinical seizure is the result of pathological hyperexcitable and
hypersynchronous neuronal activity (Engel and Pedley 2008). A hypothetical generalized
framework for discussing the pathophysiological mechanisms of seizure activity proposes two
conditions which may predispose individuals to seizure. First, there must be a population of
intrinsically hyperexcitable neurons (Engel and Pedley 2008); for example, a neuronal
population which is able to produce intrinsic burst discharges (Najm et al, 2001). Second, there
must be an increase in excitatory synaptic activity, and/or a decrease in inhibitory synaptic
activity in recurrent collaterals, the local network, and/or the projection neurons (Engel and
Pedley 2008, McCormick and Contreras 2001, Najm et al 2001). This shift in the balance of
excitation and inhibition can occur as the result of a number of mechanisms including changes in
gene transcription, alterations of the synapse at the molecular level, changes in synaptic
morphology, changes in neurite morphology, and neurodegeneration (Pitkanen and Lukasiuk
2011, Engel and Pedley 2008, Pitkanen et al 2006, Chang and Lowenstein 2003).
11. 11
The involvement of ion channel mutations in heritable forms of seizure disorder
continues to be characterized (Escayg et al 2000, Sugawara et al 2001, Baulac et al 2001,
Holland et al 2008, Singh et al 2009, and Estacion et al 2010). Mutations in the genes for
voltage-gated sodium channel β1 (Wallace et al 1998), α1 (Escayg et al 2000), and α2 (Sugawara
et al 2001) subunits have been identified in heritable forms of seizure disorder, as well as a
mutation in the γ2 subunit of the inhibitory synaptic ligand-gated chloride ion channel GABAA
(Baulac et al 2001).
Introduction to Pannexin Channels
Pannexins are integral membrane proteins that form hexameric large pores of high
conductance (475-550 pS; Bao et al 2004, Locovei et al 2006) and high permeability (molecules
up to 1kDa). Panx1 is expressed in both neuronal and, controversially, glial cell populations
within the central nervous system (Iglesias et al, 2009, Huang et al, 2009; Figure 1), and is
localized at specific subcellular sites such as the postsynaptic density (Zoidl et al 2007; Figure
1). Until recently, the notion that glial cells express pannexin was controversial as it had only
been observed in expression systems (Huang et al, 2009) and not in vivo (Vogt et al, 2005).
However, Panx1 expression has since been reported to co-immunolabel with glial fibrillary
acidic protein (GFAP, a glial marker) in hippocampal CA1 astrocytes, suggesting glial Panx1
expression (Santiago et al 2011; Figure 1).
Pannexins are expressed broadly in most vertebrate tissues outside the CNS such as taste
buds (Romanov et al 2007, Romanov et al, 2008), the retina (Zoidl et al 2008), and erythrocytes
(Locovei et al 2006), as well as in thyroid, prostate, liver and kidney tissue (Bruzzone et al,
12. 12
2003). There are three known genes encoding three pannexin proteins, each with unique
functions and expression profiles (Bruzzone et al 2003, Baranova et al 2004). For example,
Panx3 may be uniquely transcribed in the skin (Bruzzone et al 2003). Pannexins are known to
have a variety of functions in diverse cell types and at particular subcellular locations. Within the
CNS, Panx1 is localizes to the principle output cells of a number of brain areas. For instance,
they have been observed in pyramidal cells of the cortex and hippocampus (Zoidl et al 2007,
Vogt et al 2005) and on Purkinje neurons within the cerebellum (Ray et al 2006). At the
subcellular level, pannexins localize to the post-synaptic density of both PV+ inhibitory
interneurons as well as GluR2+ pyramidal neurons in the hippocampal CA1 region as well as the
neocortex. (Zoidl et al 2007) implying a role for pannexins in neuronal synaptic and non-
synaptic communication in many major structures of the mammalian CNS (MacVicar and
Thompson, 2010; Figure 1).
The post-synaptic density is a specialized, protein-dense intracellular region attached to
the post-synaptic membrane. It is apposed to the pre-synaptic active zone, and permits synaptic
proteins (such as glutamate receptors and Panx1) to remain in close association with sites of
neurotransmitter release from the presynaptic terminal. Proteins are maintained at this subcellular
location with the aid of scaffold proteins which serve to functionally organize the machinery
necessary for postsynaptic signal transduction. PSD95 is one such scaffolding protein and a
member of the membrane-associated guanylate kinase (MAGUK) superfamily of proteins.
MAGUK proteins are defined in part by their inclusion of specific subdomains such as the PDZ
domain which interacts with NMDARs and anchoring them in the post-synaptic site (Cui 2007).
Also commonly found in MAGUK proteins such as PSD95 are the SH3 and guanylate kinase
domains. Although Panx1 co-immunolocalizes with PSD95 is the post-synaptic density (Zoidl et
13. 13
al, 2007), the scaffolding protein which maintains its presence in the post-synaptic density in
excitatory and inhibitory neurons in the hippocampus and neocortex, as well as the specific
common MAGUK domains which interact with Panx1 remains unidentified. However, Panx1
interaction with actin microfilaments via the Panx1 intracellular c-terminal tail was shown to be
important for trafficking to the plasma membrane and stable presence there (Bhalla-Gehi et al
2010).
Although Panx1 can be activated and opened with a number of modes including via
purinergic receptor stimulation, mechanical stimulation, increases in intracellular Ca2+,
membrane depolarization, and increases in extracellular potassium (Bao et al, 2004; Locovei,
Wang and Dahl, 2006; Reigada et al 2008; Chekeni et al, 2010), the precise biophysical and
biomolecular mechanism by which these stimuli induce Panx1 opening is not known.
Furthermore, opening can be prevented through the use of mimetic peptides (Wang et al, 2007),
and non-specific channel blockers such as probenecid, carbenoxolone, and fluefenamic acid
(Barbe, Monyer, and Bruzzone, 2006; Dando and Roper, 2009). The latter are thought to block
Panx1 function by physically blocking the pore formed by an open Panx1 channel. The former
are thought to block Panx1 opening by binding to a particular location (such as the first
extracellular loop in the case of the 10
panx mimetic peptide), thereby preventing conformational
changes in Panx1 required for channel opening from a closed state, although this mechanism has
not been verified.
14. 14
a
Figure 1. Panx1 expression in the CA1 region of the hippocampus. A) Panx1 colocalizes with
GFAP in astrocytes in the stratum radiatum (modified from Santiago et al 2011). B) Panx1
colocalizes with NeuN in pyramidal neurons in the stratum pyramidale (modified from Santiago
et al 2011). C) Panx1 colocalizes with PSD95, a postsynaptic scaffolding protein in pyramidal
neurons in CA1 of the hippocampus (modified from Zoidl et al 2007). D) Hypothesized
colocalization of Panx1, PSD95, and NMDAR.
15. 15
Discovery of Panx Channels
The Panx protein was first identified based on bioinformatic analysis by Panchin and
colleagues (2000) while attempting to clone murine cDNA based on the invertebrate gap
junction protein, innexin. It was known that gap junctions of vertebrates are composed of
connexin proteins, which had no homologous members within the invertebrate lineage. However,
invertebrates possess gap junctional proteins which have no sequence homology with vertebrate
connexins (Panchin et al, 2000). However, Panchin et al (2000) identified proteins homologous
to innexins in many taxonomic clades including vertebrates. This led them to hypothesize that
they had discovered a new putative gap junction protein in vertebrates. For this reason, they
proposed the name ‘pannexin’ (‘Panx’; from the Latin root words ‘pan’ and ‘nexus’ meaning
‘all’ and ‘bond’ respectively) to replace the previous name of ‘innexin’ (Panchin et al, 2000).
Presciently, they anticipated the relevance of Panx to psychiatric and neurological medicine,
noting that its location on the q21 band of human chromosome 11 has been associated with
schizophrenia (St Clair et al, 1990).
Early research on Panx attempted to characterize the transcriptional profile of Panx
through Northern blot analysis and in-situ hybridization (ISH) to gain insight into its functional
expression (Bruzzone et al, 2003). Panx1 and Panx2 subtypes were found to be coexpressed in
many tissues outside the CNS. The mRNA of the Panx1 and Panx2 genes appeared to localize
predominantly to the hippocampus, cortex, cerebellum, and olfactory bulbs of P15 rat pups, with
additional dense expression of Panx2 in the striatum and thalamus. mRNA for Panx2 appeared to
stain more densely in the cortex than did Panx1 at this age. Additionally, in the hippocampus,
while staining could be observed within the stratum radiatum, stratum pyrimidale, and stratum
16. 16
oriens mRNA seemed to preferentially localize to cells within the stratum pyramidale, perhaps
suggesting a unique role for Panx within the primary output cells of this structure.
The hypothesis that Panxs form gap-junctions was initially investigated in the oocyte
expression system (Bruzzone et al, 2003). Intercellular junctions (suggested to be true gap
junctions) were observed upon injection of Panx1 mRNA into Xenopus oocytes, as well as Panx1
and Panx2 mRNA coinjection. However, Panx2 mRNA injection alone did not result in the
formation intercellular junctions. The Panx1 and Panx2 coexpression system also exhibited
unique physiological characteristics compared to Panx1 or Panx2 alone, such as reduced outward
current during a depolarizing voltage step compared to Panx1 expression alone. Therefore, the
authors suggested that Panxs may form homomeric (Panx1 subunits only) or heteromeric (Panx1
+ Panx2 subunit-containing) hemichannels, which may be expressed in different cell types
and/or have unique functions (Bruzzone et al 2003, Bruzzone et al 2005). Furthermore, Lai and
colleagues (2006) reported Panx1-mediated dye coupling in C6 glioma cells, suggesting the
presence of Panx1 gap junctions. However, dye coupling has yet to be observed in Panx1
expressing cells in vivo and is likely a phenomenon of expression systems. One possibility for
the appearance of Panx1 intercellular channels in these systems is proposed to be loss of proper
post-translational modifications that would prevent Panx1 docking with an apposed hemichannel
from a neighbouring cell and forming an intercellular junction (MacVicar and Thompson, 2010).
The gap-junction hypothesis of pannexins is in dispute (Dahl and Locovei 2006,
MacVicar and Thompson 2010). The conceptual question seems to be whether pannexins
perform adaptively redundant functions to the connexins, or rather perform adaptively unique
functions that merely appear to be redundant at first glance. The frequent inability of pannexin
channels to form gap junctions when studied as well as the many alternative functions of Panx
17. 17
that have been recently elucidated seems to indicate that gap-junction formation is not likely to
occur in vivo. Perhaps the most striking piece of evidence in favour of the non-gap junction
hypothesis of Panx channels is the discovery of Panx expression on erythrocytes (Locovei et al
2006). Therefore Panx is evidently expressed in a cell type that moves freely at the individual
cell level, and does not form gap junctions for intercellular communication (Locovei et al, 2006).
This supports the notion that Panx channels perform a function unrelated to the formation of gap
junctions.
Panx channels possess unique structural components that distinguish them from
connexins. For instance, gap-junction-forming connexin-46 hemichannels are present in the
plasma membrane and are blocked by divalent cations (such as calcium and magnesium) until
they dock with an apposed connexin hemichannel in an adjacent cell (Ebihara et al 2003). Once
docked, the cation block is removed and intercellular communication is permitted. In contrast to
connexin hemichannels, Panx channels appear to be insensitive to extracellular divalent cations,
and open to the extracellular space in physiological ionic conditions (Bruzzone et al 2005),
although Panx1 channels were found to open in response to increases in extracellular potassium
(Santiago et al 2011). However, to strongly conclude that the primary function of Panx is not the
formation of gap junctions, another candidate function ought to be positively identified and
shown to have a more important physiological role.
Indeed, such candidate functions have been proposed and investigated. As previously
discussed, when opened, Panx channels are permeable to molecules up to 1 kDa (Harris et al
2007) and are therefore the candidate pore through which messenger molecules such as ATP and
IP3 are released (Iglesias et al 2009, Locovei et al 2006). Panx has been proposed as the large
pore for ATP release from cultured astrocytes (Iglesias et al, 2009). Some evidence also suggests
18. 18
that Panx1 may be coupled to purinergic receptors (P2X2 and P2X7; Pelegrin and Surprenant
2009, Locovei et al 2007) allowing the receptor to recruit Panx1 to function as a highly
permeable pore in response to an ATP or UTP ligand. However, some research has contradicted
this finding (Yan et al 2008). Panx1 has also been found to be opened by ischemic like
conditions (oxygen-glucose deprivation; OGD) and prior to membrane breakdown during
necrosis (Thompson et al, 2006).
Pannexin Channels in Seizure and Epileptogenesis
The finding that Panx1 channels increase both the frequency and amplitude of interictal
bursting modeled by acute hippocampal slices exposed to a bath solution lacking Mg2+
, is of
particular interest to a mechanistic understand of the contribution of Panx1 to seizure activity
(Thompson et al 2008; Figure 2). Low magnesium exposure causes prolonged activation of
NMDARs in neurons following binding by their endogenous ligands in the hippocampal slice.
This permits the activation of NMDARs without the requirement of other glutamatergic
receptors such as AMPARs or kainate receptors. Blocking Panx1 opening in this condition with
the mimetic peptide 10
panx (Pelegrin and Surprenant, 2007) decreased both the frequency of
interictal bursts as well as the amplitude of the burst spikes (Thompson et al 2008). The
contribution of Panx1 channels to interictal burst activity caused by NMDAR activation suggests
that Panx1 may have a role in normal synaptic physiology by amplifying synaptic responses
(Thompson et al 2008, MacVicar and Thompson 2010; Figure 2).
Interictal spikes are clinically diagnostic of epilepsy (Staley and Dudek 2006). It has been
hypothesized that the correlation between interictal spikes and seizure implies a direct role for
interictal spiking in epileptogenesis; that is, the process by which central nervous tissue becomes
19. 19
more prone to seizure over time (Staley and Dudek, 2006). Thus, the involvement of Panx1 in a
NMDAR mediated model of interictal bursting in vitro suggests a potential involvement of
Panx1 in the pathophysiology of seizure and epileptogenesis (Thompson et al 2008, MacVicar
and Thompson 2010).
Targeting Panx1 in vivo has revealed both pro- and anti-convulsant outcomes following
administration of the convulsants pilocarpine (Kim and Kang, 2011) or kainic acid (Santiago et
al, 2011), respectively. These paradoxical effects are achieved through unique underlying
mechanisms (Figure 2) Furthermore, although these in vivo studies have implicated Panx1 in
seizure susceptibility and severity (Kim and Kang, 2011; Santiago et al, 2012), the relationship
between Panx channel function and epileptogenesis has not been investigated. This is
particularly important in light of the activation of Panx1 by NMDA receptors and the
dependence of epileptogenesis on glutamate signalling (Thompson et al, 2008; Scimemi et al
2006).
20. 20
Figure 2. Four hypothesized mechanisms of Panx1 contribution to seizure activity and
excitability. 1) NMDAR stimulation induces Panx1 opening causing a secondary inward current
(I2nd; Thompson et al 2008). 2) Panx1 opening in response to decreased extracellular glucose
(not shown) releases ATP. ATP is hydrolyzed to adenosine (Ado). Adenosine binds to the A1
receptor (A1R) resulting in the opening of ATP-sensitive potassium channels, causing
hyperpolarization (Kawamura et al 2010). 3) P2X7R activation by IP3-induced intracellular
calcium release (not shown) opens Panx1 clearing IP3 and releasing more ATP to sustain its
P2X7R-induced opening, reducing excitability (not shown, Kim and Kang, 2011). 4) Increased
extracellular potassium due to status epilepticus opens Panx1 channels by an unknown
mechanism (Santiago et al, 2011).
21. 21
Modelling Seizure and Epileptogenesis
Models of disease do not perfectly reproduce every aspect of human disease. Rather,
particular characteristics or suites of characteristics of a disease are emulated. Therefore,
investigators attempt to craft research questions such that they make appropriate use of
techniques that are currently available to them (Pitkanen et al 2006). A researcher may choose a
particular model in order to 1) understand the fundamental mechanisms of a disorder, 2)
rationalize new diagnostic techniques, 3) evaluate the effectiveness of a novel therapy, or 4) to
test ways to prevent disease (Pitkanen et al 2006).
Two preparations which have proven useful in understanding the fundamental
physiological principles underlying seizure activity in the hippocampus include chronically
implanted hippocampal electrodes for in vivo electrophysiological studies such as the electrical
kindling technique (Figure 3, Figure 4) as well as acutely isolated hippocampal preparations for
in vitro electrophysiological studies (Pitkanen et al 2006, Figure 5)).
22. 22
Figure 3. Repeated kindling stimulation increases afterdischarge duration. A) Repeated
daily electrical stimulation of a brain site such as the ventral hippocampus in the limbic system
elicits (B) an electrographic afterdischarge. C) With repeated daily stimulation, afterdischarge
duration and seizure severity (not shown) increase (Adapted from Racine 1972b).
23. 23
Kindling: Phenomenon, Technique, Model
Electrical kindling is a model of seizure and epileptogenesis (McIntyre, 2006). The
kindling technique consists of the daily repeated stimulation of a particular brain area such as the
amygdala or hippocampal region (Goddard, 1969). This stimulation protocol initially elicits a
relatively limited seizure which manifests as highly synchronous EEG activity from a local
recording electrode which outlasts the initial stimulus (Racine, 1972a). This EEG activity is
therefore called an ‘afterdischarge’ (AD). As the kindling technique is applied over many
consecutive stimulation sessions the kindling phenomenon manifests, such that the AD duration
(ADD) and the seizure severity (SS) both increase (McIntyre, 2006; Racine 1972a,b; Figure 3).
It is this progressive alteration in the severity of the EEG and behavioural responses to
repeated electrical stimulation that characterizes kindling (McIntyre, 2006; Figure 3; Figure 4).
By monitoring these parameters either chronically throughout kindling (ADD or SS; Figure 4),
or acutely at various prescribed time points during the kindling process (ADT, ADD, SS), the
contribution of candidate mechanisms of seizure and epileptogenesis can be evaluated and
quantified (McIntyre et al, 2006). An electrically kindled limbic focus is a valid model of partial
seizures (Albright and Burnham, 1980), and has therefore been used to assess the effects of a
number of conventional anticonvulsants on seizure activity (Gilbert, Bharadia, and Teskey, 2001;
Gilbert and Teskey 2001; Gilbert, Corley and Teskey 2002, Serralta et al 2006, Gilbert and
Teskey 2007).
24. 24
Figure 4. Modified Racine scale of seizure severity (Racine 1972b). With repeated kindling
stimulation, rats progressively exhibit behavioural seizures comprising inclusively greater
seizure stages. Expressed seizures are progressive in that a stage 3 contains a stage 1 and 2, a
stage 4 contains a stage 3, 2, and 1, etc.
25. 25
Electrical kindling was discovered by Goddard and colleagues (1969) while they were
attempting to determine the effect of amygdala stimulation on conditioning in rats. By
happenstance, a short, high frequency train of stimulation was repeatedly delivered to the
amygdala in a number of rats, eventually inducing several behavioural seizures which outlasted
the initial stimulus in one rat. Subsequently, a series of experiments were undertaken by Goddard
and colleagues (1969) in order to determine i) the anatomical specificity of electrical kindling, ii)
its permanence, iii) the effect of alternative stimulus parameters and paradigms on kindling
outcomes, iv) the effect of minor and major electrode-tip lesions on kindling, and v) whether the
kindling stimulation-induced effects were ‘transferrable’ to a distal stimulation site (Goddard et
al 1969). Differences between strains, species and other anecdotal observations were also
included.
From these experiments, Goddard and colleagues (1969) concluded that kindling
stimulation results in a seizure threshold reduction at the site of stimulation allowing repeated
application of a subthreshold stimulus to eventually elicit a seizure at the stimulation site. With
repeated application, this stimulus goes on to elicit behavioural ‘tonic-clonic’ seizures which
become progressively more severe, and this progressive increase in severity of the behavioural
seizures was determined to be relatively permanent, relying on a trans-synaptic mechanism
resulting in a ‘complex reorganization of function’. The results from these experiments are
consistent with the hypothesis that seizures occur on a neural substrate that reads this activity and
actively responds to it with a rearrangement of its functional organization to increase
transsynaptic excitability.
26. 26
Together, Goddard and colleagues’ experiments in 1969 provided a foundation for other
researchers to assess the validity and usefulness of electrical stimulation-induced kindled
seizures as a model of seizure and epileptogenesis (Racine 1972a,b,c; Wada and Sato 1974). Not
long after Goddard and colleagues’ seminal publication (1969), the first instance of a
spontaneous seizure state resulting from the kindling technique was demonstrated in the Baboon
species Papio papio (Wada, Ozawa, and Mizoguchi, 1975). This was an important finding
because it demonstrated that a permanently increased susceptibility to seizure as seen in patients
with epilepsy could be induced using the kindling technique if applied long enough and in a
species with a neurology that is adequately labile (Wada, Ozawa, and Mizoguchi, 1975). As
well, it illustrated how a latent epilepsy-like state could be made manifest with the correct
epileptogenic insult in an otherwise normal subject. Further, Wada, Ozawa and Mizoguchi
(1975) confirmed Goddard and colleagues preliminary results from the Macaque in the Guinea
Baboon. These results support the hypothesis that the difference between a person living with
epilepsy and an otherwise normal person may only be a matter of degree of susceptibility to
seizure.
Goddard and colleagues work was prescient of the implications of the kindling model in
that their work has also contributed to our understanding of neuroplastic changes which occur as
a result of seizure (Teskey et al, 2002, Teskey et al, 2008). Somatotopic organization of the
motor cortex is altered when kindling stimulation is delivered to the amygdala (Teskey et al,
2002) or ventral hippocampus (Van Rooyen et al, 2006). People living with epilepsy may
experience a functional reorganization of motor cortex following seizures (Teskey et al, 2008).
Penfield and Rasmussen (1950) mapped the human precentral gyrus in an effort to determine the
seizure focus in patients with epilepsy, and found that the representation of the face was ‘right-
27. 27
side up’ (Teskey et al, 2008). However, Kaas and colleagues (1979) later found the sensory
representation of the face in cortex from both Old-World Monkeys and New-World Monkeys to
be upside-down. This finding was replicated in human populations by using functional MRI to
map the cortex of individuals who had no history of epilepsy, confirming that such subjects have
an upside-down representation of the face in the neocortex similar to our Simian cousins (Servos,
1999). Together these data support the hypothesis, among others, that seizures (spontaneous or
stimulation-induced) effect neuroplastic changes in cortical tissues distant from the focus or site
of kindling. They confirm Goddard and colleagues (1969) results, demonstrating a quantifiable
change in motor system function following daily repeated electrical stimulation or spontaneous
seizures throughout life. Further, these studies establish kindling as a practical technique which
can be used to both investigate the fundamental science of seizure activity, and to test therapies
with the potential to alleviate the suffering of those living with epilepsy.
Mechanisms of Kindling
The previous studies illustrate that the kindling phenomenon emerges as a result of the
application of the electrical kindling technique. Subsequent to the confirmation of this discovery,
interest in the mechanism of kindling emerged (Racine 1972a,b; Douglas and Goddard , 1975;
Racine 1986; Mody and Heinemann, 1987; Mody, Stanton, and Heinemann, 1988). Although no
single identifiable mechanism has proven sufficient to account for the kindling phenomenon,
suites of measurable electrophysiological, morphological, and molecular changes have been
investigated for their contribution and together may constitute a close approximation of the
necessary and sufficient conditions required for kindling to occur (Corcoran and Teskey, 2009).
28. 28
Electrophysiological Mechanisms
The electrophysiological mechanisms that contribute to kindling may be grouped into
three categories: 1) kindling-induced potentiation (KIP) of excitatory drive, 2) the erosion of
inhibitory drive, and 3) the emergence of the kindling-induced burst response.
I. Kindling-induced Potentiation
Kindling-induced potentiation (KIP) is an enhancement of the excitatory post-synaptic
potential or population spike that can occur in a secondary site following an application of the
electrical kindling technique (Maru et al, 2002; Corcoran and Teskey, 2009). There is some
controversy as to whether KIP is necessary for the development of the kindling phenomenon
(Corcoran and Teskey, 2009) particularly in the hippocampus (Maru et al, 2002). However, KIP
has been identified in secondary sites when kindling outside of the limbic system (Racine et al,
1991). Thus, although inconsistently expressed, KIP remains a manifestation of AD-induced
plasticity and epileptogenesis.
Long-term potentiation (or LTP) is another phenomenon related to neural plasticity that
shares some striking conceptual similarities with KIP (Racine et al, 1991; Cain, 1989). LTP
consists of an increase in synaptic efficacy following the delivery of a high frequency train of
stimulation to synaptic afferents. It was first observed and described by Terje Lomo in 1966,
three years prior to Graham Goddard’s description of the kindling phenomenon. Lomo (1966)
recorded evoked potentials (EP) from granule cells in the dentate gyrus of the hippocampus of
cats before and after stimulation of the perforant pathway. An increase in EP amplitude from
granule cells of the dentate gyrus following the stimulus was observed. Both kindling and LTP
involve an increase in neural activity following a high frequency stimulus. Although there are
29. 29
many important differences between the two phenomena (Cain, 1989), both appear to be
expression of the Hebb Rule.
The Hebb Rule was proposed by the eminent Neuroscientist Donald O. Hebb in 1949.
Hebb (1949) postulated that mechanisms might exist which would allow synaptic strength to
increase as a function of the temporal synchrony with which a presynaptic neuron fires onto its
postsynaptic partner. This hypothesis is often summarized as ‘neurons that fire together wire
together’. Since both LTP and kindling may be mediated by Hebb-like processes, the possibility
that similar processes underlie their function was the basis of early investigations into the
mechanisms of kindling (Cain, 1989).
It is clear that both KIP and LTP are useful models for researchers interested in
understanding the mechanisms of neural plasticity. However, there are important conceptual
differences between the two phenomena (Cain, 1989). Most notably, KIP requires the elicitation
of afterdischarge, and potentiates this activity with repeated induction (Racine, 1978), whereas
LTP does not require the elicitation of epileptiform activity, nor does it cause any such events
(Cain, 1989).
II. Disinhibition
The corollary of KIP of excitatory synaptic drive would be a converse erosion of synaptic
GABAergic inhibitory drive at secondary efferent sites (Corcoran and Teskey, 2009; Leung et al
2005). Indeed, blocking fast GABAergic neurotransmission can be a useful experimental tool in
revealing enhancement of excitatory synaptic drive (Esclapez et al, 1999). Similar to the
hypothesis predicting increased excitatory drive, the decrease in inhibitory drive does not appear
equally in all secondary sites following kindling (Schinnick-Gallagher et al, 1998, Leung, et al
30. 30
2005). However, erosion of inhibition does appear to apply within the hippocampus and
amygdala (Schinnick-Gallagher et al 1998, Leung et al, 2005). Inconsistent GABAergic
disinhibition is also apparent in chronic temporal lobe epilepsy, with some sites demonstrating
robust and even enhanced GABAergic drive, while others show a decrease (Bernard et al, 2000;
Cossart et al, 2001; Esclapez et al, 1998).
III. Bursting
Neurons isolated from a seizure focus often exhibit an electrical ‘bursting’ response
(Westbrook, 2000). Bursting is a form of periodic neural activity that consists of a group of
action potentials which occur due to a neural event known as a ‘paroxysmal depolarizing shift’
(PDS; Johnston and Brown, 1984). The PDS is a depolarization of the neuronal membrane of
about 20-40 mV from the resting potential, lasting on the order of 50 to 200 ms (Johnston and
Brown, 1984; Westbrook, 2000; Wong et al, 1984). A train of action potentials known as a burst
is often produced at the peak of depolarization during a PDS (Westbrook, 2000). Bursting within
PDSs can be measured from single and populations of neurons both intracellularly and from field
potentials, respectively (Johnston and Brown, 1984). Intracellularly, the PDS is often
immediately followed by a hyperpolarization. The early depolarization component is caused
predominantly by AMPAR and NMDAR-mediated excitatory signals and is correlated with a
relatively high calcium ion conductance (Lothman, 1993a; Wong et al, 1984). The late
hyperpolarization phase which limits the duration of the early phase is often mediated by
GABAR inhibitory signals and is correlated with a relatively high potassium ion conductance
(Wong et al, 1984). Along with neurons isolated from a seizure focus, healthy neurons from
particular areas of the brain exhibit PDS under normal conditions, and a similar
31. 31
excitatory/inhibitory response pairing can be observed in cortical neurons when evoked by an
excitatory stimulus (Lothman, 1993a; Westbrook, 2000).
PDS-induced burst discharging is not only a property of neurons at a seizure focus, as
well as a limited number of healthy neurons, but importantly, though perhaps not surprisingly, is
associated with kindled neurons (Johnston and Brown, 1984). Bursting can be observed
prominently in the amygdala, piriform cortex, the substantia nigra (Corcoran and Teskey, 2009).
Bursting is a neural correlate of the interictal discharge observable between seizures events on
the EEG of patients with epilepsy, as well as the electrical activity at the level of individual cells
that occurs during an AD and is therefore considered an important component in the mechanism
of kindling (Corcoran and Teskey, 2009). Increased PDS-induced bursting is thought to be
predominantly the result of changes at the network level rather than the level of individual
neurons (the exception being changes in the intrinsic properties of granule cells within the
dentate gyrus; Johnston and Brown, 1984). That is, kindling may induce changes in excitation
and inhibition as well as changes in network properties to permit larger populations to exhibit
PDSs with greater frequency and synchrony. This may result in an increase in the frequency of
interictal burst firing as well as a progressive increase in the duration and character of AD that is
seen in kindling.
Morphological Mechanisms
Kindling does not result in, and does not depend on any significant neural damage
(Corcoran and Teskey, 2009). Rather, changes in synaptic morphology have been proposed to
account for the electrophysiological and behavioural phenomena associated with kindling (Sutula
et al, 1989; Geinisman et al, 1992; Henry et al, 2008). Specifically, an increase in the number of
32. 32
perforated synapses has been observed in the dentate gyrus following hippocampal kindling
(Geinisman et al, 1992), as well in layer V of the sensorimotor neocortex following kindling of
the corpus callosum (Henry et al, 2008) demonstrating the generalisability of the morphological
mechanisms of kindling from the hippocampus to the neocortex.
Molecular Mechanisms
Particular neurotransmitter systems seem to be uniquely involved in mediating the
kindling phenomenon. GABAergic tone, as previously mentioned, is variably decreased
following kindling as is the contribution of noradrenergic function (Giorgi et al, 2004). Indeed
data from numerous experimental lines indicate an anti-epileptic role for noradrenaline, such that
kindling rates are increased when the noradrenergic system is interfered with (Giorgi et al,
2004). On the other hand, excitatory (calcium-ion dependent glutamatergic) drive appears to be
augmented following kindling (Jarvie et al, 1990). As well, the trkB receptor and its ligand
‘brain-derived neurotrophic factor’ (BDNF) appear to be critical factors in kindling
epileptogenesis, such that when trkB receptor is knocked out epileptogenesis ceases (He et al,
2004). As BDNF is a neurotrophic molecule, this may implicate its downstream neurotrophic
effects on neuronal morphology and dendritic outgrowth, in kindling epileptogenesis (He et al,
2004).
These results concerning the mechanisms of kindled seizures and epileptogenesis
highlight its continued importance in therapeutic discovery, its insight into human epilepsy, and
the insight it offers into the fundamentals of neural function. Epilepsy research using the kindling
phenomenon as a model of epileptogenic neural plasticity is a prime example of the overlap
between basic and translational research.
33. 33
Pilocarpine Model
The pilocarpine model of acute seizure, status epilepticus and epileptogenesis has
experienced increasing popularity in recent years (Cavalheiro et al, 2006). It shares some
characteristics with electrical kindling, in that pilocarpine induces epileptogenesis which results
in a chronic increased susceptibility to seizure following a latent period, and that it is generally
considered to model some aspects of human temporal lobe epilepsy (Curia et al, 2008). Indeed,
the pilocarpine model which consists of a single systemic injection of the muscarinic
acetylcholine receptor agonist (Cavalheiro et al, 2006) results in acute seizure activity which may
progress from motor arrest to fully generalized tonic-clonic convulsions, similar to the
epileptogenic progression seen across kindling sessions (Curia et al, 2008). If the experimental
subject survives the acute phase, it can be followed by recovery and a ‘latent’ period wherein no
behavioural seizures are observed, which in turn is followed by a ‘chronic’ phase characterized
by increasingly severe spontaneous behavioural seizures (Curia et al, 2008). Behavioural
seizures induced by pilocarpine are accompanied by AD at a limbic focus (Young et al, 2009),
and hippocampal sclerosis in the chronic phase (Curia et al, 2008) similar to known
characteristics of temporal lobe epilepsy and consistent with its use as a model of that syndrome
(Curia et al, 2008).
However, there exist important differences between the electrical kindling and
pilocarpine models. As opposed to kindling, the pilocarpine model causes seizure-induced
damage and subsequent network reorganization more quickly. Induced primarily by sustained
status epilepticus lasting several hours during the acute phase, histopathological changes such as
necrosis, atrophy and dendritic sprouting are observable in the olfactory cortex, amygdala,
thalamus, hippocampus, and neocortex following pilocarpine treatment (Curia et al, 2008). Thus,
34. 34
as opposed to kindling, the pilocarpine model necessarily results in greater CNS damage causing
epileptogenesis.
Electrical kindling is known to result in a functional reorganization of afferents of the
seizure focus (Henry et al, 2006; Teskey et al, 2002). Kindling with primary stimulation sites in
the corpus callosum, the amygdala, and the hippocampus are all known to persistently alter the
functional organization of the sensorimotor neocortex following seizure propagation to this site
(Teskey et al, 2002; Van Rooyen et al, 2006). Similarly persistent changes in the sensorimotor
neocortex have been noted following administration of pilocarpine in Wistar rats, and transiently
following pilocarpine-induced SE in Long-Evans rats (Young et al, 2009). This strain difference
presents the possibility that genetic variability may account for differences in neuroplastic
functional reorganization following seizure. Furthermore, it highlights important similarities
between electrical kindling and pilocarpine as models of temporal lobe epilepsy and all of its
neuroplastic sequelae.
In vitro Modelling – The Acute Slice
At around the same time that Bliss and Lomo were first investigating the long-term
potentiation (LTP) model of neural plasticity (1973), the development of a technique to remove
and acutely analyse brain slices was being explored for the first time (Yamamoto and McIlwain,
1966). This reduced preparation allowed relatively easy experimentation with hitherto
inaccessible systems and circuits, all the while preserving much of the local circuitry intact
(Bernard, 2006). There are some advantages to studying electrophysiology in acute slices.
Although field potentials can be recorded in vivo as in early LTP studies (Lomo, 1966) these
extracellular recordings can be obtained with a greater degree of control and accuracy in the
35. 35
acute slice setting (Bernard, 2006). Furthermore, slice techniques allow visual access to
individual neurons within local brain sites of interest and allows the precise positioning of
stimulating and recording electrodes necessary for intracellular recording techniques such as the
patch clamp (Hamill et al, 1981; Figure 5).
Despite these advantages, there are some inherent limitations to the technique. Care must
be taken to remove slices thin enough to allow tissue perfusion of oxygen and artificial
cerebrospinal fluid, but thick enough to preserve circuitry and function (Bernard, 2006).
Furthermore the tight spatiotemporal coupling of neural activity and cerebral blood flow
(Chaigneau et al, 2003) is completely lost in the slice technique. However, it is perhaps unfair to
criticise an experimental technique when its supposed advantages overlap with its disadvantages.
Acute slice preparations are inherently reductionist (Bernard, 2006). A technique is not
absolutely better or worse, rather it is more or less appropriate to the research question to which
it is applied. When a research question can best be answered by a reduced model, such as
questions concerning intrinsic properties of individual neurons, or concerning local excitability
of pools of neurons with stereotyped synapses, then it is appropriate to use such a model. Thus, if
extrahippocampal circuitry is critical to the type of seizure activity being studied, then the acute
slice may not be appropriate (Bernard, 2006). Nonetheless, interictal and ictal epileptiform
discharges can be studied and compared across experimental or therapeutic manipulations
(Anderson et al, 1986; Traynelis and Dingledine, 1988; Cohen et al, 2002; Thompson et al,
2008).
There are a number of ways to study epilepsy acutely in the hippocampal slice
preparation. In general however, the strategies can be lumped into one of two categories: 1) acute
slices prepared from normal (non-epileptic) animals which are manipulated in vitro to exhibit
36. 36
epileptiform activity, or 2) acute slices obtained from patients with epilepsy, or from animals
with genetic epilepsies, seizure disorders, or rendered epileptic using experimental models of
seizure disorders such as have been described above (Bernard, 2006). Field potentials or
intracellular electrophysiology can be studied passively or in response to electrical or chemical
stimulation (Bernard, 2006; Figure 5).
Epileptiform activity can be elicited from acute slices using a number of techniques. A
method of naively kindling acute slices can be accomplished by delivering tetanic, high-
frequency stimulation to the Schafer collaterals in the stratum radiatum of CA1 in the
hippocampus (Rafiq et al, 1993). A model of electroencephalographic AD can be produced by
this stimulation-induced bursting (Bragin et al, 1997; Rafiq et al, 1993). Eliciting AD in acute
slices provides the opportunity to study local circuit synchronization; in particular the
involvement of mediators of excitatory signals such as NMDARs, as well as inhibitory mediators
such as GABAR (Stasheff et al, 1993; Bracci et al, 1999).
Removing magnesium ions from artificial cerebrospinal fluid (aCSF) allows NMDARs to
respond directly to glutamatergic stimulation, and eventually comes to elicit stable, spontaneous
interictal-like discharges from acute slice being studied (Mody et al, 1987). Low magnesium has
been reported from patients with epilepsy (Durlach, 1967) suggesting clinical implications for
this particular acute slice model. Ictal discharges can be observed with an in toto preparation of
the hippocampus exposed to magnesium ion-free aCSF (Quilichini et al, 2002). These
preparations are highly useful in testing anti-epileptic or anti-convulsant therapies as the
dependent variables (burst frequency and amplitude) are readily recordable before and after a
particular treatment (Bernard, 2006).
37. 37
Figure 5. Stimulation protocol for eliciting evoked field potentials in acute hippocampal
slices. A) A wave form of known, brief duration (0.1msec) and variable amplitude is applied to
the (B) Schaffer collaterals via a glass stimulating electrode. C) The stimulation induces an
excitatory postsynaptic field potential recorded from the dendritic arbor of CA1 pyramidale
neurons in the stratum radiatum of CA1. D) The evoked potential recording is made up of the
field potential (measured as the slope of the rising phase in red), and the population spike
(measured as the potential difference between the PS trough and a line drawn between its
flanking peaks.
38. 38
Bathing slices in aCSF containing high potassium ion concentration (~8.5mM) results in
the appearance of spontaneous epileptiform activity (Traynelis and Dingledine, 1988). This
increase in extracellular potassium is in the same order of magnitude as increases in potassium
ions experienced by hippocampal neurons during seizure activity in vivo (Fisher and Alger,
1984). This particular technique is useful in that it increases excitability among most neural
populations and networks (Fisher and Alger, 1984). Mixed models of epileptiform activity in
acute slices can be generated by combining techniques. For example, 0 Mg2+ aCSF can be made
isotonic with regular aCSF by compensating for the lack of magnesium ions by replacing them
with potassium ions, elevating the potassium ion concentration from 3.5mM to 5.0mM
(Thompson et al, 2008).
Acute slices may also be obtained from animals treated with in vivo models of seizure,
SE and epileptogenesis, such as the electrical kindling or pilocarpine models (Bernard, 2006).
Such slices could be treated with bath solutions described in the previous paragraphs and
differences could be observed (Figure 5).
Hypotheses and Specific Aims
My long-term goal is to understand how seizure-induced Panx1 activation contributes to
epileptogenesis, and how epileptogenesis, in turn, alters the contribution of Panx1 to seizure
activity. The specific objective of this thesis was to determine how Panx1 affects
epileptogenesis, and how the contribution of Panx1 to seizure activity is altered following
epileptogenesis. The central hypothesis was that Panx1 amplifies and intensifies seizure
activity, and that interfering with this activity would attenuate measures of seizure
susceptibility and severity. This was based in part upon previous studies which suggested that
39. 39
Panx1 is recruited by epileptiform activity and amplifies such activity by increasing the
amplitude and frequency of neuronal firing through a mechanism that also mediates synaptic
plasticity (Thompson et al, 2008).
Epileptogenesis may refer to dynamic processes which alter patterns of neuronal
excitability, reorganizes connectivity, and induces critical structural changes which ultimately
result in increases in seizure susceptibility, severity or duration (Pitkanen and Lukasiuk, 2011).
The rationale for pursuing this hypothesis in the experiments that follow is that once it is known
how and to what extent Panx1 can influence the course of epileptogenesis, therapeutic strategies
based on interfering with Panx1-mediated mechanism can be pursued in order to improve the
quality of life of people living with epilepsy. Further, by understanding how Panx1 contributions
to measures of seizure activity are changed following epileptogenesis, and the population-level
changes in excitability that correlate with these changes, we will better be able to determine how
Panx1 contributes dynamically to the mechanisms underlying the generation and spread of
seizures in people already diagnosed with epilepsy.
I pursued these unexplored opportunities with the following novel aims:
Specific Aim 1. To determine the contribution of the Panx1 channel to the process of
epileptogenesis. Hypothesis: That the Panx1 channel mediates a seizure activity-dependent
mechanism that is important for epileptogenesis.
a) Determine the contribution of Panx1 channel-opening to kindling epileptogenesis by
blocking this activity with a specific peptide and recording the number of kindling
stimulation sessions required to for the animal to experience 3 stage 5 seizures as well as
the rate at which rats from each group reach this criterion.
40. 40
Specific Aim 2. To determine the contribution of Panx1 channel to acute seizure activity
and local field potential excitability and following epileptogenesis. Hypothesis: That the
Panx1 channel-opening is mediated by seizure activity, and that this effect mediates a
mechanism that is important in increasing seizure susceptibility
a) Determine the differential contribution of Panx1 to acute seizure activity following
epileptogenesis by blocking Panx1 with a specific peptide prior to administration of
kindling stimulation-induced seizures in rats naïve to stimulation, in partially kindled rats
(having experienced a single stage 3 seizure), and in fully kindled rats (having
experienced 3 stage 5 seizures).
b) Determine the differential contribution of Panx1 to local field excitability and short-term
plasticity near the seizure focus in fully-kindled rats compared to age-matched controls
by stimulating acute hippocampal slices with a paired-pulse protocol at increasing
stimulation intensities and recording the amplitude and paired-pulse ratio of the slope of
the field excitatory postsynaptic potential as well as the resulting population spike.
c) Determine the contribution of the Panx1 channel to acute seizure activity in the seizure-
naïve state in vivo by blocking Panx1 with a specific peptide prior to administration of
the chemical convulsant Pilocarpine and recording the latency, duration and severity of
the resulting behavioural seizure.
41. 41
CHAPTER 2 – EMPIRICAL PAPER
Introduction
Epileptogenesis is defined as the process by which neuronal excitability is progressively
altered, rendering nervous tissue more prone to seizure (Goddard, 1969; Pitkanen and Lukasiuk,
2011). This process occurs following any of a variety of events or insults (Pitkanen and
Lukusiak, 2011), even by seizure activity itself (Dudek and Shao, 2003). Researchers have
explored the molecular mechanisms of epileptogenic plasticity with the goal of preventing
epileptogenesis (Temkin, 2001; Pitkanen et al, 2005; Pitkanen et al, 2007; Bortel et al, 2010) .
However, the prophylactic prevention of epileptogenesis remains an elusive therapeutic target for
the 50 million people who suffer from epilepsy worldwide (Pitkanen and Lukasiuk, 2011).
Pannexin1 (Panx1) is a large pore high conductance (~500pS) ion channel that has been
implicated in experimental models of seizure activity both in vitro (Thompson et al, 2008) and in
vivo (Kim and Kang, 2011; Santiago et al, 2011). Panx1 block was shown to reduce the
amplitude and frequency of NMDAR-activation dependent epileptiform activity in vitro
(Thompson et al, 2008). Furthermore, Panx1 block differentially increases and decreases seizure
severity in vivo resulting from pilocarpine (Kim and Kang, 2011) and kainic acid (Santiago et al,
2011) administration, respectively, in murine models. Further, in addition to these conflicting
findings, the relationship between Panx1 function and epileptogenesis has not been investigated.
This is particularly important in light of the activation of Panx1 by NMDA receptors and the
dependence of epileptogenesis on glutamate signalling (Thompson et al, 2008; El-Hassar et al,
2007).
42. 42
The slowly progressive nature of electrical kindling allows for a finely controlled study
of the process of epileptogenesis. In this model, repeated electrical stimulation of a central
nervous system structure elicits electrographic afterdischarges and behavioural seizures that
become progressively longer and more severe (Goddard, 1969; McIntyre, 2006; Racine, 1972b).
In rats, kindling stimulation initially elicits seizures having behavioural characteristics which are
similar to the characteristic motor symptoms associated with focal partial seizures in humans
(McIntyre, 1970; McIntyre, 1979). Following successive daily stimulation, the elicited
behavioural seizures become more similar to characteristic motor involvement and severity of
complex partial seizures with secondary generalization (McIntyre, 1970; McIntyre, 1979).
Furthermore, the kindling and pilocarpine models induce neuroplastic and subsequent functional
changes at efferent motor sites that are correlated with the severity of the stimulus induced by the
convulsants (Teskey et al, 2002; Young et al, 2009) These results suggest that electrical kindling
and the pilocarpine-induced seizures model the epileptogenesis of secondary generalization from
a primary seizure focus.
The present study explores the hypothesis that Panx1 contributes directly to the process
of epileptogenesis itself, as well as to seizure activity differentially following epileptogenesis.
These innovative results suggest that a single early treatment with Panx1 blocker is sufficient to
significantly retard kindling epileptogenesis, as well as to acutely reduce seizure susceptibility
and severity. Further, they indicate that Panx1 blockade affects seizure severity and seizure
susceptibility differentially following epileptogenesis via a mechanism that does not rely on
Panx1-mediated changes in the neuronal excitability of the seizure focus.
43. 43
METHODS
Animals
Adult male Wistar (n= 32; 250-400 grams) and Long-Evans (n= 30, 235-320 grams) were
obtained from Charles River (St Constant, PQ, Canada). Wistar rats were housed in groups of
three in standard shoebox cages in a colony room that was maintained on a 12 hour light cycle,
with lights on at 7:00 am. Long-Evans rats were individually housed in the same conditions. All
experiments were conducted during the light phase. Rats received food and water ad libitum and
were handled and housed according to the Canadian Council for Animal Care (CCAC)
guidelines and approved by the local Health Sciences Animal Care Committee.
Electrical Kindling Experiments
Electrode and cannula implantation
Adult male Long-Evans rats were each chronically implanted on the right side with a
stimulating/recording electrode and an infusion cannula. Twisted bipolar stimulating/recording
electrodes were constructed from Teflon-coated stainless steel wires having a diameter of 178
µm. The uninsulated ends of exposed wire were attached to male gold amphenol pins and the
electrode tips at the opposing end were separated by at least 0.5mm. Rats were anaesthetized
using isoflurane (5% induction, 2% maintenance), then mounted into the stereotaxic frame. 2%
Lidocaine was administered subcutaneously beneath the incision site prior to making the 1.5 inch
incision. The stimulating/recording electrode was implanted in the ventral hippocampus
according to the stereotaxic coordinates of Paxinos and Watson (1982; 4.5 mm posterior to
bregma, 4.5mm lateral to midline, and 7.0mm ventral from brain surface). The cannula was
44. 44
implanted into the right lateral ventricle (0.8mm posterior to bregma, 1.5 mm lateral to midline,
and 3.7 mm ventral from brain surface). 5 anchoring screws and 1 ground screw were placed in
the frontal, parietal and temporal bones, and the electrode and cannula were cemented to the
screws using dental acrylic. The free male amphenol pins were inserted into a nine-pin McIntyre
connector plug (Ginder Scientific, Ottawa, Ontario, Canada). Rats were allowed to recover from
surgery for a minimum of 7 days prior to afterdischarge determination.
The positioning of the electrode was confirmed by post-hoc visual inspection. For the
latter, rats were perfused with physiological phosphate-buffered saline (PBS) followed by 4%
w/v paraformaldehyde (PFA). Fixed brains were then excised and hand-sliced with a razor for
coarse visual inspection. The approximate location of the terminal end of the bipolar electrode
track was recorded.
Afterdischarge determination
On the first day of kindling, the afterdischarge (AD) threshold (ADT) was determined for
every subject. The ADT is defined as the minimum amount of current required to elicit an AD
which lasts longer than 4 seconds. Current was delivered through the ventral hippocampal
electrode and recordings were made from the same. Stimulation consisted of a one second train
of 60 Hz biphasic square wave pulses, each leading and lagging pulses both 1 ms in duration,
with the lagging pulse entrained to initiate 2 ms after the initiation of the leading pulse.
Afterdischarge threshold determination began at 50 μA , and was increased in 25 µA increments
delivered every minute until an AD which lasts longer than 4 seconds was observed. Kindling
stimulation eliciting an afterdischarge was delivered twice daily at a current intensity 100 μA
greater than the determined ADT with at least 4 hours separating each stimulation session. EEG
45. 45
was recorded from the ventral hippocampal electrode to determine afterdischarge duration.
Seizure behaviour was scored according to a 5 point ordinal scale of motor seizure progression
(Racine, 1972b).
Kindling
Afterdischarge threshold was determined at three time points throughout the extent of
each rat’s kindling lifetime. The ADT was evaluated the day following their initial ADT
determination (‘Naive’), the day following their first stage 3 seizure (‘Partially Kindled’), and
the day following their third stage 5 seizure according to the Racine scale (‘Fully Kindled’,
Racine 1972b). On each day, prior to ADT determination, subjects received 5.0 uL
intracerebroventricular administration of 7.5 mg/mL 10
panx in 0.9% saline or 0.9% saline
vehicle. The treatment was injected using a 10 µL Hamilton syringe and microdrive system at a
rate of 1.0 μL/min for 5 minutes. Following the 5 minute injection time, the treatment infused for
3 additional minutes, then the injection cannula was removed, and the dummy cannula inserted
into the guide cannula. The rat was then placed in a faraday cage, and the ADT determined. The
seizure stage and ADD was also recorded.
Pilocarpine Experiments
10
panx – selective inhibitor of Panx1 hemichannels
In order to assess the effect of the Panx1 channel block on acute seizure and status
epilepticus, the selective Panx1 pore blocker ‘10
panx’ was administered prior to delivery of the
chemical convulsant pilocarpine. TAT-10
panx panx is a mimetic peptide which binds to the first
46. 46
extracellular loop of Panx1 selectively, thereby blocking the flux of permeant molecules
(Pelegrin and Surprenant 2007). Wistar rats were treated in pairs with 10
panx (7.5 mg/kg, Tocris
Bioscience) or saline vehicle. 10
panx was administered intraperitoneally (i.p.) in sterile 0.9%
saline 30 minutes prior to injection with pilocarpine (see below).
Pilocarpine-induced seizures and status epilepticus
All rats received atropine methyl-nitrate (10 mg/kg) injected intraperitoneal (i.p.) 30
minutes prior to pilocarpine or saline treatment in order to block the effects of pilocarpine on
peripheral muscarinic receptors. Pilocarpine (360 mg/kg) or physiological saline was
administered intraperitoneally. The dosages were selected based on previous reports (Hort et al.,
1999, 2000; Šroubek et al., 2001; Dykstra et al., 2007).
Following drug administration, seizure behaviour was video recorded for 60 minutes,
observed and scored during both the recording session and post hoc. The measured parameters
were 1) latency to behavioural seizure, 2) seizure duration, and 3) seizure severity. The seizure
severity scale consists of the following stages: Stage 1 – mouth and facial movements, Stage 2 –
head nodding, Stage 3 – unilateral forelimb clonus, Stage 4 – bilateral forelimb clonus and
rearing, Stage 5 – a stage 4 with falling episodes (Racine, 1972). Diazepam (10 mg/kg, i.p.) was
administered either after SE or 45 minutes following convulsant injection to attenuate seizure
activity and promote survival. All rats were also given 5 ml of 5% dextrose in physiological
saline to replace electrolytes and facilitate recovery until brain extraction could occur. Later that
same day, the rats were humanely euthanized with a 1 mL injection of Euthanyl, perfused with
phosphate-buffered saline followed by a 4% paraformaldehyde solution and their brains
extracted.
47. 47
In Vitro Experiments
Acute Hippocampal Slices
To determine differences in the excitability of kindled compared to non-kindled
hippocampal foci, acutely isolated hippocampal slices were prepared from Long-Evans rats
(“Naive”, n=8), from Long-Evans rats (“Sham Controls”, n=5), and from rats that had exhibited
three stage 5 behavioural seizures as scored according to the Racine scale (‘Fully Kindled’,
Racine 1972b) male Long-Evans rats (n=5). Under isoflurane anesthesia, brains were removed
and placed in cold slicing solution consisting of (in mM) 215 NaCl, 2.5 KCl, 26 NaHCO3, 10
MgCl2, 1.6 NaH2PO4, 10 D-glucose, and 215 sucrose, that was bubbled with 5% CO2/95% O2.
Horizontal hippocampal slices (370 μm) were cut using a vibratome and maintained for 45 min
in a warm (37°C) recovery solution composed of artificial CSF (aCSF) (in mM): 120 NaCl, 26
NaHCO3, 3 KCl, 10 Glucose, 1.25 NaH2PO4, 1.3 MgSO4, and 2 CaCl2, and continuously
bubbled with 5% CO2/95% O2 to maintain a pH of 7.4. After a minimum of 45 minutes, slices
were transferred to a recording chamber that was continuously perfused with aCSF at 32°C.
Extracellular field potentials were recorded with glass micropipettes filled with aCSF (2–3 MΩ)
and signals were acquired using an Axopatch 200B amplifier (Molecular Devices; low-pass
filter, 10 kHz; high-pass filter, 1 Hz; acquisition frequency, 10 kHz; gain, 500×). Evoked field
EPSPs (fEPSPs) were elicited in the CA1 stratum radiatum region of the hippocampus by
electrically stimulating the Schaffer collaterals (CA3 collateral axonal projections to CA1) with a
monopolar glass pipette electrode filled with aCSF at 50% maximal stimulation (the point at
48. 48
which the fEPSP slope did not increase further with increasing stimulation was taken as 100%
maximal stimulation).
Statistics
The alpha levelfor all statistical tests listed below is p<0.05.
Acute Electrical Kindling Experiments
The difference in ADT and ADD between naive, partially-kindled, and fully-kindled
Panx-blocked and control rats was analysed using two-tailed unpaired t-tests. However, when
analysing the ADTs of fully kindled rats, the data failed the Kolmogorov-Smirnov test for
normality likely due to a floor effect in the Panx1-blocked rats. The Mann-Whitney test, a non-
parametric test, was used in lieu of the t-test in this instance.
The difference in SS between Panx1-blocked and controls rats was also analysed using
two-tailed unpaired t-tests. Again, when analysing the SS of fully kindled rats, the data failed the
Kolmogorov-Smirnov test for normality likely due to a ceiling effect in control rats. The Mann-
Whitney test, a non-parametric test, was used in lieu of the t-test in this instance.
Pilocarpine Experiments
The difference in the severity of the first seizure between Panx1-blocked and control rats
was analysed using a two-tailed t-test. The difference in the severity of the most severe seizure
between Panx1-blocked and control rats was analysed using a one-tailed t-test.
The difference in seizure latency between Panx1-blocked and control rats was analysed
using a one-tailed t-test. As well, the difference in seizure duration of both the first observed
49. 49
seizure and the cumulative total seizure duration during the one hour observation period between
Panx1-blocked and control rats was analysed using a two-tailed t-test.
In vitro Experiments
The differences in standardized fEPSP slope between kindled and sham-kindled slices
were determined across a range of stimulus intensities (100µA, 150µA, 200µA, 300µA, 500µA,
750µA, 1000µA) to derive a plot of input stimulus intensity versus output fEPSP slope. This
protocol was performed in normal aCSF, after the peptide Panx1 blocker 10
panx was washed on,
and again in normal aCSF to wash off the 10
panx. The input/output curve was analysed using a
6x3 ANOVA with follow up Tukey’s tests between all columns. A paired-pulse protocol was
used to elicit fEPSPs. The paired pulse ratio (PPR) was calculated as the quotient of the
standardized slope of the fEPSP resulting from the second stimulus over the first. PPRs were
analysed across the same range of stimulus intensities as the fEPSP slopes (discussed above).
The differences in standardized population spike amplitude between kindled and sham-
kindled slices were also determined across a range of stimulus intensities (100µA, 200µA,
300µA, 500µA, 750µA, 1000µA) to derive a plot of input stimulus intensity versus output
population spike amplitude. This protocol was performed in normal aCSF, after the peptide
Panx1 blocker 10
panx was washed on, and again in normal aCSF to wash off the peptide blocker.
The input/output curve was analysed using a 6x3 ANOVA with follow up Tukey’s tests between
all columns. A paired-pulse protocol was used to elicit population spikes. The paired pulse ratio
(PPR) was calculated as the quotient of the population spike amplitude resulting from the second
stimulus over that elicited by the first. Population spike amplitude and PPRs were analysed
across the same range of stimulus intensities as the fEPSP slopes (discussed above).
50. 50
Epileptogenesis in Electrical Kindling
The difference in the number of days required to fully kindle rats treated with Panx
blocker as compared to control rats was analysed using a two-tailed unpaired t-tests. The
differences in kindling rates between these two groups were compared using survival analysis,
with the third stage 5 seizure elicited as the termination criterion. The subsequent significant
hazard ratio from this analysis was calculated as the ratio of the drop-out rate of control rats to
that of Panx blocker-treated rats.
51. 51
RESULTS
A single central infusion of Panx1 blocker chronically attenuates epileptogenesis
Epileptogenesis was measured as the number of stimulation induced seizures required to
elicit three stage 5 seizures as scored according to the Racine scale (‘fully kindled’; Racine,
1972b), as well as the rate at which subjects became fully kindled. A single administration of
Panx1 blocker prior to assessing the afterdischarge threshold in naive rats significantly increased
the mean (+/- SEM) number of kindling sessions required for rats to have their third stage 5
seizure (58.71 +/- 15.06 kindling sessions) compared to vehicle treated controls (29.86 +/- 4.53
kindling sessions; t=1.835, df=12, p=0.0457; Figure 1a).
Furthermore, survival analysis indicates that rats receiving a single treatment with Panx1
blocker (median survival = 47 kindling sessions) become fully kindled at a significantly slower
rate than did vehicle-treated controls (median survival = 27 kindling sessions, median survival
ratio = 0.57; Mantel-Cox Test, χ2
= 3.865, df = 1, p=0.0495; Figure 1b). The rate at which
subjects became fully kindled (that is, the ‘hazard ratio’ for epileptogenesis to 3 stage 5 seizures)
was 3.74 times higher in vehicle controls compared to centrally Panx1 blocked rats.
52. 52
Figure 1. A single central infusion of Panx1 blocking peptide (7.5mg/kg) has the chronic effect
of (A) increasing the number of kindling sessions required for the rat to exhibit three stage 5
seizures(‘fully kindled’), and (B) decreasing the rate at which rats become fully kindled
compared to vehicle control.
53. 53
Acutely, Panx1 block has pro- and anti-convulsant effects in the electrical kindling model
In the electrical kindling model (Figure 2a), the afterdischarge threshold (ADT) was
determined following administration of the Panx1 blocker in naive rats, in partially kindled rats,
and in fully kindled rats (Figure 2b,c). Panx1 block did not significantly change the mean (+/-
SEM) ADT (t=0.048, df=15, p=0.9620) in naive rats (146.9 +/-32.88 µA) compared to vehicle-
treated controls (144.4 +/- 37.22 µA), nor in partially kindled rats (t=0.75, df=9, p=0.4742; 100.0
+/-17.68 µA) compared to vehicle-treated controls (125.0+/-26.61 µA). However, Panx1 block
significantly decreased the ADT (KS distance = 0.3588, p<0.05; U = 4.00, p=0.0409) in fully
kindled rats (62.50 +/- 12.50 µA) relative to vehicle-treated controls (115.6 +/20.01 µA).
The AD duration (ADD) was determined from the ADs elicited when determining the
ADT in naive, partially kindled and fully kindled rats (Figure 2d). Panx1 block did not
significantly change the mean (+/- SEM) ADD in naive rats (41.13 +/- 9.76 seconds) compared
to vehicle-treated controls (46.33 +/- 14.87 seconds; unpaired two-tailed t-test, t=0.285, df=15,
p=0.7797), in partially kindled rats (32.00 +/- 7.24 seconds) compared to vehicle controls (55.00
+/- 14.18 seconds; unpaired two-tailed t-test, t=1.354, df=9, p=0.2087), or in fully kindled rats
(65.75 +/- 10.23 seconds) compared to vehicle controls (68.88 +/- 10.58 seconds; t= 0.186,
df=10, p=0.8562).
The seizure severity (SS) was determined from the behavioural seizure concomitant with
the AD elicited when determining the ADT in naive, partially kindled and fully kindled rats, and
scored according to a five-point ordinal scale (Figure 2e). Panx1 block did not change the mean
(+/- SEM) SS in naive rats (stage 0.88 +/- 0.35) compared to vehicle-treated controls (0.56 +/-
0.34; two-tailed unpaired t-test, p=0.3356), or in partially kindled rats (stage 1.4 +/- 0.40)
compared to vehicle-treated controls (stage 1.6 +/- 0.48; U = 17.00, p=1.000). However, Panx1
54. 54
block significantly decreased the SS in fully kindled rats (stage 5.0 +/- 0.0) compared to vehicle-
treated controls (stage 3.5 +/- 0.87; U = 8.00, p=0.0492).
55. 55
Figure 2. Acute Panx1 block decreases seizure severity and afterdischarge threshold (ADT) but
only in fully kindled rats. A) With repeated daily stimulation of the ventral hippocampus, the
elicited AD becomes longer. B) Sample traces of ADs elicited while determining the ADT under
control conditions and under Panx1 block in fully kindled rats. C) Panx1 block significantly
decreases ADT but only in fully kindled rat. D) Panx1 block has no effect on AD duration
(ADD). E) Panx1 block significantly decreases behavioural seizure severity, but only in fully
kindled rats.
56. 56
Acute Panx1 block is anti-convulsant in the pilocarpine model
In order to determine the contribution of Panx1 to acute behavioural seizure, a Panx1
blocker (10
panx) or 0.9% saline vehicle were administered to 24 adult male Long-Evans rats
followed by the convulsant pilocarpine. Pilocarpine injection induced seizures in fewer rats
treated with 10
panx (4 of 12 total) than in rats treated only with saline vehicle (8 of 12). Of the
rats that exhibited seizures, administration of the Panx1 blocker significantly increased the mean
(+/- SEM; t=2.220, df=10, p=0.0253) latency to the first seizure (1642 +/- 422.4 seconds, N=4)
induced by pilocarpine, compared to the mean seizure latency of vehicle treated controls (815 +/-
166.8 seconds, N=8; Figure 3a). Further, treatment with Panx1 blocker significantly decreased
the mean (+/- SEM; t=2.236, df=10, p=0.0492) severity of the first seizure observed (stage 3 +/-
0.58, N=4) compared to vehicle controls (stage 4.5 +/- 0.4, N=8), and significantly decreased the
mean severity (t=2.066, df=10, p=0.0392) of the most severe recorded seizure (stage 3.8 +/-
0.6), compared to vehicle controls (stage 4.8 +/- 0.16; Figure 3c).
The Panx1 blocker did not significantly change the mean duration (+/- SEM) of either the
first seizure (t=0.1060, df=10, p=0.9177) or the sum duration of all seizure bouts in the total 60
minutes of observation (t=0.1004, df=10, p=0.9220) in rats treated with 10
panx (317.8 +/- 294.1
seconds and 650.3 +/-321.6 seconds, respectively), compared to vehicle controls (286.8 +/- 149.1
seconds and 692.1 +/- 246.3 seconds, respectively; Figure 3b).
57. 57
Figure 3. Panx1 significantly increases seizure latency and decreases seizure severity in the
pilocarpine model of seizure and status epilepticus. A) The latency to the first observed
behavioural seizure is increased under acute Panx1 block. B) The severity of both the first
seizure and the most severe recorded seizure of only those rats exhibiting behavioural seizure are
decreased under acute Panx1 block. C) Panx1 block does not change the duration of the first
seizure nor the sum duration of all observed seizure bouts.
58. 58
Panx1 block alters short-term plasticity but not excitability at SC-CA1 synapses
Field potentials were evoked by paired-pulse stimulation of the Schaffer collaterals
across a range of stimulus intensities in hippocampal slices obtained from both kindled and
sham-kindled rats. The percent increase in fEPSP slope with increasing stimulus intensity was
used as a measure of evoked potential magnitude standardized across samples by allowing the
rising slope of the potential evoked at 1000uA to be 100%. These results were analyzed with a
6x2 ANOVA to compare kindled and sham-kindled rats across the range of stimulus intensities
(Figure 4), and using a 6x3 ANOVA in kindled and sham-kindled rats (Figure 5).
Kindling does not alter evoked field potentials
A two-way ANOVA was conducted to examine the effect of increasing stimulation
intensity and kindling on the amplitude of the evoked field excitatory postsynaptic potential
(fEPSP) measured as the percent change in fEPSP slope from baseline. No significant
interaction between the two factors was observed (F(5,48)=0.07794, p=0.9953). Neither was
there a significant effect of stimulation intensity (F(1,48)=1.072, p=0.3058) or kindling (F(5,
48)=1.029, p=0.4114) on the percent change in fEPSP slope (Figure 4a,b).
Kindling does not alter evoked fEPSP PPR
A two-way ANOVA was conducted to examine the effect of increasing stimulation
intensity and kindling on the paired-pulse ratio of the evoked fEPSP as calculated by the quotient
of the percent increase of the second fEPSP slope over that of the first. No significant interaction
between the two factors was observed (F(5,48)=1.060, p=0.9318). Neither was there a significant
effect of stimulation intensity (F(1,48)=0.6552, p=0.2442) or kindling (F(5, 48)=1.167,
p=0.4079) on the percent change in fEPSP PPR (Figure 4c).
59. 59
Figure 4. Kindling does not significantly change the amplitude of the field excitatory
postsynaptic potential (fEPSP) standardized to the slope of the stimulus-evoked potential at 1000
uA compared to sham-kindled controls across a range of stimulation intensities(B).
Representative traces of evoked potentials elicited with 300 uA stimulation are shown in A. C)
Kindling does not significantly alter the PPR of the standardized fEPSP slope across a range of
stimulation amplitudes.
60. 60
Panx1 block does not alter evoked field excitability in kindled or sham-kindled slices
A two-way ANOVA was conducted to examine the effect of increasing stimulation and
Panx1 block on the percent change in fEPSP slope from baseline in sham-kindled rats. No
significant interaction between the two factors was observed (F(10,71)=0.04903, p=1.000).
Neither was there a significant effect of stimulation intensity (F(5,71)=1.124, p=0.338) or Panx1
block (F(2, 71)=1.234, p=0.3025) on the percent change in fEPSP slope (Figure 5a).
A two-way ANOVA was conducted to examine the effect of increasing stimulation and
Panx1 block on the percent change in fEPSP slope from baseline in kindled rats. No significant
interaction between the two factors was observed (F(10,64)=0.1094, p=0.9997). Neither was
there a significant effect of stimulation intensity (F(5,64)=1.791, p=0.5260) or Panx1 block
(F(2,64)=0.6490, p=0.1272) on the percent change in fEPSP slope (Figure 5b).
Panx1 block does not alter evoked fEPSP PPR in kindled or sham-kindled slices
A two-way ANOVA was conducted to examine the effect of increasing stimulation and
Panx1 block on the percent change in fEPSP slope PPR from baseline in sham-kindled rats. No
significant interaction between the two factors was observed (F(10,65)=0.9947, p=0.8838).
Neither was there a significant effect of stimulation intensity (F(5,65)=0.8164, p=0.3778) or
Panx1 block (F(2, 65)=0.3960, p=0.9608) on the PPR of the percent change in fEPSP slope
(Figure 5c).
A two-way ANOVA was conducted to examine the effect of increasing stimulation and
Panx1 block on the percent change in fEPSP slope PPR from baseline in kindled rats. No
significant interaction between the two factors was observed (F(10,64)=0.8887, p=0.4056).
Neither was there a significant effect of stimulation intensity (F(5,64)=0.6273, p=0.3351) or
62. 62
Figure 5. Panx1 block does not significantly alter the slope of the standardized fEPSP compared
to normal aCSF in either slices obtained from (A) sham-kindled or (B) kindled rats across a
range of stimulus intensities. Panx1 block does not alter the PPR of the slope of the standardized
fEPSP in either slices obtained from (C) sham-kindled or (D) kindled rats across a range of
stimulus intensities.
63. 63
Neither kindling nor Panx1 block alter population spike amplitude
A two-way ANOVA was conducted to examine the effect of kindling and Panx1 block on
population spike amplitude elicited by a 200µA stimulus. No significant interaction between the
two factors was observed (F(2, 21)=0.1299, p=0.8789). Neither was there a significant effect of
kindling (F(1, 20)=0.4356, p=0.6526) nor Panx1 block (F(2,20)=0.7285, p=0.4030) on
population spike amplitude (Figure 6a).
Panx1 block but not kindling alters the population spike amplitude PPR
A two-way ANOVA was conducted to examine the effect of kindling and Panx1 block on
population spike amplitude PPR elicited by two 200µA stimuli delivered consecutively with a 50
msec interval. No significant interaction between the two main effects was observed
(F(2,20)=0.1353, p=0.8743). Further, kindling did not significantly alter population spike PPR
(F(1,20)=1.740, p=0.0395). However, a significant main effect of Panx1 block was observed
(F(2,20)=3.814, p=0.2020). Although Bonferroni-corrected follow-up tests failed to determine a
difference in any of the three comparisons (aCSF vs aCSF+10
panx, aCSF vs aCSF wash, or
aCSF+10
panx vs aCSF wash), the largest difference was between aCSF + 10
panx and aCSF wash
in kindled slices (t=1.991, p>0.05; Figure 6b).
64. 64
Figure 6. A) Representative traces demonstrating paired-pulse facilitation under Panx1 block. B)
Panx1 block does not significantly change the amplitude of the population spike elicited at a
stimulation intensity of 200uA compared to those elicited under normal aCSF in slices either
obtained from control (sham-kindled) nor kindled slices. C) A significant main effect of
treatment with Panx1 blocker (10
panx) on the paired-pulse ratio of the population spike elicited in
acute hippocampal slices obtained from both control (sham-kindled) and kindled rats
65. 65
DISCUSSION
I have discovered entirely novel contributions of the Panx1 channel to both seizure and
epileptogenesis. Here, I report the pioneering finding that the contribution of Panx1 to seizure
activity is not only changed following epileptogenesis, but that Panx1 also contributes to the
process of epileptogenesis itself. Further, the current study confirms the anti-convulsant
properties of Panx1 block in the pilocarpine model of seizure and status epilepticus. Finally, a
role for Panx1 in the short term plasticity of synchronous firing at SC-CA1 synapses was
revealed, but cannot explain the increase in excitability observed following Panx1 block in
kindled animals. However, further studies are required to elucidate the underlying mechanisms
which explain the many reported contributions of Panx1 to excitability at the population level
within the focus. As well, understanding the mechanism whereby Panx1 interference results in a
persistent decrease in epileptogenesis could offer many ways to prevent the ‘kindling’ of
epileptic tissue from initially mild convulsant insults.
Previous studies have explored the molecular basis for the observed role of Panx1 in
seizure activity in a number of models. Panx1 contributes to an in vitro model of interictal
bursting dependent on NDMAR-potentiation by removal of Mg2+
from the aCSF bath solution
alleviating the NMDAR pore-block by that ion. When Panx1 was blocked in this model, the
frequency and amplitude of the burst firing was reduced but not eliminated. The frequency and
amplitude of burst firing partially increased again upon wash off of the Panx1 peptide blocker
(Thompson et al, 2008). These results are consistent with a model in which Panx1 partially
mediates synchrony and excitability at the population level. They are also consistent with the
possibility that Panx1-mediated effect may be the result of a contribution of Panx1 to synaptic
activity following NMDAR stimulation. NMDAR stimulation results in a secondary inward
66. 66
current that is mediated by Panx1 (Thompson et al, 2008), and although both Panx1 and
NMDAR are expressed extrasynaptically, the co-expression of Panx1 with PSD95 (a post-
synaptic scaffolding protein associated with NMDAR; Zoidl et al 2007) is consistent with a
model where NMDAR and Panx1 are closely associated at that subcellular location. This line of
evidence and reasoning was the initial impetus for subsequent studies to investigate the
contribution of Panx1 to seizure activity in vivo, including the present studies (Kawamura et al,
2010, Kim and Kang 2011, Santiago et al 2011).
Panx1 plays a role in mediating a multi-step mechanism of autocrine regulation in
hyperexcitable neurons (Kawamura et al 2010). In a series of experiments the researchers
demonstrate that Panx1 mediates a mechanism of purinergic autoregulation of CA3 neuronal
excitability. In conditions where sufficient ATP is present in neurons, Panx1 mediates a release
of ATP in response to a reduction of extracellular glucose (such as high neuronal activity). ATP
is hydrolysed to adenosine, and adenosine activates A1 receptors on the neuron releasing ATP as
well as its neighbours. Through G-protein coupled action, the A1R opens ATP-sensitive K+
channels at the post-synaptic site, causing a local hyperpolarization of neurons, reducing their
firing rate (Kawamura et al 2010). This is a potential mechanism of anti-convulsive action of the
ketogenic diet, since one of the conditions under which a reduction in extracellular glucose might
not result in a decrease in intracellular ATP is when carbohydrates are replaced by a high-fat,
high-protein diet producing ketone bodies (Kawamura et al, 2010). However, this study did not
explicitly investigate a mechanism whereby low extracellular glucose might open Panx1
channels (Kawamura et al 2010).
Despite these results demonstrating a role for Panx1 in decreasing neuronal excitability,
Panx1-mediated augmentation of aberrant, ictal-like burst firing via a mechanism whereby it
67. 67
induces a secondary depolarizing inward current remained uncontested on the population and
single cell level (Thompson et al, 2008). Thus, further studies investigated the involvement of
Panx1 in seizure activity in vivo (Kim and Kang, 2011; Santiago et al 2011). The P2X7 receptor
is a member of a family of ionotropic receptors which open in response to ATP-binding
(‘purinergic’, Locovei et al 2007, Pelegrin and Surprenant 2006). It also uniquely responds to
purinergic stimulation by gradually opening the Panx1 channel (Pelegrin and Surprenant, 2006).
Using pharmacological and genetics interventions in mice, Kim and Kang (2011) demonstrated
that P2X7R inhibition increase seizure susceptibility in the Pilocarpine model of seizure and
status epilepticus. Furthermore, the researchers demonstrated that Panx1 block increased
behavioural seizure severity and EEG afterdischarge power, but did not do so above and beyond
the increased severity seen with P2X7R inhibition (Kim and Kang 2011). They propose a
mechanism whereby muscarinic acetylcholine M1 receptor stimulation by pilocarpine releases
inositol triphosphate (IP3) into the cytoplasm where it binds to IP3 receptors on the endoplasmic
reticulum to increase intracellular calcium resulting in Panx1 opening. Panx1 opening may
permit the efflux of IP3, limiting the M1 receptor-mediated responses. Furthermore, ATP efflux
through Panx1 activates P2X7Rs which both desensitizes M1Rs to limit the duration of IP3
production, and maintain Panx1 opening for the efflux of IP3 from the cell (Kim and Kang,
2011).
The effect of Panx1 targeting with a non-specific blocker as well as genetic knockouts
have been studied in an additional model of seizure and status epilepticus using the chemical
convulsant kainic acid (KA, Santiago et al 2011). In this series of experiments, elevated
extracellular potassium, such as is known to occur during seizure, was found to open Panx1
channels. The novel finding that glia express Panx1 in vivo was confirmed by immunolabelling
68. 68
of Panx1 and glial fibrillary acidic protein (GFAP; a glial marker) in astrocytes in hippocampal
CA1 (Santiago et al 2011). Panx1-mediated dye flux was shown to increase during status
epilepticus, confirming that Panx1 opens during prolonged seizure activity. Finally, Panx1 block
was found to decrease seizure severity but not latency to forelimb clonus following KA
administration. These results are consistent with a proconvulsant role of Panx1 channels as
hypothesized from the early in vitro work (Santiago et al 2011, Thompson et al 2008) However,
results presented in this chapter demonstrate an increase in the latency to behavioural seizure
which was not observed by Santiago and colleagues (2011) which may be attributable to the
different criteria used by the studies. Whereas, Santiago and colleagues (2011) used forelimb
clonus onset as a metric of behavioural seizure susceptibility, the method adopted in this chapter
uses motor arrest as a metric, a behaviour which commonly occurs prior to unilateral forelimb
clonus (Racine 1972b). Motor arrest may thus be a more sensitive measure of behavioural
seizure initiation than forelimb clonus.
Here we report for the first time, the novel finding that a single administration of Panx1-
blocker is sufficient to significantly and persistently reduce kindling epileptogenesis. This
finding is particularly important for two reasons: 1) clinically-relevant anti-epileptogenic
therapies are currently an unmet clinical need and 2) no previous studies have successfully
identified an anti-epileptogenic effect following a single dose of anti-convulsant (see review
Pitkanen and Lukasiuk, 2011). The notion that some types of seizure disorders are progressive in
nature (that ‘seizures beget seizures’; Gowers, 1881), has been confirmed both in animal models
of seizure and epileptogenesis as well as in human populations (see review Pitkanen and Sutula,
2003;Ben-Ari et al 2008). Symptomatically-progressive epilepsy is most common in patients
experiencing seizures of temporal-lobe origin (Hauser, 2008). In one study, nearly 50% of
69. 69
patients receiving anti-convulsant therapies continued to experience uncontrolled seizures after 3
years of therapeutic intervention, a subset of these patients experiencing progressive,
epileptogenic features such as increased seizure frequency and cognitive decline (Collaborative
Group for the Study of Epilepsy, 1994). These results presented in this chapter suggest that a
therapeutic intervention targeting the complex process of epileptogenesis can persistently
attenuate the progressive nature of seizure disorders, and may have the potential to improve the
quality of life for those continuing to live with this condition.
The mechanism by which Panx1 could mediate such a robust response remains an
important target of understanding for future research. The discovery and more detailed
description of the precise mechanism by which Panx1 acts to alter the course of epileptogenic
neuroplasticity not only has the potential to offer many possible therapeutic targets in addition to
the one described here, but also to offer a more precise understanding of the process itself. It will
be interesting to determine if Panx1 block of kindling epileptogenesis generalizes to other
models involving status epilepticus and traumatic brain injury. If this effect is confirmed in
additional models of epileptogenesis, it is likely to increase not only the clinical relevance of
these findings, but also provide additional platforms for the investigation of the specific
mechanism facilitating this novel effect.
It is likely that Panx1 mediates some mechanism which reads increases in neuronal
activity such as occur during seizure activity and translates them into persistent reorganization of
circuitry at the synaptic or population levels (Pitkanen and Lukasiuk, 2011). Future studies will
also investigate the mechanism by which Panx1 comes to be functionally recruited at the focus
and efferent circuits to provide its anti -and pro-convulsant modes, respectively. From the results
of the current study, the notion that Panx1 block can decrease seizure severity in the naïve state
70. 70
and that this effect is increased following epileptogenesis are now established. However, the
system-level mechanism by which Panx1 differentially contributes seizure spread is currently
unknown.
CONCLUSION
The present study reveals the important and entirely novel finding that a single
administration of Panx1 blocker is sufficient to significantly and persistently attenuate the course
of kindling epileptogenesis. Further, it provides the additional novel insights that Panx1 block
decreases seizure severity in the pilocarpine model of seizure and status epilepticus as well as in
the kindling model following epileptogenesis. Panx1 was also found to both decrease and
increase seizure susceptibility, in the pilocarpine model and the kindling model following
epileptogenesis, respectively. Finally, kindling did not cause a change in Panx1-dependent
excitability or short-term plasticity at CA3-CA1 synapses. However, a novel Panx1-mediated
decrease in short-term plasticity of the population spike was discovered in slices from both
kindled and sham kindled rats. Together these results argue for the importance of Panx1 in
epileptogenesis as well as in acute seizure activity and synaptic plasticity.
71. 71
CHAPTER 3 - GENERAL DISCUSSION
A researcher often attempts to rationalize the best model with which to study a particular
disease process in order to improve his or her odds of finding a result that has value and may be
published. Although researchers may simply attempt to pick the model which is most likely to
reveal a positive results, more often an attempt is made to pick a model which can clearly
provide an answer to a research question which is important or interesting regardless of whether
or not the null hypothesis is rejected. This can help to mitigate the risk of engaging in research
into important unknowns.
Of the experiments discussed in Chapter 2, by far the greatest risk was in investigating
the effect of Panx1 block on kindling epileptogenesis. No previous studies had investigated this
phenomenon, which made the positive result difficult to predict. Furthermore, the labour-
intensive nature of electrical kindling requires a commitment of one to two months of twice daily
stimulation sessions lasting approximately 10 minutes per animal. The animals must also survive
an early surgery in order to place a chronically implanted electrode and cannula into the
hippocampus and ventricle, respectively. The dental acrylic headcap that is generated is then
stressed daily with torsion when connecting the headcap to the EEG lead, as well as when the
animal experiences a stage 5 seizure. The animal repeatedly falls due to the loss of postural
control, oftentimes stressing the headcap, which can lead to electrode displacement, or loss of the
headcap entirely.
Specific strategies have been developed to mitigate some of the aforementioned risks. For
example, the use of a headcap having 6 anchoring screws helps to limit further restrict the range
of motion of the implanted electrode and prevents headcap loss. Other times, risks are absorbed
by the researcher. Regardless, the opportunity to investigate epileptogenesis in this finely
