the role of gap junctions in brain glucose …...1.2 mechanisms of glucose metabolism in the brain...

The Role of Gap Junctions in Brain Glucose Deprivationand Glucose Reperfusion

by

Sonia Rebecca Sugumar

A thesis submitted in conformity with the requirementsfor the degree of Master of Science

Graduate Department of PhysiologyUniversity of Toronto

c© Copyright 2014 by Sonia Rebecca Sugumar

Abstract

The Role of Gap Junctions in Brain Glucose Deprivation and Glucose Reperfusion

Sonia Rebecca Sugumar

Master of Science

Graduate Department of Physiology

University of Toronto

2014

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population

and can result in various neurological sequalae including seizures, coma, and brain

death. There is still a limited understanding of the generation and propagation of

hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal dam-

age. Moreover, glucose reperfusion after a period of transient hypoglycemia has been

shown to cause neuronal hyperexcitability which can have further damaging effects.

Gap junctional communication can be involved in the spread of hypoglycemic injury

in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily

propagate and 2) by engaging the astrocytic network in metabolic compensation as

well as enhancing astrocytic buffering of K+. This study aims to investigate the

role that gap junctions play during brain energy deprivation. Results from these ex-

periments show that gap junction blockade can have a neuroprotective role during

hypoglycemia and glucose reperfusion.

ii

Acknowledgements

I would like to first and foremost wholeheartedly thank my supervisor, Dr. Peter

Carlen, for his unending encouragement, thoughtful advice, and eternal optimism

during my time as a graduate student in his lab.

Many thanks to my supervisory committee member and advisor Dr. Liang Zhang

for his valuable input into every aspect of this project from planning experiments to

developing the hypothesis and editing this thesis.

A big thank you also to Dr. Adria Giacca for her perspectives and guidance as a

member of my supervisory committee.

This work would not have been possible without the past and present members

of the Carlen Lab who have become like a second family. Thank you all for your aca-

demic insights, hours of interesting conversation, and moral support. In particular,

I would like to thank Dr. Shanthini Mylvaganam not only for her tireless adminis-

trative support but also for her valued life advice. A special thanks to Dr. Carlos

Florez and Dr. Victor Lukankin for teaching me various experimental techniques, to

Joshua Dian for teaching me everything I know about computer programming and

electronics, and to Margaret Maheandiran for sharing her hypoglycemia expertise.

Lastly, I would like to sincerely thank my parents, sister, and friends for their

steadfast support and devotion during this time.

iii

Contents

1 Introduction 1

1.1 Causes and Effects of Brain Glucose Deprivation . . . . . . . . . . . . 2

1.1.1 Common Causes of Brain Glucose Deprivation . . . . . . . . . 2

1.1.2 Clinical Symptoms of Brain Glucose Deprivation . . . . . . . . 3

1.2 Mechanisms of Glucose Metabolism in the Brain . . . . . . . . . . . . 4

1.2.1 Physiological Mechanisms . . . . . . . . . . . . . . . . . . . . 4

1.2.2 Cellular Mechanisms of Brain Glucose Deprivation . . . . . . . 4

1.2.3 Glucose Reperfusion Injury . . . . . . . . . . . . . . . . . . . 8

1.3 Characterization of Hypoglycemic Seizures . . . . . . . . . . . . . . . 9

1.3.1 Brief Description of Seizures . . . . . . . . . . . . . . . . . . . 9

1.3.2 Clinical Trials and Case Studies . . . . . . . . . . . . . . . . . 10

1.3.3 In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3.4 In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.4 Brief Description of Gap Junctions (GJs) . . . . . . . . . . . . . . . . 16

1.4.1 Composition and Expression . . . . . . . . . . . . . . . . . . . 16

1.4.2 Gap Junction Pore Characteristics . . . . . . . . . . . . . . . 16

1.4.3 Connexin (Cx) and Pannexin (Px) Hemichannels . . . . . . . 17

1.4.4 Gap Junctions in Ischemia . . . . . . . . . . . . . . . . . . . . 18

iv

1.4.5 Characteristics of GJ Blockers Used . . . . . . . . . . . . . . . 18

1.4.6 Role of GJs in Seizures . . . . . . . . . . . . . . . . . . . . . . 21

1.4.7 Possible Involvement of Connexin and Pannexin Hemichannels 23

1.5 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1.5.1 Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1.5.2 Outline of Experiments . . . . . . . . . . . . . . . . . . . . . . 24

2 Rationale and Hypotheses 26

2.1 Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.2 Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3 Materials and Methods 28

3.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.2 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.2.1 Tissue Preparation . . . . . . . . . . . . . . . . . . . . . . . . 29

3.3 Extracellular Electrophysiology . . . . . . . . . . . . . . . . . . . . . 30

3.3.1 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.3.2 Induction of Hypoglycemia and Blocker Application . . . . . . 31

3.3.3 Glucose Reperfusion and Blocker Application . . . . . . . . . 32

3.4 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.4.1 Slice Inclusion Criteria . . . . . . . . . . . . . . . . . . . . . . 32

3.4.2 Seizure Inclusion Criteria . . . . . . . . . . . . . . . . . . . . . 32

3.4.3 Spectrogram Production . . . . . . . . . . . . . . . . . . . . . 33

3.4.4 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . 33

4 Results 34

4.1 Characterization of Hypoglycemic Seizures . . . . . . . . . . . . . . . 34

v

4.1.1 Electrographic Seizure and Evoked Potentials . . . . . . . . . 34

4.1.2 Spectral Features of Hypoglycemic Seizures . . . . . . . . . . 36

4.2 Effects of Gap Junction Blockers on Hypoglycemic Seizures . . . . . . 38

4.2.1 Electrographic Features and Evoked Potentials . . . . . . . . . 38

4.3 Effect of Pannexin Blockade on Hypoglycemic Seizures . . . . . . . . 39

4.4 Characterization of Glucose Reperfusion Seizures . . . . . . . . . . . 40

4.5 Effects of Gap Junction Blockers on Glucose Reperfusion Seizures . . 41

5 Discussion and Conclusions 57

5.1 Seizures and Synaptic Failure . . . . . . . . . . . . . . . . . . . . . . 57

5.2 Controversial Role of Gap Junctions in Seizures . . . . . . . . . . . . 60

5.3 Possible Mechanisms of Glucose Reperfusion Seizures . . . . . . . . . 61

5.4 Mefloquine as a Selective Blocker . . . . . . . . . . . . . . . . . . . . 63

5.5 Conclusions and Future Experiments . . . . . . . . . . . . . . . . . . 63

Bibliography 65

vi

List of Figures

1.1 Glucose transport from blood to neurons and astrocytes . . . . . . . 5

1.2 Mechanism for hypoglycemia-induced neuronal death . . . . . . . . . 7

4.1 Continuous LFP recording of hypoglycemic seizure. . . . . . . . . . . 36

4.2 Propagation of DC Shift . . . . . . . . . . . . . . . . . . . . . . . . . 37

4.3 Evoked field potentials during hypoglycemia . . . . . . . . . . . . . . 42

4.4 Spectrogram of hypoglycemic seizure . . . . . . . . . . . . . . . . . . 43

4.5 Continuous LFP recording during low-glucose + octanol . . . . . . . 45

4.6 Evoked field potentials during hypoglycemia + octanol . . . . . . . . 47

4.7 Continuous LFP recording during low-glucose + mefloquine . . . . . 49

4.8 Evoked field potentials during hypoglycemia + mefloquine . . . . . . 51

4.9 Summary of experimental protocols . . . . . . . . . . . . . . . . . . . 52

4.10 Quantitative features of seizures . . . . . . . . . . . . . . . . . . . . . 52

4.11 Continuous LFP trace showing glucose reperfusion seizures . . . . . . 53

4.12 Evoked field potentials during reperfusion experiment . . . . . . . . . 55

4.13 Continuous LFP trace showing glucose reperfusion + CBX . . . . . . 55

vii

Chapter 1

Introduction

Hypoglycemia as a result of excess insulin can have severe neurological sequalae in-

cluding seizures, coma, and brain death. Children and adolescents are particularly

susceptible to hypoglycemic seizures, which can cause irreversible neurological deficits

throughout development. Gap junctions are not only implicated in the generation

and propagation of seizures, but also hypoglycemia-induced excitotoxicity leading to

neurodegeneration. This thesis aims to characterize hypoglycemic and glucose reper-

fusion seizures in a new in vitro brain slice model and test the effects of gap junction

blockade in both these conditions.

The following introduction will provide a focused review of the relevant hypothe-

ses, studies, and mechanisms used to generate the objectives of this thesis.

1

Chapter 1. Introduction 2

1.1 Causes and Effects of Brain Glucose Depriva-

tion

The body’s ability to rigorously control insulin levels is critical for the homeostasis

of blood and tissue glucose, which in turn is important for proper body function[34].

The breakdown of this tight regulation can lead to either over or undersupply of glu-

cose, resulting in a spectrum of morbidities caused by both of these non-euglycemic

conditions. Hyperglycemia, the most commonly recognized and researched symptom

of Type I Diabetes Mellitus, can be treated with aggressive insulin therapy, however

this method has the associated risk of causing hypoglycemia, which, when left unat-

tended, can be fatal[44, 37]. The damaging effects of severe hypoglycemia on the brain

ultimately complicates the otherwise straightforward management of diabetes[32].

1.1.1 Common Causes of Brain Glucose Deprivation

The most common cause of hypoglycemia is excess insulin[33], either endogenous (eg.

tumours in insulin-secreting pancreatic cells, congenital hyperinsulinism) or exoge-

nous (insulin overdose)[91, 73]. However, other disorders involving the malfunction

of glucose transporters can also lead to this low-glucose condition. Many Type I di-

abetic patients can be asymptomatically hypoglycemic up to 10% of the time, and

exhibit symptoms of hypoglycemia twice a week, with an average of one severe episode

per year[35]. Hypoglycemia-related deaths have been known to occur in 2-4% of the

Type I diabetic patient population.


1.1.2 Clinical Symptoms of Brain Glucose Deprivation

Physiologically, the body uses glucose as a carbon-based energy substrate, which it

then metabolizes to produce ATP, the main transporter of chemical energy[73]. Al-

though it only accounts for 2% of the body weight, the brain uses 25%[93] of the circu-

lating glucose pool because of its high energetic demand and low capacity for energy

synthesis or storage. Astrocytes and neurons require high levels of ATP, primarily

to support synaptic activity, which requires proper functioning of energy-dependent

enzymes and transporters, and replenishment of the metabolite pool and ion gradi-

ents disrupted by postsynaptic potentials[4]. Thus, it is one of the organs that is

the most acutely influenced by fluctuations in blood glucose levels. Normally, blood

glucose is maintained between 3.9 and 7.1 mM (0.8-2.3 mM in the brain)[63, 45, 8].

During episodes of hypoglycemia, when blood glucose drops below 2 mM, the cerebral

glucose concentration reduces to nearly zero[26], resulting in an array of neurologi-

cal complications. Sensations of warmth, weakness, fatigue, difficulty thinking, and

confusion are all neuroglycopenic symptoms that accompany mild to moderate hy-

poglycemia, whereas more prolonged or intense degrees of glucose deprivation can

cause irreversible changes in brain function and can result in seizures, coma, and

death[35, 95]. Moreover, recurrent hypoglycemia can lead to hypoglycemia unaware-

ness during which glucose counter-regulation is impaired and clinical indicators of

moderate hypoglycemia are absent, increasing the probability of severe hypoglycemic

episodes[35].


1.2 Mechanisms of Glucose Metabolism in the Brain

1.2.1 Physiological Mechanisms

During the resting state, glucose travelling through the blood and entering the cen-

tral nervous tissue first crosses the blood-brain-barrier by facilitated diffusion via

the glucose transporter 1 (GLUT-1) into the extracellular space, through which it

navigates to reach its neuronal and glial membrane targets[92, 151]. Studies on neu-

rovascular coupling have demonstrated that energy dependent glucose uptake occurs

through a strong association between neuronal activity, local blood flow and glucose

metabolism[90]. It has been established that glycolysis, the first step of glucose catal-

ysis, is primarily performed in the astrocytes[15, 110], which take up glucose from the

extracellular space through GLUT-1 transporters[99]. Pyruvate, the end-product of

this process, is then converted into lactate and transported through monocarboxylase

transporters (MCTs) into the extracellular space, once again, in an energy-dependent

fashion[78]. Lactate from astrocytes and glucose from the extracellular space are both

taken up by neurons through MCT-1, 2 and GLUT-3 transporters, respectively (See

Figure 1.1)[90]. Here, these energetic substrates continue to be catalyzed through the

tricarboxylic acid cycle followed by oxidative phosphorylation in the electron trans-

port chain to produce ATP.

1.2.2 Cellular Mechanisms of Brain Glucose Deprivation

Hypoglycemia has been shown to elicit a variety of pancreatic and hormonal counter-

regulatory responses in the body[34], but these mechanisms are beyond the scope of

this review. Failure of blood glucose homeostasis can lead to episodes of brain glu-

cose deprivation, which can result in a variety of morbidities depending on the sever-


Figure 1.1: Glucose transport from blood to neurons and astrocytes. Adapted fromLeybaert et al. 2005. Glucose transporter GLUT-1 transports glucose across en-dothelial cells and also across astrocytic membranes into the astrocyte where it getsconverted into pyruvate first, then lactate. This lactate is transported out of the as-trocyte through the monocarboxylate transporter MCT-1 and into neurons througheither the MCT-1 or 2 where it gets converted back into pyruvate for use in the Kreb’scycle. Glucose can also be directly transported to neurons through neuronal GLUT-3transporters.


ity and duration of the pathological epoch. Moderate glucose deprivation, thought

to be detected by glucosensing neurons[73], first kick-starts compensatory cascades

in the brain to conserve the limited energy reserves, primarily by halting synap-

tic transmission[4] and preventing neuronal death due to energy depletion. Both

adenosine release by neurons and increase in intracellular pH caused by ammonia ac-

cumulation resulting from amino acid metabolism during low glucose conditions can

suppress neuronal activity[148, 21, 5].

Abrupt depletion of ATP is characteristic of severe hypoglycemia and can cause neu-

ronal depolarization, which leads to excitotoxicity, and ultimately, neuronal death,

identified in vivo by the accompanying electroencephalographic (EEG) isoelectric-

ity and in vitro by histological analysis of neuronal damage[6, 5]. The maintenance

of the resting membrane potential by ATP-dependent ion channels is compromised

during hypoglycemia due to the lack of ATP. This leads to the dissipation of the

potassium gradient, visualized by in vitro patch clamping as a slow depolarization

of the neuronal membrane, and changes in intracellular [Ca2+][159, 65]. This de-

polarization of the membrane causes vesicular release of neurotransmitters into the

synaptic cleft, which, when detected by surrounding astrocytes, leads to an increase in

intra-astrocytic [Ca2+][46]. The mechanism of gliotransmitter release in response to

increase in astrocytic [Ca2+]i is still under debate, but if true, could cause the excess

extracellular glutamate/aspartate accumulation observed by many during ischemia.

Given the pathological concentrations of excitatory neurotransmitters released dur-

ing hypoglycemia, excitotoxicity leading to neuronal death is inevitable. Dong et al.

(2013)[46] recently described a mechanism in which extrasynaptic NMDA receptors

were activated due to the overflow of neurotransmitters from the synaptic region, lead-


Figure 1.2: Mechanism for hypoglycemia-induced neuronal death. Adapted from Suhet al. 2007[136]. Hypoglycemia-induced neuronal depolarization leads to release ofzinc and glutamate into synaptic cleft where it gets taken up by post-synaptic neuronto cause mitochondrial ROS production which leads to DNA damage and ultimatelyneuronal death.

ing neurons into the well-known extrasynaptic NMDAR-dependent cell-death path-

way. Others have described another such pathway, activated by zinc release and influx

from pre- into post-synaptic neurons, which induces the production of reactive oxygen

species (ROS) from mitochondria[135, 137](Figure 1.2). Although most symptoms of

hypoglycemia are typically subdued by delivering a bolus injection of glucose into the

system, the damage caused by ROS is irreversible[34, 4]. DNA damage, non-specific

mitochondrial membrane permeability transition, and poly(ADP-ribose) polymerase-

1 (PARP-1) activation all contribute to ROS-related cell death[127, 82, 103]. In addi-

tion to these mechanisms of necrotic cell death, apoptotic cell death is also stimulated

by the decrease in intracellular K+, which activates caspases[73].

Many of these pathological mechanisms have been targeted in an effort to prevent


or reduce hypoglycemia-related injury. The blockade of A1 adenosine receptors[148],

delivery of alternative energy substrates[4], and inhibition of NADPH oxidase[136]

are examples of treatments that were shown to be neuroprotective during the hypo-

glycemic condition. Others have directly pursued the attenuation of excitotoxicity, a

key state that can trigger many pathways leading to neurodegradation and death, by

blocking NMDA receptors and electrographic seizures[2, 128, 158, 107]. This thesis

aims to, in part, characterize the hypoglycemic seizure in vitro due to the crucial role

it plays in the generation of excitotoxic cell death during this low glucose condition.

1.2.3 Glucose Reperfusion Injury

Reperfusion injury is a common concept for those who study stroke. The mito-

chondrium, the main bioenergetic organelle, is drastically affected by the cascade of

events stimulated by re-establishment of blood flow[115]. This mitochondrial dys-

function results in the production of ROS which in turn leads to further disruptions

in mitochondrial function. This increases this organelle’s permeability to ions and

large molecules (< 1500 kDa), ultimately leading to an imbalance in the mitochon-

drial membrane potential and loss of function, even when the glucose levels have been

restored. Similar events have been observed during glucose reperfusion by Suh and

colleagues[136]. They found that there was increased production of neuronal superox-

ide through a NADPH oxidase-dependent pathway during reperfusion rather than the

hypoglycemic episode itself. Since zinc is known to activate NADPH oxidase, it is not

surprising that the hippocampal areas that are the most susceptible to hypoglycemia-

induced damage also contain high levels of presynaptic vesicular zinc[131]. A clinical

study looking at the effects of normoglycemic versus hyperglycemic reperfusion after a

hypoglycemic episode found that markers of stress and inflammation were significantly


higher in subjects whose hypoglycemic symptoms were treated with a hyperglycemic

solution[23], indicating that there is a potential for neuronal damage during the glu-

cose reperfusion stage. Another study on insulin-injected rats confirmed that there

is an increase in apoptotic and necrotic cell loss during glucose reperfusion[27], but

the mechanism of action is still under debate. This thesis also contains observations

regarding the state of the murine brain slice during glucose reperfusion.

1.3 Characterization of Hypoglycemic Seizures

1.3.1 Brief Description of Seizures

Definition and biochemical causes of seizures

An epileptic seizure is defined as “a transient occurrence of signs and/or symptoms

due to abnormal excessive or synchronous neuronal activity in the brain”[48]. Al-

though they do not always coincide, the start and termination of an epileptic seizure

are often defined by behavioural symptoms and the presence or absence of EEG

ictal discharges. This disease state can have numerous clinical manifestations de-

pending on factors such as origin of pathological discharges, propagation pattern,

and maturity of the brain. The most well-received hypothesis for a seizure initiation

mechanism is a disruption of the balance between excitatory and inhibitory neuronal

activation patterns that typically governs physiological brain function[165]. Conse-

quently, malfunctions in membrane channels that are involved in synaptic signalling

(NMDA-, AMPA-, GABA-receptors) and maintaining ion homeostasis (Ca2+, K+,

voltage-gated-Na2+-channels), can contribute to the overexcitation of neurons which

leads to the hypersynchronous discharges typically observed during seizures[102].


Seizure-induced cell death

Epilepsy is one of the most common neurological disorders[11] and occurs more fre-

quently in children than in adults. Because of the expanding body of evidence

suggesting that childhood seizure experiences can cause long-term developmental

issues[70, 141], the study of neurological damage induced by seizures has gained value.

Holopainen[69] describes pathological seizure-effects on the brain as a three stage pro-

cess: 1) excess glutamate-induced excitotoxicity resulting in rapid neuronal death, 2)

initiation of counter-regulatory responses such as activation of trophic factors as well

as further cell loss due to inflammatory processes and stimulation of apoptosis, and

3) long-lasting changes in synaptic connectivity, functional reorganization of the net-

work to support unprovoked, and spontaneous seizure activity. Electrophysiologically,

cell-death and neuronal injury is indicated by an irreversible loss in evoked synaptic

local field potentials, representing synaptic failure. Astrocytes and microglia have

also been known to play a role in the pathophysiology of seizures[42].

Given the discussion regarding neuronal death caused by hypoglycemia per se in the

above sections, this thesis investigates the likely cause of this hypoglycemia-induced

functional brain-failure and whether or not seizures play a role in these pathological

mechanisms.

1.3.2 Clinical Trials and Case Studies

Early experiments conducted by Banting and colleagues[7] (1922) showed the ability

of insulin-induced hypoglycemia to provoke convulsions in non-diabetic rabbits. This


study was followed up several years later by small and large scale clinical trials con-

firming that patients presenting hypoglycemia, as a side-effect of therapeutic insulin

injection[95] or due to excessive alcohol consumption[51, 66], often had associated

convulsions. One such trial was conducted by Davis et al.[37] (1997) who observed

episodes of hypoglycemia in 657 children and adolescents with Type I Diabetes. Ac-

cording to this study, children under 6 years of age were especially susceptible to se-

vere episodes of hypoglycemia, with 90% of all severe episodes manifesting as seizures.

In addition to these clinical trials[44], individual cases of hypoglycemia leading to

seizures (Gibbs and Murray, 1954) have also been reported. Morano[98] (1970)

and Tolis[146] (1965) described patients who were admitted because of hypoglycemic

seizures due to alcohol consumption. Werder et al.[157] (1971) and Blasetti et al.[14]

(2007) present case studies where patients presented with hypoglycemia-induced seizures

caused by an islet tumor and an insulinoma, respectively. A case study by Lapenta[87]

and colleagues (2010) of a 61-year old diabetic patient demonstrates a well-documented

instance of an insulin-induced focal hypoglycemic seizure, a relatively rare form of

seizure, compared to the more common generalized tonic-clonic seizure. These au-

thors then emphasize the complex nature of this metabolic phenomenon by noting

that the seizure might not be a direct effect of low glucose, rather, it might a result of

temporal lobe recruitment, this brain region being one which is especially susceptible

to seizures.

It is apparent based on the clinical trials and case studies that there is certainly

a positive correlation between the prevalence of severe hypoglycemia and the onset

of seizures, yet based on these studies alone, no mechanism of seizure initiation could

be elucidated. Thus, it became imperative to establish suitable animal models and


use alternate methods of scientific inquiry, both invasive in vivo and in vitro tech-

niques, to more clearly understand the role that low glucose plays in the prevalence

of seizures.

1.3.3 In Vivo Studies

Insulin-induced seizures in animals

Banting and Best’s landmark discovery that pancreatic extract (insulin) could drasti-

cally lower blood glucose levels also led the question of clinical symptoms as a result

of pathologically low blood glucose. In their study of insulin-induced hypoglycemia

in rabbits, Banting and colleagues[7] noted that after a period of moderate hypo-

glycemia, associated with behaviours of hunger and thirst, these rabbits frequently

deteriorated into a convulsive state. These seizures, which typically lasted for 2 - 3

minutes and involved the whole body, ceased with the administration of dextrose, but

recurred every 15 minutes spontaneously if this treatment was not provided. A study

by Kirchner[83] showed that rats that were intraperitoneally injected with insulin

developed violent tonic-clonic seizures consisting of jumps and barrel rotations when

blood glucose reached 1.0 mM. Other groups also found that they could reliably in-

duce hypoglycemic seizures with similar behavioural characteristics by administering

insulin to rats[106, 24]. This large body of evidence convincingly shows that there is

a causal relationship between insulin-induced hypoglycemia and seizure onset.


Treatment of hypoglycemic seizures

Most in vivo studies have treated hypoglycemic seizures with bolus injections of

glucose[7, 83, 106, 24, 152]. In fact, the current treatment strategy for patients

with iatrogenic hypoglycemia is an injection of 25-30% glucose dissolved in saline,

although studies have shown that the production of reactive oxygen species dur-

ing hypoglycemia could exacerbate neuronal damage during glucose reperfusion[136].

A paradoxical report by Puente[114] showed that recurrent moderate hypoglycemia

could reduce the extent of neuronal damage caused by severe episodes of hypoglycemia

in the rat brain. In addition, more traditional methods such as blockade of AMPA

and NMDA receptors to minimize excitotoxicity have also been successful in reduc-

ing hypoglycemia-induced neuronal damage[100, 24]. Lesions of the ventromedial

hypothalamus[79], fasting[140](later countered by Velisek et al.[152]), which increases

ketone bodies (an alternate energy substrate), and acetate injections[149] have been

shown to reduce hypoglycemic convulsions in vivo.

Origin of hypoglycemic seizures

Interest in the mechanisms of hypoglycemic seizures escalated due to the ongoing

debate over the role of seizures in exacerbating neuronal damage during energy de-

privation. Since focal epilepsies have been easier to measure and treat compared

to generalized ones, a considerable effort was expended in order to locate the low-

glucose induced seizure initiation zone. A study by Goodwin[61] et al. in 1940 showed

that there were no significant changes in cortical EEG patterns preceding the ob-

served hypoglycemic convulsions, suggesting a subcortical origin for seizure initiation.


Later, Tokizane and Sawyer[145] (1957) used rabbits in their EEG studies to conclude

that hypoglycemic seizures originated in the amygdala and hippocampus, suggesting

that these structures were more prone to seizures and hypoglycemia-induced neu-

ronal damage[67]. These findings were later confirmed by a study on memory prob-

lems in patients experiencing hypoglycemic episodes[125] and other groups who have

reported the involvement of the hippocampus during hypoglycemia[85] and hypo-

glycemic seizures[24].

Structures involved in hypoglycemic seizures

As is the case with most metabolic disorders, results of an experimental challenge can

vary due to numerous issues including the model system used, experimental protocols,

and type of equipment used; this invariably leads to controversies surrounding spe-

cific aspects of pathological mechanisms including seizure onset zone. Although there

is much evidence supporting the claim that hypoglycemic seizures tend to originate

in the most seizure-susceptible structures, like the hippocampus and amygdala, del

Campo et al.[41] found a lack of correlation between ictal EEG patterns of the hip-

pocampus and right frontal cortex and the observed behavioural seizures. Conversely,

Velisek[152] (2008) showed a strong correlation between hypoglycemia-induced con-

vulsions and ictal discharges in EEG recordings of the substantia nigra pars reticulata

(SNR) and the hippocampus. Although the origin of the hypoglycemic seizure was

not determined in this paper, the researchers concluded the SNR plays a crucial role

in hypoglycemic seizure control in vivo. The controversy surrounding this subject

demonstrates the need for further in-depth mechanistic studies.


1.3.4 In Vitro Studies

Based on clinical and in vivo studies, it is clear that severe hypoglycemia (defined as a

blood glucose value less than 25 mg/dL[1 mM][150]) precedes seizure onset, suggesting

that brain glucose deprivation leads to neuronal excitation and increased synchronous

firing. Contrastingly, in vitro studies have found that hypoglycemia can attenuate

basal synaptic transmission in the hippocampus and dorsolateral septal nucleus[147].

This dichotomy once again reflects the mechanistic complexities which result from a

metabolic insult such as brain glucose deprivation.

Many groups have tested the effects of glucose deprivation in other models of seizures

such as the low [Mg2+]i[83] and kainate[124] models, but until the study by Abdelmalik[2]

in 2007, there were no reproducible in vitro models of seizures that could be elicited

by a hypoglycemic solution without priming with a convulsant. In this study, the

group perfused isolated intact hippocampi from young mice (postnatal day 8 - 13)

with solutions containing various concentrations of glucose. Using this dose-response

study, they found that they could reliably induce spontaneous ictal discharges, fol-

lowed by depressed synaptic transmission when glucose concentrations in the artificial

cerebral spinal fluid (ACSF) were dropped to below 2 mM. These in vitro seizures

were blocked by NMDA- and AMPA-receptor antagonists, but not by GABA agonism

or anticonvulsant administration. Velisek and colleagues[152], who subjected brain

slices to low-glucose (2 mM) ACSF, found that patch-clamped GABAergic cells in

the SNR responded with a more depolarized resting membrane potential, resulting

in a higher firing frequency as glucose concentrations were decreased. These results

show that hypoglycemic seizures can be effectively modeled in vitro using murine

brain slices.


1.4 Brief Description of Gap Junctions (GJs)

1.4.1 Composition and Expression

Gap junctions (GJs) are direct intercellular cytoplasmic connections permeable to

ions, small molecules (< 1 kDa), and hydrophilic molecules (e.g., ATP, glucose

triphosphate inositol), which are formed by two juxtapositing hemi-channels present

on membranes of adjacent cells[1]. Each of these hemichannels or connexons, is com-

posed of six connexin (Cx) subunits (arranged circularly around a central pore[18]), of

which there are 21 types, classified by molecular weight. The central nervous system

(CNS) is extensively coupled by 11 different types of GJs expressed on most brain

cell types, which can be joined either homo- or heterocellularly. It is evident that

gap junctions are largely present in glia, especially astrocytes[120], however, there

are also several reports of gap junctional coupling of interneurons[97]. Cx43 in astro-

cytes, and Cx36 in neurons are two of the most well-studied connexin proteins. In

addition to forming the GJ pore as a duo, unapposed connexons can also be present

on membranes. These hemichannels, which remain closed under physiological condi-

tion to retain ion gradients and membrane integrity, have been known to open during

metabolic inhibition[29, 143] and could exacerbate neuronal injury[120].

1.4.2 Gap Junction Pore Characteristics

Various gating systems are in place to finely regulate GJ permeability in the short

and long-term: voltage, phosphorylation, intracellular calcium and pH, adhesion pro-

teins, extracellular matrix, and hormones[1, 17]. A variety of electrophysiological and

imaging techniques have been used to measure channel characteristics of GJs includ-

ing dye-transfer techniques (e.g., fluorescence recovery after photobleaching [FRAP],


microinjection) and conductance measurements by dual patch clamp[1]. Tables 2 and

3 in a review by Rouach et al.[120] summarizes effectors which increase or decrease GJ

communication in astrocytes and neurons, respectively. In particular, acidification of

the intracellular milieu[108], administration of alcohols[54, 89, 52] and glycyrrhetinic

acid derivatives[153, 13, 59], all act to inhibit GJs, whereas depolarization[47, 39, 62],

alkalization, and decreased Ca2+[113] increase GJ communication.

GJs have been described as “fast-track intercellular communication routes”[28]

because of their ability to connect various cell types in order to establish a cytoplas-

mic continuum between them. In the heart, GJs form pores between cardiomyocytes

and play a major role in propagating the cardiac action potential to adjacent cells

within the required time-scale[118]. In the CNS, GJs not only intercellularly couple

together neurons, astrocytes, oligodendrocytes, and ependymal cells, but there is also

evidence of neuron-astrocyte and astrocyte-oligodendrocyte coupling[139].

1.4.3 Connexin (Cx) and Pannexin (Px) Hemichannels

The idea that connexin proteins present on neuronal and glial cell membranes can

form functional (open) hemichannels, allowing for increased cell permeability, is rel-

atively new[56]. The once central dogma that the assembling of connexin proteins

was for the sole purpose of constructing gap junctional pores was challenged by ob-

servations which found that large depolarizations[109], the lowering of extracellular

Ca2+ concentration[43], and metabolic inhibition[29] all resulted in the opening of un-

apposed connexin hemichannels. Similarly, pannexins, which are orthologues to the

invertebrate gap-junction forming innexin proteins, also formed functional “hemichan-

nels” that were able to increase membrane permeabilization[104].


1.4.4 Gap Junctions in Ischemia

As interest in the role of astroctyes in the brain escalated, studies showed that these

extensively coupled cells are involved in processes that take advantage of this intercon-

nected network; e.g., K+ and neurotransmitter buffering[80, 163, 64] from areas of high

neuronal activity and redistribution to distal astrocytes; activity-dependent redistri-

bution of energetic substrates throughout the astrocytic network[57, 121]. GJs have

been thoroughly studied in the context of hypoxia-ischemia because they are known to

remain open and active during this condition[30]. Several groups report that blocking

or knocking out GJs in this condition decreases neuronal damage[116, 117, 40, 49, 50].

Astrocytic calcium waves, known to increase astrocytic glutamate release, leading

into the classical glutamate-induced excitotoxcity pathway, is thought to be medi-

ated by inter-astrocytic GJs[46]. Rouach et al.[121] also found that intra-astrocytic

energy substrates (usually lactate) can travel via GJs to sustain synaptic transmis-

sion and epileptiform activity during exogenous glucose deprivation. Although GJs

in astrocytes and neurons play a major role during metabolic inhibition, there is still

controversy surrounding their exact mechanism of action. For example, Cotrina’s

study[30], which seems to indicate that GJs remain open during ischemic conditions,

contradicts Contreras’ study which presents evidence supporting reduced GJ com-

munication during energy deprivation[29]. This controversy illustrates the need for

more detailed mechanistic elucidations of the state and role of GJs during metabolic

inhibition in the brain.

1.4.5 Characteristics of GJ Blockers Used

GJ blockers, typically used in in vitro characterizations of GJ function, are chem-

icals that can disrupt electrical coupling between or dye transfer to neighbouring


cells[77]. Blockade of GJs results in metabolic and electrical uncoupling of previ-

ously GJ-associated cells. This can be a protective action to isolate unhealthy cells

and prevent them from spreading cell-death signals to their metabolically coupled

neighbours[111]. GJ blockers perform their action by three different mechanisms:

1) binding to the channel and affecting GJ conductance, 2) changing the lipid en-

vironment around the channels, and 3) indirectly by changing intracellular pH or

[Ca2+][122]. A few relevant classes of blockers include glycyrrhetinic acid derivatives

(carbenoxolone [CBX]), quinine derivatives (mefloquine), and long-chain alcohols (oc-

tanol). The characteristics of these three compounds will be individually examined.

Carbenoxolone

Despite being called the “dirty drug” because of its notorious non-specificity, car-

benoxolone (CBX) is one of the most commonly used GJ blockers[28]. This com-

pound, which has been used clinically to treat gastric ulcers, has a steroid-like struc-

ture, is water-soluble[122], and can almost completely block GJ conductances at con-

centrations of 50-100µM. The reason CBX is said to be non-specific is two-fold: not

only does it block several types of connexins[36, 132], it reportedly also blocks voltage-

gated Ca2+ channels[154], p2x7 receptors[134], and NMDA currents[25]. The mech-

anism of CBX action to block GJs is still largely unknown, although it is thought

to act indirectly by activating protein kinases which could affect the phorphorylated

state of the pore, inducing it to change into an open/closed configuration[76].


Mefloquine

Mefloquine, a quinine derivative, was initially used as a treatment for malaria[77].

It has also been shown to potently block GJs with a greater level of specificity than

CBX[31]. Mefloquine is more useful for the study of GJs compared to its parent

compound, quinine, because of the latter’s tendency to affect numerous voltage and

ligand-gated channels in addition to its action on GJs[161, 129]. The increase in

lipophilicity by substituting a quinucludine ring with a piperidine ring is thought

to play a role in this increase in potency of mefloquine versus quinine[31]. At the

lowest effective dose of 100 nM, mefloquine almost completely abolished pannexin

hemichannel currents[71], while at higher doses of 3 µM or 30 µM, it completely

blocked only the neuronal connexin (Cx36) or both neuronal and astrocytic connex-

ins (Cx43, Cx26, Cx32), respectively.

Octanol

Octanol, being a long-chain alcohol, has hydrophobic properties, which renders it

capable of easily integrating into lipid membranes[122]. The resulting change in the

fluidity of the membrane, either increased bulk fluidity[138] or decreased fluidity in

cholesterol-rich domains[9], closes membrane-embedded connexin hemichannels, dis-

assembling GJs. At concentrations between 0.1 and 3 mM, octanol has been shown

to rapidly, reversibly, and completely inhibit GJ communication[19]. However, like

CBX, octanol is not strongly selective for one particular type of connexin and has

been shown to also block T-type calcium channels[144] (EC50 = 122 µM) and par-

tially inhibit NMDA-, AMPA-, and kainate receptor responses[77].


Controls for gap junction blockade

Aside from treatment of GJs with specially designed mimetic peptides for targeted

blockade, there are a number of control techniques that can be used to demonstrate the

involvement of specific GJ. One such method is to use compounds that are structurally

and chemically similar to the above-mentioned blockers, but without the ability to

disrupt GJ communication[77]. It may also be useful to repeat experiments using

multiple blockers with different mechanisms of action, but which are all capable of

GJ blockade. Although most available GJ blockers are not selective and could bind

to non-GJ targets, they can be useful tools in neuroscience, particularly because of

the increasing interest in the study of the function of electrical synapses in disease

states such as epilepsy.

1.4.6 Role of GJs in Seizures

Due to their role in providing a cytoplasmic continuum for the fast propagation of cur-

rents, GJs are hypothesized to enhance the synchrony necessary for the development,

maintenance, and propagation of seizures, which can be visualized electrographically

as local or large-scale synchronized oscillations[120, 28]. The facilitation of seizure

activity by chemical synapses was long examined in the field of epilepsy before exper-

imental and theoretical evidence supported the involvement of GJ communication in

the synchrony and stabilization of bursting firing patterns[112, 120]. Several in vitro

and in vivo studies have reported that GJ blockers abolish spontaneous ictal bursting

present in various model of seizures, induced by bicuculline[38], low magnesium[84],

4-aminopyridine[119], and high potassium[96].

Though the majority of studies agree that blockade of GJs generally has an anti-


convulsant effect, other reports have also found that administration of GJ blockers

can have the opposite effect. Voss et al. showed increase in seizure-like activity in

slices subjected to low magnesium ACSF with the addition of carbenoxolone, meflo-

quine, quinine, and quinidine[155]. However, this could be due to non-GJ effects

of these blockers[10]. In addition to electrophysiological studies, genetic studies us-

ing connexin knockouts have also found that absence of these GJs decreases seizure

activity[94]. These contradictory effects of GJ blockade are reviewed further in Jin

and Chen (2011)[76].

The role of astrocytic GJs during seizures also seems to be paradoxical. They are

known to play a role in potassium buffering from the extracellular mileu during

seizures and redistribution within the GJ-coupled astrocytic network[130]. The block-

ade of astrocytic Cx43 has been shown to block spontaneous seizure-like-events (SLEs)

in organotypic slice cultures[123]. This is thought to be a homeostatic mechanism[112]

whereby blockade of astrocytic GJs stops the redistribution of activity-dependent lo-

cal increases in potassium, ultimately leading to a seizure-terminating depolarization

block, resulting in a postictal depression of the EEG[16]. In addition, Rouach[121] et

al. (2008) found that energetic substrates travelled through astrocytic GJs to loca-

tions of high energy demand and that GJ blockers could have an anticonvulsant effect

by preventing metabolite delivery to “hungry” epileptic neurons. Contrastingly, ex-

periments in hippocampal slices of Cx43 knockout versus control mice show increased

spontaneous epileptiform activity[156], suggesting a time and state-dependent dy-

namic role for astrocytic GJs. Short-term, reversible block of astrocytic GJs appear

to have anticonvulsant effects, whereas chronic removal of GJ communication seems

to be pro-epileptic[112].


1.4.7 Possible Involvement of Connexin and Pannexin Hemichan-

nels

There is a growing body of evidence for the presence of functional connexin and pan-

nexin (Px) hemichannels and their relevance in the study of neurological disorders[22].

Under physiological conditions, these hemichannels remain closed to retain membrane

integrity and ion gradients, but have been shown to open during metabolic stress[143],

depolarization, and increased intracellular calcium[55]. Many of the conditions for

opening of hemichannels are met in epileptic tissue, suggesting their involvement in

this tissue state. Studies have shown that ATP and glutamate, which have have

pro-epileptic effects, can be released through astrocytic hemichannels[133]. The re-

lease of ATP is thought to mobilize astrocytic calcium waves, which supports hy-

persynchronization and maintains a stable seizure environment[86]. In fact, blocking

hemichannels using a Px1 blocking peptide decreased spike amplitude and seemed to

reduce aberrant bursting caused by low magnesium perfusion[142]. In a bicuculline

slice culture model of epilepsy, a connexin mimetic peptide, which mainly blocked

hemichannels, had protective effects[162]. These data suggest the involvement of both

gap junctions and hemichannels in metabolic stress as well as seizures. The percent

contribution of each to these pathological mechanisms has not yet been elucidated.

1.5 Experimental Design

1.5.1 Model

This study uses an in vitro juvenile mouse thick hippocampal slice to examine the role

of gap junctions during brain glucose deprivation. Juvenile mice (postnatal day [PD]

14-21) are used for a number of reasons. There have been several studies reporting


the increased susceptibility of young human brains to hypoglycemic/hypoxic injury

compared to adults[37, 75]. In addition, the juvenile hippocampal structure seems to

be at higher risk of developing seizures compared to the adult brain[12, 160, 67]. Ab-

delmalik et al.[2] used immature mice for their study of in vitro hypoglycemic seizures

(PD 8-13). However, studies have shown that seizure-susceptibility in the immature

brain is due to the excitatory action of GABA and that the switch from excitatory

to inhibitory GABA occurs at PD 13.5[81]. This renders the juvenile brain an ideal

candidate for the study of hypoglycemic seizures.

Previous attempts to illicit hypoglycemic seizures by dropping glucose concentrations

to 2 mM down from 10 mM using 400 µm brain slices have failed[83]. Abdelma-

lik et al.[2] effectively induced hypoglycemic seizures by using an immature intact

hippocampus preparation. One of the reasons that this preparation was successful

could have been because of the availability of more intact circuitry in a whole versus

thinly sliced hippocampus. However, as previously mentioned, mice under PD 13

are not ideal candidates for hypoglycemic seizure experiments. Wu et al.[160] de-

scribed a method of preparing a “thick” hippocampal slice (up to 1 mm), which is

adequately perfused, oxygenated, while preserving enough circuitry to generate intrin-

sic hippocampal network rhythms. This model was used for all electrophysiological

experiments in this study.

1.5.2 Outline of Experiments

This thesis will first explore the characteristics of hypoglycemic seizures in the murine

thick hippocampal slice, measured as local field potentials using a standard electro-

physiology set-up. The effects of various gap junctional blockers on hypoglycemic


seizures and synaptic activity will then be discussed. This will be followed by results

surrounding the development of a novel model of recurrent seizures during glucose

reperfusion on which the effects of gap junction blockade will also be tested. Overall

this thesis aims to provide a clearer picture of the role of gap junctions during hy-

poglycemic and glucose reperfusion seizures in the brain with an in-depth discussion

regarding the involvement of seizures in hypoglycemia-induced neuronal damage.

Chapter 2

Rationale and Hypotheses

2.1 Rationale

Hypoglycemia is a serious side effect of insulin overdose in the diabetic population

and often results in seizures, particularly in the juvenile population. Gap junctions

have been implicated in the initiation and propagation of seizures in various in vitro

models. In addition, they have been known to participate in hypoglycemia-induced

neurodegenerative mechanisms in both neurons and astrocytes. The goal of this

thesis was not only to test whether gap junctions play a role in the onset or propaga-

tion of hypoglycemic seizures, but also to provide further evidence for the discussion

regarding the necessity and sufficiency of the presence of seizures in causing neurode-

generation.

Glucose reperfusion injury is also gaining recognition in scientific literature as well as

in emergency care clinics that treat patients with iatrogenic hypoglycemia. The role of

gap junctions was also examined in a novel model of glucose reperfusion seizures with

the goal of linking the mechanisms of hypoglycemic and glucose reperfusion-induced

neuronal degeneration and examining possible gap junction targeting therapies.

26

Chapter 2. Rationale and Hypotheses 27

2.2 Hypotheses

Previous experiments that examined the generation of hypoglycemic seizures found

that the presence of seizures exacerbated neuronal injury[2]. One of the main thoughts

regarding the mechanism of seizure generation and propagation is through the cyto-

plasmic continuity provided by open gap junctions connecting adjacent neurons. As-

trocytic gap junctions have also been implicated in the buffering action of astrocytes

to redistribute accumulating ions from the active site through the astrocytic network

to other areas, potentially resulting in prolonged seizure activity. In addition, gap

junctions have been shown to mediate activity-dependent metabolic supply during

oxygen-glucose-deprivation[121] and provide an interastrocytic route for intraastro-

cytic calcium waves which are known to cause excitotoxic damage. Thus, it can be

hypothesized that the blockade of gap junctions will be neuroprotective during hypo-

glycemia, not only because of its anticonvulsant activity, but also due to its potential

in reducing hypoglycemia-mediate damage per se.

Although it is known that glucose reperfusion can also lead to neuronal damage,

this mechanism of action has not been fully elucidated. This thesis aims to, in part,

explore the electrophysiological characteristics of brain tissue during glucose reper-

fusion after a transient episode of severe hypoglycemia in a novel model of glucose

reperfusion.

Chapter 3

Materials and Methods

3.1 Chemicals

Sucrose, D-glucose, sodium chloride (NaCl), potassium chloride (KCl), calcium chlo-

ride (CaCl2), magnesium sulphate (MgSO4), sodium bicarbonate (NaHCO3), sodium

phosphate dibasic (NaH2PO4), 3β-hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate

(carbenoxolone disodium salt [CBX], 100 µM), 1-octanol (OCT, 200 µM) were all ac-

quired from Sigma. OCT was dissolved directly into the artificial cerebral spinal fluid

(ACSF). CBX was first dissolved in sterile water to a concentration of 100 mM and

serially diluted to the final concentration in ACSF.

(±)-erythro-(R*/S*)-mefloquine was obtained from Bioblocks. This was dissolved

first in DMSO to a concentration of 10 mM then diluted in ACSF to a final concen-

tration of either 100 nM or 300 nM.

28

Chapter 3. Materials and Methods 29

3.2 Animals

All animal studies were approved by and conducted in accordance with the Animal

Research Council guidelines at the University Health Network (Toronto, Canada).

C57/BL6 male mouse pups were obtained from Charles River Breeding Farms (Mon-

treal, Quebec, Canada) in litters at postnatal day (PD) 12. Mice of PD 14 - 21 were

used for all in vitro experiments. The pups were housed with their littermates and

dam, who had ad libitum access to sterilized rodent chow and water. They were kept

in a temperature-controlled environment, held constant at 23±1 Celcius, with a 12-h

day/night cycle.

3.2.1 Tissue Preparation

Tissues were prepared by decapitating the animal anesthetized under 70mg/kg pen-

tobarbital following an intracardiac perfusion with sucrose-based ACSF. The sucrose-

based ACSF consisted of (in mM): 218 Sucrose, 2 KCl, 3 MgSO4, 1 CaCl2, 26

NaHCO3, 1.25 NaH2PO4, and 10 D-glucose, with a final osmolarity between 340

and 350 mOsm. The brain was then quickly removed from the skull and kept in

ice-cold, oxygenated (saturated with carbogen: 5% CO2 and 95% O2) sucrose-based

ACSF for 2-5 min before removal of the cerebellum and hemisection (division of the

left and right hemispheres along the midsagittal plane) on a frozen dissecting surface.

The remaining brainstem tissue at the caudal end of each of the lobes was removed to

expose the ventral side of the hippocampus. A total of 10 12 brain slices of 800 µm

thicknesses were obtained by gluing (with cryocyanate glue) the brain tissue onto an

agar block and cutting along the transverse plane of the middle-dorsal hippocampus

using a vibratome sectioning system (Vibratome 1000 plus; St. Louis, MO, USA).


After slicing, adjacent cortical tissue was cut away, leaving only slices of the hip-

pocampus, which were initially kept in oxygenated ACSF at 36 Celcius for 15 min

before lowering the holding temperature to 25 Celcius for 1-6 h and transferring

to a recording chamber. The ACSF contained (in mM): 123 NaCl, 25 NaHCO3, 10

D-Glucose, 4 KCl, 1 MgSO4, 1.2 NaH2PO4, 1 CaCl2, with a final osmolarity between

280-300 mOsm.

Thick hippocampal slices were kept in a BSK-4 slice keeper (Scientific Systems Design

Inc.), which allowed for “interface-like” holding conditions in a closed atmosphere sat-

urated with carbogen for maximal oxygen perfusion. For recording, the slices, placed

on lens paper, were transferred to an “interface” type recording chamber and secured

with a loose mesh. Warmed, oxygenated ACSF was then continuously perfused over

the slice at a rate of 2 mL/min. The temperature of the perfusate was controlled at 37

Celcius using a temperature controller. The Hass chamber bath was also oxygenated

and maintained at 37 Celcius to maximally humidify and oxygenate the slice.

3.3 Extracellular Electrophysiology

3.3.1 Data Acquisition

Extracellular field potentials were evoked by stimulating the mossy fibers of the den-

tate gyrus. An insulated bipolar 60 µm twisted nichrome wire was used to deliver

the biphasic square wave stimulation pulses, which had a duration of 0.1 ms and

frequency of 1 per 1-2 mins. This stimulating electrode was attached to a Grass

S88 stimulus generator (Grass Telefactor, West Warwick, RI, USA). Extracellular

local field potentials were recorded from the strata radiatum of the CA1 and CA3


regions of the hippocampus using electrodes pulled (Narishige PP-83 two-stage puller

[Narishige, Tokyo, Japan]) from glass pipettes (1.5 MΩ) filled with freshly prepared

ACSF. The solution-containing electrode surrounded a chlorided silver wire. The

recordings were monopolar and were referenced to a distant ground immersed in

ACSF, also made of chlorided silver wire. Signals were amplified using either Ax-

oclamp 2B (hypoglycemia only) or Axopatch 700A (glucose reperfusion) [Molecular

Devices, Sunnyvale, CA, USA] and data, acquired at 10 KHz, were digitized using

Axon Digidata 1322A (Molecular Devices). The data were analyzed offline using

pClamp V8 (Molecular Devices) and MATLAB (MathWorks).

3.3.2 Induction of Hypoglycemia and Blocker Application

Brain glucose deprivation was modeled by decreasing the ACSF’s D-glucose concen-

tration to 0 or 0.5 mM from the original 10 mM. Baseline local field potentials in

normal ACSF were collected for 10 min to ensure stabilization of the signal, after

which the normal ACSF was substituted with low-glucose ACSF. Local field poten-

tials (LFPs) were subsequently recorded throughout the 30 min glucose deprivation

challenge followed by a 10 min normal ACSF washout period. During baseline con-

ditions, 10 consecutive paired-pulse evoked potential recordings were obtained at in-

creasing stimulus voltages to construct an I0 curve. The voltage at which the second

evoked potential was at 75% of the maximum was determined and used for further

stimulations.

To monitor effects of gap junctional blockers, LFPs, acquired once again from the

strata radiatum layers of the CA1 and CA3 regions of the hippocampus, were recorded

for at least 5 min in normal ACSF once the optimal stimulus intensity was determined.


The blockers (200 µM OCT, 100 nM mefloquine, or 300 nM mefloquine) were then

added to the ACSF for pre-perfusion of the slice for 5 min, after which the hypo-

glycemia+blockers solution was perfused for a 30 minute challenge. The slice was

then washed out with normal ACSF for 10 min.

3.3.3 Glucose Reperfusion and Blocker Application

Baseline LFPs were recorded for 10 minutes followed by the perfusion of low-glucose

ACSF into the slice. As soon as the first hypoglycemic seizure was observed, low-

glucose ACSF was immediately replaced with normal ACSF and monitored for an

additional 30 min. For the gap junction blocker experiments, carbenoxolone (100

µM CBX) was added into the normal ACSF and applied simultaneously with glucose

reperfusion.

3.4 Data Analysis

3.4.1 Slice Inclusion Criteria

The data were analyzed offline using pClamp V8 (Molecular Devices) and MATLAB

(MathWorks). Only slices whose initial field response amplitudes were ≥ 0.5 mV were

included for further analyses, with the amplitude measured as the vertical distance

between average baseline and trough/peak of the synaptic component of the evoked

potential.

3.4.2 Seizure Inclusion Criteria

Since many hypoglycemic seizure-like electrographic discharges were relatively short

(i.e. < 20 s), a full seizure was measured to include what others might call “preictal”


activity[164]. The presence of a seizure was confirmed using Clampfit (Molecular

Devices) power spectrum analysis tools. The power of frequencies between 20 and 40

Hz was much higher within seizure zones compared to baseline.

3.4.3 Spectrogram Production

After a visual inspection, a region of interest was selected, imported into MATLAB,

and notch-filtered at 60 Hz. A continuous wavelet transform analysis technique was

applied to the region of interest and normalized to the baseline. These transformations

were presented as colour-coded frequency vs time plots, with the warmer end of the

spectrum indicating higher power. Frequencies between 1 and 200 Hz were analyzed

and presented in the spectrogram.

3.4.4 Statistical Analysis

All statistical analyses were performed using MATLAB. The two-tailed two propor-

tion z-test was used for compared differences between proportions and the t test was

used for comparing the averages of data sets. The data was considered significant

if the p-value was lower than 0.05. All error bars on graphs and errors on charts

represent ± S.E.M.

Chapter 4

Results

4.1 Characterization of Hypoglycemic Seizures

4.1.1 Electrographic Seizure and Evoked Potentials

Wu et al.[160] described an in vitro brain slice preparation method in which intrinsic

hippocampal rhythms could persist for several hours. There has also been a report of

an in vitro model for hypoglycemic seizures in isolated, intact hippocampi[2], where

the reduction of ACSF glucose concentration from 15 mM to 2 mM or 0 mM could

reliably induce electrographic seizures in 7/9 or 8/8 slices, respectively. This study

reports a novel model of brain glucose deprivation using 800µm “thick” hippocampal

slices, in which hypoglycemic seizures are induced by decreasing ACSF glucose con-

centrations to 0.5 mM. Figure 4.1A depicts the local field potentials recorded in the

CA3 dendritic layer of the hippocampus. After 10 minutes of recording in baseline

conditions to ensure stability of the slice in its new environment, the perfusion of a

low glucose ACSF elicited ictal activity in 21/25 hippocampal slices (for definition of

ictal activity, please refer to Methods subsection 3.4.2). An expansion of the boxed

34

Chapter 4. Results 35

area in Figure 4.1A can be found in Figure 4.1B. The latter shows a typical elec-

trographic hippocampal seizure with its characteristic baseline (DC: direct current)

shift, noted as such if the baseline shifts by more than 5 mV[2]. The typical latency

to seizure onset was 12 ± 1 min after the perfusion of low glucose ACSF, with seizure

durations of 48 ± 9 s. This the DC shift was associated with 37% of all analyzed

hypoglycemic seizures (31/84) and was initiated a nearly equal number of instances

in both the CA1 and CA3 regions of the hippocampus, with 7% of DC shifts starting

in the CA3 and being unable to propagate to the CA1. These data could suggest

that the CA3 region of the hippocampus is more vulnerable to hypoglycemia-induced

seizure-like events than the CA1 region in this model.

Field EPSPs (fEPSPs) were evoked by stimulation of the dentate gyrus mossy

fibres and recorded in the CA1 and CA3 strata radiatum. The amplitude of the

dendritic fEPSP is thought to be an accurate reflection of synaptic input to the

CA1 and CA3 regions[74], and thus, an indicator of the state of tissue health. A

comparison of the evoked potentials recorded during the baseline with those recorded

after 30 minutes of hypoglycemia followed by 10 minutes of washout (re-introduction

of normal ACSF) showed that evoked potentials were irreversibly depressed to less

than 10% of the baseline (Figure 4.3A). fEPSPs measured in one minute intervals

throughout the hypoglycemia protocol in 12 slices (evoked potentials binned every

two minutes and normalized to average baseline) show that this is a reproducible

phenomenon (Figure 4.3B), with synaptic depression beginning after 7 minutes of

glucose deprivation.


5 10 15 20 25 30−20

−15

−10

−5

0

5Hypoglycemia

Time(min)

mV

1040 1050 1060 1070 1080 1090 1100 1110 1120 1130−15

−10

−5

0

5Close−up of Hypoglycemic Seizure

Time(s)

mV

Baseline

Figure 4.1: Continuous local field recording of hypoglycemic seizure. LFPs were

recorded in the CA3 dendritic layer with mossy-fibre-stimulated fEPSPs measured

every 1 minute. A. Full trace of experimental protocol including 10 min baseline

recording followed by 30 min low-glucose ACSF challenge. This slice showed repetitive

seizure-like activity associated with DC shifts during hypoglycemia. These phenom-

ena were reproduced in 21/25 slices subjected to the same challenge. B. Gray boxed

region of A is expanded to show the waveform of a typical hypoglycemic seizure. DC

shift is bracketed by >20 Hz ictal activity.

4.1.2 Spectral Features of Hypoglycemic Seizures

The use of the continuous wavelet transform analysis technique to produce frequency-

time plots (spectrograms) has gained popularity in recent years, being commonly

employed for predicting seizure-onset zones/times given human and rodent EEG

data[53, 72, 105]. In this study, spectral features are used to more thoroughly charac-


Figure 4.2: Propagation of DC shift associated with hypoglycemic seizures between

CA1 and CA3 dendritic layers. 84 separate hypoglycemic seizures were analyzed. Of

these, 31 seizures (37%) were associated with the characteristic shift of the baseline,

called a DC shift when baseline changed by >5 mV. Of these 31 events, roughly 40%

spread from either the CA1 to CA3 or CA3 to CA1. The remaining 7% of DC shifts

were present in the CA3 but did not spread to CA1.

terize hypoglycemic seizures and compare them with those of other in vitro models of

seizures. Panel A of Figure 4.4 shows a typical 40 second segment of a baseline LFP

recording. The ictus-containing segment in Panel B of Figure 4.4 was highpass filtered

at 1 Hz and notch-filtered at 60 Hz to remove electrical noise. The seizure magnified

from the box in Panel B was used to produce the spectrogram visualized in Panel D,

which shows a transformation of the wave phenomena from the beginning to the end

of the ictal event. The high-frequency components nested within the lower-frequency

wave seem to broaden as the seizure progresses. These features are reminiscent of fre-

quency vs time plots of multi electrode array-recorded 4-aminopyridine seizures[60].


4.2 Effects of Gap Junction Blockers on Hypo-

glycemic Seizures

4.2.1 Electrographic Features and Evoked Potentials

To observe the effects of gap junction blockade during brain glucose deprivation,

slices were pre-perfused with 200 µM octanol for 5 minutes before normal ACSF

was substituted with low-glucose ACSF. Octanol treatment blocked hypoglycemic

seizures in 9/10 slices. Statistical analysis using a two-tailed two-proportion z-test

showed that there was a significant difference between the number of seizures during

hypoglycemia and during hypoglycemia with gap junctional blockade (p < 0.01). The

top panel of Figure 4.5 shows the effect of hypoglycemia on the hippocampal slice

for comparison with the bottom panels. Figure 4.5B shows the effect of perfusing a

sample slice with octanol during hypoglycemia. Note the lack of a DC shift during

the entire challenge. Spontaneous non-ictal (mostly single-unit) activity increased in

amplitude as shown in the bottom panel, which expands the contents of the box in

Figure 4.5B.

During hypoglycemia only, an irreversible depression of synaptic activity was ob-

served. When octanol was present during the challenge, however, the fEPSPs mea-

sured after the 10 minute washout period were consistently greater than the initial

baseline fEPSP (Figure 4.6A) by nearly 150% (n = 7). As seen in Figure 4.6B, the

onset of depression of synaptic activity is delayed compared with the hypoglycemic

condition, with evoked potentials decreasing to only ∼60% of the baseline. As soon

as normal glucose was reintroduced to the slice, evoked potentials quickly rose to


surpass baseline levels.

4.3 Effect of Pannexin Blockade on Hypoglycemic

Seizures

Octanol is known to non-selectively block several connexin gap junctions as well as

pannexin hemichannels[144]. We wanted to test the contribution levels of gap junction

and pannexin hemichannel blockade on the observed octanol effect. Mefloquine, a

drug commonly used for the treatment of malaria can selectively block pannexin

hemichannels in a dose-dependent manner[71]. A mefloquine concentration of 100 nM

can block pannexin hemichannels, whereas a higher concentration of 300 nM can block

neuronal connexins. Thus, a dose of 100 nM mefloquine was used for all pannexin

experiments. Panels A and B of Figure 4.7 compare the effects of hypoglycemia per se

and hypoglycemia with pre-perfused mefloquine, respectively. Unlike octanol, this low

dosage of mefloquine was unable to block hypoglycemic seizures in 4/4 hippocampal

slices. Use of the two-tailed two-proportioned z-test to compare the pannexin blockade

against hypoglycemia per se showed that there was no significant difference in the

presence of seizures between these two conditions. The gray and orange boxes in panel

B are expanded in panels C and D, respectively. C shows a hypoglycemic seizure

with its characteristic baseline shift bracketed by ictal activity even while pannexin

hemichannels were being blocked by low-dose mefloquine, suggesting that octanol’s

effect on hypoglycemic seizures was through a gap junction-dependent mechanism.

Also, unlike octanol, mefloquine was unable to prevent the irreversible depression of

synaptic activity that is typical of the hypoglycemic condition (Figure 4.8).


Figure 4.9 shows a summary bar chart of the three experimental protocols exam-

ined so far. Briefly, when hippocampal slices were perfused with a low-glucose ACSF,

84% of these slices had electrographic seizures as measured via recording electrodes

in the dendritic layers. Ictal activity was accompanied by irreversible synaptic de-

pression within ∼7 min of glucose deprivation. When slices were preincubated with

octanol, a gap junction blocker, for 5 minutes prior to low-glucose ACSF perfusion,

ictal activity was no longer present and there was no irreversible loss of fEPSPs. In-

stead, there was a brief, less drastic reduction in evoked potential amplitude, which

was recovered and exceeded the baseline during the washout period. The effect of

octanol can be attributed to its role in blocking gap junctions but not pannexins. Pan-

nexin blockade per se using a selective blocker was unable to prevent hypoglycemic

seizures and the accompanying loss of evoked potentials.

4.4 Characterization of Glucose Reperfusion Seizures

The idea of glucose reperfusion injury is fairly new and was briefly described by

Suh et al.[136], but its relative, ischemic reperfusion injury, has long been studied

in stroke research and is known to cause mitochondrial injury leading to cell death.

We were also interested in understanding the underlying mechanisms behind glu-

cose reperfusion injury and postulated that cell-death in this condition could occur

through a seizure-dependent pathway. To test this, a glucose reperfusion model, once

again using juvenile murine thick hippocampal acute slices, was established wherein

slices were subjected to low-glucose ACSF only long enough to experience one hypo-

glycemic seizure at which point, they were immediately rescued with normal glucose

ACSF and monitored for a period of up to 30 min. 9/10 slices that were rescued im-

mediately after the first hypoglycemic seizure went on to have subsequent repetitive


seizures during glucose reperfusion. One representative instance of this experiment

is shown in Figure 4.11, panel A. B expands the gray box to show the hypoglycemic

seizure. Immediately after this, the slice was rescued with 10 mM glucose ACSF and

proceeded to seizure continuously as shown in panels C and D. Figure 4.12 shows

an example of evoked field potentials taken during three instances of the glucose

reperfusion experiment. Interestingly, the synaptic activity is enhanced when slices

are immediately rescued with normal ACSF. This increase in excitation could be re-

sponsible for the reperfusion seizures observed, which eventually cause an irreversible

decrease in fEPSPs.

4.5 Effects of Gap Junction Blockers on Glucose

Reperfusion Seizures

Connexin channel activity is implicated in the spread of ischemic reperfusion injury

in the heart[126]. In order to test the involvement of gap junctions during glucose

reperfusion, 100 µM carbenoxolone was added to normal ACSF that was used to

rescue slices after their first hypoglycemic seizure. A sample LFP recording of this

experimental protocol can be found in Figure 4.13. The addition of CBX blocked

reperfusion seizures in 3 out of 4 slices, but although there is a trend suggesting that

gap junction blockade is protective during reperfusion, p-value was greater than 0.05

using a two-tailed two-proportion z-test.


−5 0 5 10 15 20 25 30−20

−15

−10

−5

0

5

10

15

20

25m

V

Time (ms)

Evoked Potentials

Before hypo

After 30 min hypo

0

0.5

1

1.5

2

-20 -10 0 10 20 30 40 50 60 70

No

rma

lize

d F

ield

Re

sp

on

se

Time (min)

BaselineLow Glucose WashoutB

A

Figure 4.3: Evoked field potentials during hypoglycemia. A. Sample evokes acquired

during hypoglycemia protocol. “Before” and “after” evoked potentials were acquired

during the 10 minute baseline period and the 10 minute washout period, respectively.

A near complete abolishment of evoked potentials after 30 minutes of hypoglycemia.

B. Normalized average of evoked potentials acquired in 12 slices. The decrease in

synaptic activity begins after ∼7 min of low-glucose and decreases to nearly 10%

of baseline levels during hypoglycemic challenge. The loss of this synaptic activity

is irreversible, as visualized by average evoked potentials acquired during 20 minute

washout period. All error bars indicate ± S.E.M.


Figure 4.4 (following page): Spectrogram of hypoglycemic seizure. A. Sample traceof baseline LFP recording B. Hypoglycemic seizure surrounded by pre- and post-ictalbursts. Trace was high-pass filtered at 1 Hz to remove baseline shifts and notch filteredat 60 Hz to remove electronic noise. C. Expansion of gray box in B showing a close-up of the filtered hypoglycemic seizure for comparison with the spectrogram in D.D. Frequency-time plot constructed using the continuous wavelet transform (CWT)analysis technique, showing spectral changes as seizure progresses. Movement towardsred represents higher power at a particular frequency. Each ictal burst within theseizure has a low-frequency component with an associated high-frequency component,similar to other in vitro seizure models.


Figure 4.5 (following page): Continuous LFP recording during low-glucose + octanol.A. For comparison, the continuous LFP recording containing a hypoglycemic chal-lenge is presented. B. Continuous LFP in the CA3 during low glucose + octanolexperiment. After 10 minutes of baseline recording in normal ACSF, 200 µM octanolis added to the perfusing solution for a 5 minute pre-incubation period. Then, theslice is subjected to low-glucose ACSF containing the same concentration of octanolto test the effect of gap junction blockade during hypoglycemia. Perfusion with oc-tanol stops hypoglycemic seizures in 9/10 slices. C. Close up of thick region in octanoltrace to show that there is no ictal activity, rather the thickening of the LFP is dueto single unit activity.


510

15

20

25

30

35

40

20

10 0

Hypoglycemia

mVmVmV

Baseline


Figure 4.6 (following page): Evoked field potentials during hypoglycemia + octanol.A. “Before” and “after” evoked potentials were acquired during the 10 minute baselineperiod and the 10 minute washout period, respectively. Perfusion of the slice withoctanol during hypoglycemia prevents the irreversible depression of synaptic activityand enhances it by 150% during the washout period. B. Normalized average of evokedpotentials acquired in 12 slices in hypoglycemia only condition compared to 7 slices inhypoglycemia + octanol. The depression of synaptic activity starts later during thelatter condition and is less severe compared to hypoglycemia. During the washoutperiod, evoked potentials recover and surpass baseline levels. All error bars indicate± S.E.M.


Figure 4.7 (following page): Continuous LFP recording during low-glucose + meflo-quine. A. For comparison, the continuous LFP recording containing a hypoglycemicchallenge is presented. B. Continuous LFP in the CA3 during low glucose + meflo-quine experiment. After 10 minutes of baseline recording in normal ACSF, 100 nMmefloquine is added to the perfusing solution for a 5 minute pre-incubation period.Then, the slice is subjected to low-glucose ACSF containing the same concentrationof mefloquine to test the effect of pannexin blockade during hypoglycemia. Hypo-glycemic seizure persist when pannexin hemichannels are blocked by low-dose meflo-quine. C. Close-up of hypoglycemic seizure in pannexin blockade during hypoglycemiacondition. Seizure morphology is similar to hypoglycemic seizures and is associatedwith the characteristic baseline shift. D. Close-up of activity after DC shift. Ictalactivity persists throughout hypoglycemic insult.


100 nM M

eflo


−5 0 5 10 15 20 25 30−5

−4

−3

−2

−1

0

1

2

3

4

5

mV

Time (ms)

Evoked Potentials

Before hypoAfter 30 min hypo

Figure 4.8: Evoked field potentials during hypoglycemia + mefloquine. A. “Before”

and “after” evoked potentials were acquired during the 10 minute baseline period and

the 10 minute washout period, respectively. Perfusion of the slice with mefloquine

during hypoglycemia does not prevent the irreversible depression of synaptic activity.


84

10

100

16

90

0

HYPO HYPO + OCT HYPO + MEFLO

PE

RC

EN

TA

GE

PREVALENCE OF SEIZURESSeizure No Seizure

*

Figure 4.9: Summary of experimental protocols. Bar chart shows the prevalence of

seizures in each of the three experimental conditions: hypoglycemia, hypoglycemia +

octanol, and hypoglycemia + mefloquine. There was a significant difference between

the number of seizures between the low-glucose condition per se and when octanol

was added, as measured by a two-tailed two-proportion z-test (p < 0.01). Using

the same statistical test, it was determined that there was no significant difference

between the hypoglycemia and mefloquine conditions.

Figure 4.10: Quantitative features of seizures in different protocols. Difference in

latency and duration of seizures between the hypoglycemia condition compared to

hypoglycemia + mefloquine condition was not significant at p < 0.05 level. This

result was acquired using the Student’s t-test. All errors indicate ± S.E.M.


Figure 4.11 (following page): Continuous LFP trace showing glucose reperfusionseizures. A. Continuous LFP trace of experimental protocol including 10 min base-line, low-glucose, and reperfusion with normal glucose ACSF immediately after firsthypoglycemic seizure. 9/10 slices subjected to the same conditions had repetitiveseizures during glucose reperfusion. B. The hypoglycemic seizure is expanded fromthe gray box in A. C. Close-up of continuous glucose reperfusion seizure D. Furtherzoom of ictal activity shown in C.


Glucose R

eperfusion


−5 0 5 10 15 20 25 30−5

−4

−3

−2

−1

0

1

2

3

4

5

mV

Time (ms)

Evoked Potentials

BaselineAfter hypo seizureAfter glucose reperfusion

Figure 4.12: Evoked field potentials during reperfusion experiment. Evoked field

potentials are acquired at three different times during the reperfusion protocol and

layered to compare slice health throughout the experiment. The dark gray trace

shows an fEPSP acquired immediately after glucose reperfusion following the first

hypoglycemic seizure. The increase in amplitude reflects the increased excitatory

state of the slice during glucose reperfusion which could be a main factor in inducing

glucose reperfusion seizures. However, after 30 min of glucose reperfusion, fEPSP

amplitude was significantly smaller than baseline conditions.

Figure 4.13 (following page): Continuous LFP trace showing glucose reperfusion +CBX. A. For comparison, sample reperfusion protocol trace is presented. B. A hypo-glycemic seizure followed by glucose reperfusion with the addition of 100 µM CBX.Reperfusion seizures which were present 90% of the time in control conditions wereblocked by addition of CBX in 3/4 slices. Although there is a trend which suggestsgap junction involvement in the pathophysiology of glucose reperfusion, statisticalanalysis using two-tailed two-proportion z-test showed that this result was not sig-nificant at the p = 0.05 level. C. Close up of the initial hypoglycemic seizure beforeglucose reperfusion from panel B.

Chapter 5

Discussion and Conclusions

5.1 Seizures and Synaptic Failure

The disruption of the delicate balance between excitatory and inhibitory drive in the

brain is implicated in seizure generation and has been extensively studied in various

seizure models[69, 164, 165]. Paradoxically, depriving the brain of its main energy

source, can lead to the genesis of this energy-expensive neurological state through var-

ious mechanisms that increase excitation and synchrony in the brain. These include:

1) the dissipation of electrochemical gradients across cell membranes resulting in ex-

citotoxic depolarization due to malfunction of gradient-sustaining, energy-dependent

transporters; 2) the accumulation of excitatory neuro- and gliotransmitters in the

extracellular space due to changes in intracellular calcium concentration and impaired

astrocytic buffering; 3) the opening of GJs due to the use of alternative fuels (amino

acids), producing ammonia and increasing intracellular pH[137].

The combination of both increased excitation and increased synchrony renders this

brain state ideal for the generation of ictal activity. Many studies have explored the

57

Chapter 5. Discussion and Conclusions 58

permanent neuropathological effects of seizures and have found that they can lead

to acute necrosis, apoptosis, and eventually, synaptic remodelling that can result in

spontaneous seizure onset[69, 67]. However, reports have said that the mechanism

underlying hypoglycemia-induced neurodegeneration is not necessarily mediated by

seizures. A review by Suh et al. (2007)[137] outlines a neurodegenerative mecha-

nism triggered by severe brain glucose deprivation whereby neuronal depolarization

can lead to non-seizure-dependent excitotoxic pathways involving the release and

subsequent uptake of glutamate and zinc by pre- and post-synaptic neurons, and

ultimately, ROS-mediated DNA damage resulting in neuronal death. In addition, a

study by Dong et al.. (2013)[46] outlines an extrasynaptic NMDAR-dependent path-

way of cell death during ischemia which does not explicitly depend on the presence

of seizures.

In this study, gap junction blockade during hippocampal glucose deprivation was

able to abolish hypoglycemic seizures in a statistically significant way, while prevent-

ing the irreversible loss of synaptic activity that is characteristic of prolonged, severe

hypoglycemia. This finding adds fuel to the hypothesis of a seizure-dependent mech-

anism for hypoglycemia-induced neurodegeneration. Also in support of this postula-

tion, Abdelmalik et al.. (2007)[2] found that the complete abolition of hypoglycemic

seizures using NMDA-receptor antagonists as well as select anticonvulsants always

led to neuroprotection visualized electrographically as the maintenance of synaptic

transmission.

It is perhaps because of the limited number of techniques available to measure ic-

tal activity in humans and rodents compared to the vast array of molecular analysis

techniques, that the role of seizures in neuronal injury during brain glucose depriva-


tion has not been as thoroughly studied. In fact many of the pathological pathways

linked to hypoglycemia-induced injury studied using molecular techniques are also

triggered by seizures; e.g initiation of the apoptotic cascade, mitochondrial damage,

ROS production[68]. Howevever, since the presence of seizures was not tested in these

studies, their involvement in hypoglycemic neuronal damage could not be confirmed.

Additionally, the generation of seizures in in vitro models requires sufficient tissue

health and network connectivity. This could be a reason why experiments to test the

neuronal outcomes of brain glucose deprivation using standard slices have failed to

elicit seizures[83] and are unable to lend support to the involvement of seizures in

hypoglycemic injury.

The GJ blockade during hypoglycemia experiments strongly suggest the necessity

of seizures to mediate hypoglycemia-induced permanent synaptic failure, especially

since irreversible synaptic depression always followed seizure termination. However,

previous experiments have shown a reduction in synaptic transmission in the absence

of seizures[74]. In fact, even in the present study, there was a slight reduction of

fEPSP amplitude during gap junction blockade, which was ameliorated upon glucose

reperfusion. Instead of being an indicator for neuronal damage, this transient synap-

tic depression could reflect the action of compensatory mechanisms, which conserve

energy by preventing energy expensive processes such as synaptic activity. Thus,

it is likely that the presence of seizures exacerbates pathological mechanisms that

had already initiated due to energy deprivation and abolishes the effects of energy-

conserving compensatory mechanisms to hasten neuronal death.


5.2 Controversial Role of Gap Junctions in Seizures

Gap junctions, although well-studied in the heart and brain, are still relatively poorly

understood compared to other types of channels and transporters. This is due, in part,

to the lack of compounds and techniques available to selectively block these pores in

order to specifically test the properties of the several isoforms present. Many past

experiments used molecular biology techniques to quantify the number of GJs in a

preparation until it was discovered that GJ activity was more tightly correlated to

GJ phosphorylation than to GJ number[111]. Genetic knockout models of specific

types of connexins have also always been accompanied by the problem of functional

compensation by other subtypes of connexins, and non-specific effects due to the

ubiquitous nature of connexin expression. Conditionally knocking out connexins has

gained favour due to its higher level of spatial and temporal specificity. Gap junction

blockers and dual patch clamp are heavily used for the electrophysiological study of

gap junctions. However, as mentioned previously, gap junction blockers are noto-

riously non-selective not only blocking several connexin isoforms, but also blocking

other ion channels[28]. Imaging techniques and dyes are also gaining popularity for

the study of GJs, but it is also possible for dye-transfer to occur through a non-GJ

mediated pathway. For this reason, literature on gap junctions is complex and even

contradictory, especially in the context of seizures.

A review by Carlen (2012)[22] discusses the intricacies of GJ involvement in seizures.

They have different roles depending on the type of cell they are expressed on. Neu-

ronal GJs seem to play a role in synchronizing electrochemical waves, thereby facili-

tating seizure propagation. Astrocytic gap junctions are implicated in the buffering

of ions from the extracellular space during seizure activity, preventing the tissue from


entering a depolarization block and prolonging the seizure. Several in vitro and in vivo

seizure models have reported anticonvulsant effects of gap junction blockers. However,

paradoxical studies also report pro-convulsant effects of gap junction blockers. The

results of the current study show several instances of diminished seizure activity due

to GJ blockade, supporting the hypothesis of the anticonvulsant and neuroprotective

effect of gap junction blockers. In the hypoglycemic model, this could be due to one or

a combination of several factors: 1) GJ blockade limits seizure initiation and propaga-

tion by reducing synchrony of neuronal activity; 2) GJ blockade stops the propagation

of apoptotic and excitotoxic factors; 3) GJ blockade limits intra-astrocytic calcium

waves that have been implicated in the release of excitatory gliotransmitters. Future

experiments using a combination of techniques should be used to further disentangle

the mechanistic role of gap junctions in the generation and propagation of seizures

and their role in the spread of hypoglycemia-related injury.

5.3 Possible Mechanisms of Glucose Reperfusion

Seizures

Reperfusion injury has been well described in stroke literature, but reports of neu-

ronal injury after glucose reperfusion in insulin-induced hypoglycemic humans and

rats have drawn attention to reperfusion injury in the context of glucose deprivation

per se. A landmark article by Suh et al. (2007)[136] described a possible mechanism

of neuronal injury during glucose reperfusion. Oxidative stress and ROS production

is a primary factor involved in neurodegeneration during hypoglycemia. However,

Suh et al. found that superoxide generation mediated by the NADPH oxidase en-

zyme, was higher during glucose reperfusion compared to hypoglycemia. The results


of the present study indicate that glucose reperfusion is associated with excitotoxic-

ity that manifests as repetitive seizures. This is reminiscent of ischemia reperfusion

injury during which glutamate excitotoxicity has been observed[115]. Thus, it can be

hypothesized that the mechanisms involved in excitotoxic seizure generation during

hypoglycemia and glucose reperfusion could be similar. The main difference between

these two states in terms of ictal activity is that the latter condition has an unlimited

supply of glucose, allowing for prolonged, energy-dependent seizures. In addition, it

is possible that compensatory mechanisms that were initiated during hypoglycemia

to maximize energy usage were not terminated in time during glucose reperfusion,

potentially causing over-excitation.

The mechanism of gap junction action during glucose reperfusion is still unclear.

It is possible that the involvement of gap junction blockade only extends as far as

limiting seizure propagation, and thereby preventing seizure-induced neuronal death.

On the other hand, it is also conceivable that the blockade of gap junctions stops the

spread of ROS to adjacent cells, containing ROS-induced damage to a smaller area,

and preventing the commonly described “bystander effect”. However, recent litera-

ture suggests that astrocytic gap junctions are important for resistance to oxidative

stress[88]. Following this line of thought, the blockade of gap junctions would actually

exacerbate oxidative stress-induced damage. More focused studies are required to re-

solve the mechanisms of seizure initiation and neuronal degeneration during glucose

reperfusion.


5.4 Mefloquine as a Selective Blocker

Although the use of mefloquine to selectively block pannexin hemichannels is useful

in order to untangle the function of gap junctions versus hemichannels, the drug

itself has certain drawbacks. It is commonly prescribed as a medication for malaria,

but is contraindicated in patients with epilepsy. In the quest to identify the seizure

initiation mechanism of mefloquine, Amabeoku and colleagues[3] injected mice with

different doses of the drug and found that GABA-related pathways could play a role.

Other epileptogenic hypotheses include the ability of high doses of mefloquine to

disrupt neuronal calcium homeostasis, and paradoxically, its interference in normal

gap junction function[101]. In control experiments conducted for the present study, a

dosage of mefloquine which was sufficient to block neuronal connexins ilicited seizures

per se without the addition of any other convulsant. For this reason, a proper dose-

response curve for the effects of mefloquine on hypoglycemic seizures could not be

generated. The area of gap junction study still has room for growth in terms of

pharmacological manipulations because of the lack of selective blockers. A drug

screen to analyze blocking efficacy might be beneficial for this field.

5.5 Conclusions and Future Experiments

This thesis aimed to present a new model of brain glucose deprivation in mouse hip-

pocampal thick slices in order to understand the contribution of seizure activity to

hypoglycemia-mediated neuronal injury. Since gap junctions are implicated in seizure

genesis and are thought to participate in various mechanisms of hypoglycemic brain

damage, they were useful tools for the study of hypoglycemic seizures. We found that

gap junction blockade significantly reduced the prevalence of seizures during hypo-


glycemia and protected the slice from undergoing the irreversible loss of evoked field

potentials that is typical during the hypoglycemic state. In addition, a novel model of

glucose reperfusion was presented, also using murine thick slices. The slice relapsed

into a state of repetitive seizures during glucose reperfusion, but ictal activity was

prevented with the addition of gap junction blockers, suggesting the involvement of

gap junctions during both hypoglycemia and glucose reperfusion. The results of these

experiments indicate that the blockade of gap junctions during hypoglycemia and glu-

cose reperfusion is neuroprotective, possibly through a seizure-dependent mechanism.

More evidence for the neuroprotective role of gap junctions can be gathered by us-

ing histological techniques to stain experimentally challenged tissue. Nissl staining

can be used to quantify the amount of neuronal damage during hypoglycemia, hypo-

glycemia + gap junction blockade, glucose reperfusion, and glucose reperfusion + gap

junction blockade. Additionally, optical and fluorescence techniques in combination

with electrophysiological recordings could be used to directly test the activity of gap

junctions during these different metabolic challenges. A series of fluorescence recov-

ery after photobleaching (FRAP) experiments are currently being conducted using an

astrocyte-specific fluorescent dye to visualize the closed/open state of gap junctions.

This can be a potentially useful technique to further disentangle the involvement of

neuronal versus astrocytic gap junctions or hemichannels. The use of conditional

connexin knockouts in conjunction with these fluorescent and electrophysiological

techniques could have the ability to present powerful evidence for the action of gap

junctions during hypoglycemia.

Bibliography

[1] M Abbaci, M Barberi-Heyob, W Blondel, F Guillemin, and J Didelon. Advan-

tages and limitations of commonly used methods to assay the molecular perme-

ability of gap junctional intercellular communication. Biotechniques, 45:33–62,

2008.

[2] PA Abdelmalik, P Shannon, A Yiu, P Liang, Y Adamchik, M Weisspapir,

M Samoilova, WM Burnham, and PL Carlen. Hypoglycemic seizures during

transient hypoglycemia exacerbate hippocampal dysfunction. Neurobiology of

Disease, 26:646–660, 2007.

[3] GJ Amabeoki and CC Farmer. Gamma-aminobutyric acid and mefloquine-

induced seizures in mice. Prog Neuro Psychopharm Bio Psych, 29:917–921,

2005.

[4] AI Amaral. Effects of hypoglycaemia on neuronal metabolism in the adult brain:

role of alternative substrates to glucose. J Inherit Metab Dis, 36:621–634, 2013.

[5] RN Auer. Hypoglycemic brain damage. Metab Brain Dis, 19:169–175, 2004.

[6] RN Auer, Y Olsson, and BJ Siesjo. Hypoglycemic brain injury in the rat. corre-

lation of density of brain damage with the EEG isoelectric time: a quantitative

study. Diabetes, 33:1090–1098, 1984.

65

Bibliography 66

[7] FG Banting, CH Best, JB Collip, JJR Macleod, and EC Noble. The effect of

pancreatic extract (insulin) on normal rabbits. Am J Physiol, 62:162–176, 1922.

[8] LF Barros, CX Bittner, A Loaiza, and OH Porras. A quantitative overview of

glucose dynamics in the gliovascular unit. Glia, 55:1222–1237, 2007.

[9] EM Bastiaanse, HG Jongsma, A van der Laarse, and BR Takens-Kwak.

Heptanol-induced decrease in cardiac gap junctional conductance is mediated

by a decrease in the fluidity of mebranous cholesterol-rich domains. J Membr

Biol, 136:135–145, 1993.

[10] M Beaumon and G Maccaferri. Is connexin36 critical for GABAergic hypersyn-

chronization in the hippocampus. J Physiol, 589:1663–1680, 2011.

[11] H Beck and E Elger. Epilepsy research: a window onto function and dysfunction

of the human brain. Dialogues Clin Neurosci, 10:7–15, 2008.

[12] Y Ben-Ari and GL Holmes. Effects of seizures on developmental processes in

the immature brain. Lancet Neurol, 5:1055–1063, 2006.

[13] EM Blanc, AJ Bruce-Keller, and MP Mattson. Astrocytic gap junctional com-

munication decreases neuronal vulnerability to oxidative stress-induced disrup-

tion of Ca2+ homeostasis and cell death. Neurochem, 70:958–970, 1998.

[14] A Blasetti, L Di Pietro, G di Corcia, E Franzoni, F Chiarelli, and Verrotti

A. Can insulinoma cause generalised epilepsy. J Pediatr Endocrinol Metab,

20:837–840, 2007.

[15] AK Bouzier-Sore and L Pellerin. Unraveling the complex metabolic nature of

astrocytes. Front Cell Neurosci, 7:179, 2013.

Bibliography 67

[16] A Bragin, M Penttonen, and G Buzsaki. Termination of epileptic afterdischarge

in the hippocampus. J Neurosci, 17:2567–2579, 1997.

[17] R Bruzzone, TW White, and DL Paul. Connections with connexins: the molec-

ular basis of direct intercellular signaling. Eur. J. Biochem, 238:1–27, 1996.

[18] F Bukauskas and C Peracchia. Two distinct gating mechanisms in gap junction

channels: CO2-sensitive and voltage-sensitive. Biophysical Journal, 72:2137–

2142, 1997.

[19] JM Burt and DC Spray. Single channel events and gating behavior of the

cardiac gap junction channel. Proc Natl Acad Sci, 85:3431–3434, 1988.

[20] J Byrne and J Roberts, editors. From Molecules to Networks. An Introduction

to Cellular and Molecular Neuroscience. Elsevier Academic Press, San Antonio,

Texas, USA, 2004.

[21] P Calabresi, D Centonze, A Pisani, and G Bernardi. A possible mechanism for

the aglycemia-induced depression of glutamatergic excitation in the striatum.

J Cereb Blood Flow Metab, 17:1121–1126, 1997.

[22] PL Carlen. Curious and contradictory roles of glial connexins and pannexins in

epilepsy. Brain Research, 1487:54–60, 2012.

[23] A Ceriello, A Novials, and D Giugliano. Evidence that hyperglylcemia after re-

covery from hypoglycemia worsens endothelial function and increases oxidative

stress and inflammation in healthy control subjects and subjects with type1

diabetes. Diabetes, 6:2993–2997, 2012.

Bibliography 68

[24] AG Chapman, B Engelsen, and BS Meldrum. 2-amino-7-phosphono-heptanoic

acid inhibits insulin-induced convulsions and striatal aspartate accumulation in

rats with frontal cortical ablation. J Neurochem, 49:121–127, 1987.

[25] AN Chepkova, OA Sergeeva, and HL Haas. Carbenoxolone impairs LTP and

blocks NMDA receptors in murine hippocampus. Neuropharmacology, 55:139–

147, 2008.

[26] IY Choi, SP Lee, SG Kim, and R Gruetter. In vivo measurements of brain

glucose transport using the reversible Michaelis-Menten model and simultaneous

measurements of cerebral blood flow changes during hypoglycemia. J Cereb

Blood Flow Metab, 21:653–663, 2001.

[27] X Chu, Y Zhao, F Liu, Y Mi, J Shen, X Wang, J Liu, and W Jin. Rapidly raise

blood sugar will aggravate brain damage after severe hypoglycemia in rats. Cell

Biochem Biophys, 2013.

[28] BW Connors. Tales of a dirty drug: carbenoxolone, gap junction, and seizures.

Epilepsy Currents, 12:66–68, 2011.

[29] JE Contreras, HA Sanches, EA Eugenin, D Speidel, M Theis, K Willecke,

FF Bukauskas, MVL Bennett, and JC Saez. Metabolic inhibition induces open-

ing of unapposed connexin 43 gap junction hemichannels and reduces gap junc-

tional communication in cortical astrocytes in culture. Proc Natl Acad Sci,

99:495–500, 2002.

[30] ML Cotrina, J Kang, JHC Lin, E Bueno, TW Hansen, L He, Y Liu, and M Ned-

ergaard. Astrocytic gap junctions remain open during ischemic conditions. J

Neurosci, 18:2520–2537, 1998.

Bibliography 69

[31] SJ Cruikshank, M Hopperstad, M Younger, BW Connors, DC Spray, and

M Srinivas. Potent block of Cx36 and Cx50 gap junction channels by meflo-

quine. Proc Natl Acad Sci, 101:12364–9, 2004.

[32] PE Cryer. Hypoglycemia: pathophysiology, diagnosis, and treatment. Oxford

Universiy Press, New York, 1997.

[33] PE Cryer. Diverse causes of hypoglycemia-associated autonomic failure in dia-

betes. N Engl J Med, 350:2272–2279, 2004.

[34] PE Cryer. Hypoglycemia, functional brain failure, and brain death. The Journal

of Clinical Investigation, 117:868–870, 2007.

[35] PE Cryer, SN Davis, and H Shamoon. Hypoglycemia in diabetes. Diabetes

Care, 26:1902–1912, 2003.

[36] JS Davidson, IM Baumgarten, and EH Harley. Reversible inhibition of inter-

cellular junctional communication by glycyrrhetinic acid. Biochem Biophys Res

Commun, 134:29–36, 1986.

[37] EA Davis, B Keating, GC Byrne, M Russell, and TW Jones. Hypoglycemia:

incidences and clinical predictors in a large population-based sample of children

and adolescents with iddm. Diabetes Care, 20:22–25, 1997.

[38] M De Curtis, A Manfridi, and G Biella. Activity-dependent pH shifts and

periodic recurrence of spontaneous interictal spikes in a model of focal epilep-

togenesis. J Neurosci, 18:7543–7551, 1998.

[39] MH De Pina-Benabou, M Srinivas, DC Spray, and E Scemes. Calmodulin kinase

pathway mediates the K+-induced increase in gap junctional communication

between mouse spinal cord astrocytes. J Neurosci, 21:6635–6643, 2001.

Bibliography 70

[40] MH De Pina-Benabou, V Szostak, A Kyrozia, D Rempe, D Uziel, M Urban-

Maldonado, S Benabou, DC Spray, HJ Federoff, PK Stanton, and R Rozental.

Blockade of gap junctions in vivo provides neuroprotection afterperinatal global

ischemia. Stroke, 36:2232–2237, 2005.

[41] M del Campo, PA Abdelmalik, CP Wu, PL Carlen, and L Zhang. Seizure-like

activit in the hypoglycemic rat: lack of correlation with the electroencephalo-

gram of free-moving animals. Epilepsy Research, 83:243–248, 2009.

[42] O Devinsky, A Vezzani, S Najjar, NC De Lanerolle, and MA Rogawski. Glia and

epilepsy: excitability and inflammation. Trends Neurosci, 36:174–184, 2013.

[43] SH DeVries and EA Schwartz. Hemi-gap-junction channels in solitary horizontal

cells of the catfish retina. J Physiol, 445:201–230, 1992.

[44] Control Diabetes and Complications Trial Research Group. Implementation of

treatment protocols in the diabetes control and complications trial. Diabetes

Care, 18:361–376, 1995.

[45] GA Dienel and NF Cruz. Nutrition during brain activation: does cell-to-cell

lactate shuttling contribute significantly to sweet and sour food for thought?

Neurochem Int, 45:321–351, 2004.

[46] QP Dong, JQ He, and Z Chai. Astrocytic ca2+ waves mediate activation of ex-

trasynaptic NMDA receptors in hippocampal neurons to aggravate brain dam-

age during ischemia.

[47] MO Enkvist and KD McCarthy. Astroglial gap junction communication is

increased by treatment with either glutamate or high k+ concentration. J Neu-

rochem, 62:489–495, 1994.

Bibliography 71

[48] RS Fisher, W van Emde Boas, W Blume, C Elger, P Genton, P Lee, and JJr

Engel. Epileptic seizures and epilepsy: definitions proposed by the International

League Against Epilepsy (ILAE) and the International Bureau for Epilepsy

(IBE). Epilepsia, 4:470–472, 2005.

[49] MV Frantseva, L Kokarovtseva, CG Naus, PL Carlen, D MacFabe, and

JL Perez-Velazquez. Specific gap junctions enhance the neuronal vulnerabil-

ity to brain traumatic injury. J Neurosci, 22:644–653, 2002.

[50] MV Frantseva, L Kokarovtseva, and JL Perez-Velazquez. Ischemia-induced

brain damage depends on specific gap-junctional coupling. Cereb Blood Flow

Metab, 22:453–462, 2002.

[51] N Freinkel, DL Singer, RA Arky, SJ Bleicher, JB Anderson, and CK Silbert.

Alcohol hypoglycemia. i. Carbohydrate metabolism of patients with clinical

alcohol hypoglycemia and the experimental reproduction of the syndrome with

pure ethanol. J Clin Invest, 1963:1112–1133, 1963.

[52] K Fujita, K Nakanishi, K Sobue, T Ueki, K Asai, and T Kato. Astrocytic gap

junction blockage and neuronal Ca2+ oscillation in neuron-astrocyte cocultures

in vitro. Neurochem Int, 33:41–49, 1998.

[53] K Gadhoumi, JM Lina, and J Gotman. Seizure prediction in patients with

mesial temporal lobe epilepsy using EEG measures of state similarity. Clin

Neurophysiol, 124:1745–1754, 2013.

[54] C Giaume, C Fromaget, A el Aoumari, J Cordier, J Glowinski, and D Gros. Gap

junctions in cultured astrocytes: single-channel currents and characterization

of channel-forming protein. Neuron, 6:133–143, 1991.

Bibliography 72

[55] C Giaume, A Koulakoff, L Roux, D Holcman, and N Rouach. Astroglial net-

works: a step further in neuroglial and gliovascular interactions. Nat Rev Neu-

rosci, 11:87–99, 2010.

[56] C Giaume, L Leybaert, CC Naus, and JC Saez. Connexin and pannexin

hemichannels in brain glial cells: properties, pharmacology, and roles. Front

Pharmacol, 4, 2013.

[57] C Giaume, A Tabernero, and JM Medina. Metabolic trafficking through astro-

cytic gap junctions. Glia, 21:114–123, 1997.

[58] FA Gibbs and EL Murray. Hypoglycemic convulsions; three case reports. Elec-

troencephalogr Clin Neurophysiol, 6:674–678, 1954.

[59] GS Goldberg, JF Bechberger, and CC Naus. A pre-loading method of evaluating

gap junctional communication by flurescent dye transfer. Biotechniques, 18:490–

497, 1995.

[60] A Gonzalez-Sulser, J Wang, and R Dzakpasu. The 4-aminopyridine in vitro

epilepsy model analyzed with a perforated multi-electrode array. Neurophar-

macology, 60:1142–1153, 2011.

[61] JE Goodwin, WK Kerr, and FL Lawson. Bioelectric responses in metrazol and

insulin shock. American Journal of Psychiatry, 96:1389–1405, 1940.

[62] B Granda, A Tabernero, LI Sanchez-Abarca, and JM Medina. The K-ATP

channel regulates the effect of Ca2+ on gap junction permeability in cultured

astrocytes. FEBS Lett, 427:41–45, 1998.

[63] R Gruetter, K Ugurbil, and ER Seaquist. Steady-state cerebral glucose concen-

trations and transport in the human brain. J Neurochem, 70:397–408, 1998.

Bibliography 73

[64] E Hansson, H Muyderman, J Leonova, L Allansson, J Sinclair, F Blomstrand,

T Thorlin, M Nilsson, and L Ronnback. Astroglia and glutamate in physiology

and pathology: aspects on glutamate transport, glutamate-induced cell swelling

and gap-junction communication. Neurochem Int, 37:317–329, 2000.

[65] RJ Harris, T Wieloch, L Symon, and BK Siesjo. Cerebral extracellular calcium

activity in severe hypoglycemia: relation to extracellular potassium and energy

state. J Cereb Blood Flow Metab, 4:187–193, 1984.

[66] SP Hart and BM Frier. Causes, management and morbidity of acute hypo-

glycaemia in adults requiring hospital admission. Quart J Med, 91:505–510,

1998.

[67] SR Haut, J Veliskova, and SL Moshe. Susceptibility of immature and adult

brains to seizure effects. Lancet Neurol, 3:608–617, 2004.

[68] DC Henshall and RP Simon. Epilepsy and apoptosis pathways. J Cereb Blood

Flow Metab, 25:1557–1572, 2005.

[69] IE Holopainen. Seizures in the developing brain: cellular and molecular mecha-

nisms of neuronal damage, neurogenesis and cellular reorganization. Neurochem

Int, 52:935–947, 2008.

[70] CC Huang and YC Chang. The long-term effects of febrile seizures on the

hippocampal neuronal placticity - clinical and experimental evidence. Brain

Dev, 31:383–387, 2009.

[71] R Iglesias, DC Spray, and E Scemes. Mefloquine blockade of pannexin1 currents:

resolution of a conflict. Cell Communication and Adhesion, 16:131–137, 2010.

Bibliography 74

[72] P Indic and J Narayanan. Wavelet based algorithm for the estimation of fre-

quency flow from electroencephalogram data during epileptic seizure. Clin Neu-

rophysiol, 122:680–686, 2011.

[73] NK Isaev, EV Stel’mashuk, and DB Zorov. Cellular mechanisms of brain hy-

poglycemia. Biochemistry (Moscow), 72:471–478, 2007.

[74] Y Izumi and CF Zorumski. Developmental changes in long-term potentiation

in CA1 of rat hippocampal slices. Synapse, 20:19–23, 1995.

[75] FE Jensen and C Wang. Hypoxia-induced hyperexcitability in vivo and in vitro

in the immature hippocampus. Epilepsy Res, 26:131–140, 1996.

[76] MM Jin and Z Chen. Role of gap junctions in epilepsy. Neurosci Bull, 27:389–

406, 2011.

[77] GR Juszczak and AH Swiergiel. Properties of gap junction blockers and their

behavioural, cognitive and electrophysiological effects: animal and human stud-

ies. Prog Neuro-Psychopharmacology and Bio Psych, 33:181–198, 2009.

[78] O Kann and R Kovacs. Mitochondria and neuronal activity. Am J Physiol Cell

Physiol, 292:C641–C657, 2007.

[79] KJ Kellar, AE Langley, BH Marks, and JJ O’Neill. Ventral medial hypothala-

mus: involvement in hypoglycemic convulsions. Science, 187:746–748, 1975.

[80] H Kettenmann and BR Ransom. Electrical coupling between astrocytes and

between oligodenderocytes studied in mammalian cell cultures. Glia, 1:64–73,

1988.

Bibliography 75

[81] R Khazipov, I Khalilov, R Tyzio, E Morozova, Y Ben-Ari, and GL Holmes.

Developmental changes in GABAergic actions and seizure susceptibility in the

rat hippocampus. Eur J Neurosci, 19:590–600, 2004.

[82] YH Kim, EY Kim, BJ Gwag, S Sohn, and JY Koh. Zinc-induced cortical neu-

ronal death with features of apoptosis and necrosis: mediation by free radicals.

Neuroscience, 89:175–182, 1999.

[83] A Kirchner, J Veliskova, and L Velisek. Differential effects of low glucose con-

centrations on seizures and epileptiform activity in vivo and in vitro. European

Journal of Neuroscience, 23:1512–1522, 2006.

[84] R Kohling, Sj Gladwell, E Bracci, M Vreugdenhil, and JG Jefferys. Prolonged

epileptiform bursting induced by 0-Mg2+ in rat hippocampal slices depends on

gap junctional coupling. Neuroscience, 105:579–587, 2001.

[85] Z Kokaia, M Metsis, M Kokaia, J Bengzon, E Elmer, ML Smith, T Timmusk,

BK Siesjo, H Persson, and O Lindvall. Brain insults in rats induce increased

expression of the bdnf gene through differential use of multiple promoters. Eur

J Neurosci, 6:587–596, 1994.

[86] A Kumaria, CM Tolias, and G Burnstock. ATP signalling in epilepsy. Purinergic

Signal, 4:339–346, 2008.

[87] L Lapenta, C Di Bonaventura, J Fattouch, F Bonini, S Petrucci, S Gagliardi,

S Casciato, M Manfredi, M Prencipe, and AT Giallonardo. Focal epileptic

seizure induced by transient hypoglycaemia in insulin-treated diabetes. Epilep-

tic Disord, 12:84–87, 2010.

Bibliography 76

[88] HT Le, WC Sin, S Lozinsky, J Bechberger, JL Vega, QX Guo, JC Saez, and

CC Naus. Gap junction intercellular communication mediated by connexin43

in astrocytes is essential for their resistance to oxidative stress. J Biol Chem,

2013.

[89] SH Lee, WT Kim, AH Cornell-Bell, and H Sontheimer. Astrocytes exhibit

regional specificity in gap-junction coupling. Glia, 11:315–325, 1994.

[90] L Leybaert. Neurobarrier coupling in the brain: a partner of neurovascular

and neurometabolic coupling? Journal of Cerebral Blood Flow and Metabolism,

25:2–16, 2005.

[91] K Lord, E Dzata, KE Snider, PR Gallagher, and DD De Leon. Clinical pre-

sentation and management of children with diffuse and focal hyperinsulinism:

a review of 223 cases. Journal of Clinical Endocrine Metabolism, 98:E1786–9,

2013.

[92] H Lund-Andersen. Transport of glucose from blood to brain. Physiological

Reviews, 59:305–352, 1979.

[93] P Magistretti. From Molecules to Networks. An Introduction to Cellular and

Molecular Neuroscience, chapter Brain Energy Metabolism. In Byrne and

Roberts [20], 2004.

[94] N Maier, M Guldenagel, G Sohl, H Siegmund, K Willecke, and A Draguhn.

Reduction of high-frequency network oscillations (ripples) and pathological net-

work discharges in hippocampal slices from connexin 36-deficient mice. J Phys-

iol, 541:521–528, 2002.

Bibliography 77

[95] R Malouf and JC Brust. Hypoglycemia: causes, neurological manifestations,

and outcome. Ann Neurol, 17:421–430, 1985.

[96] DG Margineanu and H Klitgaard. Can gap-junction blockade preferentially

inhibit neuronal hypersynchrony vs. excitability? Neuropharmacology, 41:377–

383, 2001.

[97] C Meier and R Dermietzel. Electrical synapses–gap junctions in the brain.

Results Probl Cell Differ, 43:99–128, 2006.

[98] J Morano, H Rios, H Morano, and E Resches. Hypoglycemic coma and con-

vulsions due to ingestion of ethyl alcohol. Arch Argent Pediatr, 68:204–207,

1970.

[99] A Nehlig and JA Coles. Cellular pathways of energy metabolism in the brain:

is glucose used by neurons or astrocytes. Glia, 55:1238–1250, 2007.

[100] B Nellgard and T Wieloch. Cerebral protection by AMPA- and NMDA-receptor

antagonists administered after severe insulin-induced hypoglycemia. Exp Brain

Res, 92:259–266, 1992.

[101] RL Nevin. Epileptogenic potential of mefloquine chemoprophylaxis: a

pathogenic hypothesis. Malaria Journal, 8:188–194, 2009.

[102] JL Noebels, M Avoli, and MA Rogawski. Jasper’s Basic Mechanisms of

the Epilepsies; 4th edition. National Center for Biotechnology Information,

Bethesda, 2012.

[103] KM Noh and JY Koh. Induction and activation by zinc of NADPH oxidase in

cultured cortical neurons and astrocytes. J Neurosci, 20:RC111, 2000.

Bibliography 78

[104] JA Orellana, PJ Saez, KF Shoji, KA Schalper, N Palacios-Prado, V Velarde,

C Giaume, MV Bennett, and JC Saez. Modulation of brain hemichannels and

gap junction channels by pro-inflammatory agents and their possible role in

neurodegeneration. Antioxid Redox Signal, 11:369–399, 2009.

[105] A Ovchinnikov, A Luttjohann, A Hramov, and G van Luijtelaar. An algorithm

for real-time detection of spike-wave discharges in rodents. J Neurosci Methods,

194:172–178, 2010.

[106] KS Panickar, K Purushotham, MA King, G Rajakumar, and JW Simpkins.

Hypoglycemia-induced seizures reduce cyclic amp response element binding pro-

tein levels in the rat hippocampus. Neuroscience, 83:1155–1160, 1998.

[107] MP Papagapiou and RN Auer. Regional neuroprotective effects of the nmda re-

ceptor antagonist mk-801 (dizocilpine) in hypoglycemic brain damage. J Cereb

Blood Flow Metab, 10:270–276, 1990.

[108] CA Pappas, MG Rioult, and BR Ransom. Octanol, a gap junction uncoupling

agent, changes intracellular [H+] in rat astrocytes. Glia, 16:7–15, 1996.

[109] DL Paul, L Ebihara, LJ Takemoto, KI Swenson, and DA Goodenough. Con-

nexin46, a novel lens gap junction protein, induces voltage-gated currents in

nonjunctional plasma membrane of xenopus oocytes. J Cell Biol., 115:1077–

1089, 1991.

[110] L Pellerin and P Magistretti. Glutamate uptake into astrocytes stimulates aer-

obic glycolysis: a mechanism coupling neuronal activity to glucose utilization.

Proc Natl Acad Sci, 91:10625–10629, 1994.

Bibliography 79

[111] C Peracchia. Chemical gating of gap junction channels. Roles of calcium, pH

and calmodulin. Biochim et Biophys Acta, 1662:61–80, 2004.

[112] JL Perez-Velazquez and PL Carlen. Gap junctions, synchrony and seizures.

Trends Neurosci, 23:68–74, 2000.

[113] JL Perez-Velazquez, TA Valiante, and PL Carlen. Modulation of gap junctional

mechanisms during calcium-free induced field burst activit: a possible role for

electrotonic coupling in epileptogenesis. J Neurosci, 14:4308–4317, 1994.

[114] EC Puente, J Silverstein, AJ Bree, DR Musikantow, DF Wozniak, S Maloney,

D Daphna-Iken, and SJ Fisher. Recurrent moderate hypoglycemia ameliorates

brain damage and cognitive dysfunction induced by severe hypoglycemia. Dia-

betes, 59:1055–1062, 2010.

[115] S Pundik, K Xu, and S Sundararajan. Reperfusion brain injury: focus on

cellular bioenergetics. Neurology, 79:S44–S51, 2012.

[116] A Rami, T Volkmann, and J Winckler. Effective reduction of neuronal death

by inhibiting gap junctional intercellular communication in a rodent model of

global transient cerebral ischemia. J Exp Neurol, 170:297–304, 2001.

[117] A Rawanduzy, A Hansen, TW Hansen, and MJ Nedergaard. Effective reduc-

tion of infact volume by gap junction blockade in a rodent model of stroke.

Neurosurg, 87:916–920, 1997.

[118] S Rohr. Role of gap junctions in the propagation of the cardiac action potential.

Cardiovasc Res, 62:309–322, 2004.

Bibliography 80

[119] FM Ross, P Gwyn, D Spanswick, and SN Davies. Carbenoxolone depresses

spontaneous epileptiform activity in the CA1 region of rat hippocampal slices.

Neuroscience, 100:789–796, 2000.

[120] N Rouach, E Avignone, W Meme, A Koulakoff, L Venance, F Blomstrand,

and C Giaume. Gap junctions and connexin expression in the normal and

pathological central nervous system. Biology of the Cell, 94:457–475, 2002.

[121] N Rouach, A Koulakoff, V Abudara, K Willecke, and C Giaume. As-

troglial metabolic networks sustain hippocampal synaptic transmission. Sci-

ence, 322:1551–1554, 2008.

[122] R Rozental, M Srinivas, and DC Spray. How to close a gap junction channel:

efficacies and potencies of uncoupling agents. Methods Mol Biol, 154:447–476,

2001.

[123] M Samoilova, K Wentland, Y Adamchik, AA Velumian, and PL Carlen. Con-

nexin 43 mimetic peptides inhibit spontaneous epileptiform activity in organ-

otypic hippocampal slice cultures. Exp Neurol, 210:762–775, 2008.

[124] PE Schauwecker. The effects of glycemic control on seizures and seizure-induced

excitotoxic cell death. BMC Neuroscience, 13:94–108, 2012.

[125] B Schultes, W Kern, K Oltmanns, A Peters, S Gais, HL Fehm, and J Born. Dif-

ferential adaptation of neurocognitive brain functions to recurrent hypoglycemia

in healthy men. Psychoneuroendocrinology, 30:149–161, 2005.

[126] R Schulz and G Heusch. Connexin 43 and ischemic preconditioning. Cadiovasc

Research, 62:335–344, 2004.

Bibliography 81

[127] SL Sensi, HZ Yin, SG Cerriedo, SS Rao, and JH Weiss. Preferential Zn2+ influx

through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochon-

drial superoxide production. Proc Natl Acad Sci, 96:2414–2419, 1999.

[128] RP Simon, JW Schmidley, BS Meldrum, JH Swan, and AG Chapman. Exci-

totoxic mechanisms in hypoglycaemic hippocampal injury. Neuropathol Appl

Neurobiol, 12:567–576, 1986.

[129] J Snyders, KM Knoth, SL Roberds, and MM Tamkun. Time-, voltage-, and

state-dependent block by quinidine of a cloned human cardiac potassium chan-

nel. Mol Pharmacol, 41:322–330, 1992.

[130] GC Somjen. Ion regulation in the brain: implications for pathophysiology.

Neuroscientist, 8:254–267, 2002.

[131] GG Somjen. Ions in the Brain: Normal Function, Seizures, and Stroke. Oxford

University Press, USA, 2004.

[132] DC Spray, R Rozental, and M Srinivas. Prospects for rational development of

pharmacological gap junction channel blockers. Current Drug Targets, 3:455–

464, 2002.

[133] C Steinhauser and G Seifert. Jasper’s Basic Mechanisms of the Epilepsies; 4th

edition, chapter Astrocyte dysfunction in epilepsy. In [102], 2012.

[134] SO Suadicani, CF Brosnan, and E Scemes. P2X7 receptors mediate ATP release

and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci, 26:1378–

1385, 2006.

Bibliography 82

[135] SW Suh, K Aoyama, and Y Chen. Hypoglycemic neuronal death and cognitive

impairment are prevented by poly(adp-ribose) polymerase inhibitors adminis-

tered after hypoglycemia. J Neuroscie, 23:10681–10690, 2003.

[136] SW Suh, ET Gum, and RA Swanson. Hypoglycemic neuronal death is triggered

by glucose reperfusion and activation of neuronal NADPH oxidase. Journal of

Clinical Investigation, 117:910–918, 2007.

[137] SW Suh, AM Hamby, and RA Swanson. Hypoglycemia, brain energetics, and

hypoglycemic neuronal death. Glia, 55:1280–1286, 2007.

[138] BR Takens-Kwak, HG Jongsma, and AC Van Ginneken. Mechanism of

heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp

study. Am J Physiol, 262:C1531–C1538, 1992.

[139] RS Talhouk, MP Zeinieh, MA Mikati, and ME El-Sabban. Gap junctional

intercellular communication in hypoxia-ischemia-induced neuronal injury. Prog

Neurobiol, 84:57–76, 2008.

[140] GC Tamasi and EJ Drenick. Resistance to insulin convulsions in fasted mice.

Endocrinology, 92:1277–1279, 1973.

[141] MP Thibeault-Eybalin, A Lortie, and L Carmant. Neonatal seizures: do they

damage the brain? Pediatr Neurol, 40:175–180, 2009.

[142] RJ Thompson, MF Jackson, M Olah, DJ Rungta, RL nd Hines, MA Beazely,

JF MacDonald, and BA MacVicar. Activation of pannexin-1 hemichannels

augments aberrant bursting in the hippocampus. Science, 322:1555–1559, 2008.

[143] RJ Thompson, N Zhou, and B MacVicar. Ischemia opens neuronal gap junction

hemichannels. Science, 312:924–927, 2006.

Bibliography 83

[144] SM Todorovic and CJ Lingle. Pharmacological properties of T-type Ca2+ cur-

rent in adult rat sensory neurons: effects of anticonvulsant and anesthetic

agents. J Neurophysiol, 79:240–252, 1998.

[145] T Tokizane and CH Sawyer. Sites of origin of hypoglycemic seizures in the

rabbit. Arch Neurol Psychiatry, 77:259–266, 1957.

[146] AD Tolis. Hypoglycemic convulsions in children after alcohol ingestion. Pediatr

Clin North Am, 12:423–425, 1965.

[147] M Tsurusaki, T Akasu, and S Shoji. Patch-clamp analysis of hypoglycemia-

induced inhibition of synaptic transmission in the rat dorsolateral septal nu-

cleus. Kurume Med J, 41:65–72, 1994.

[148] CP Turner, MR Blackburn, and SA Rivkees. A1 adenosine receptors mediate

hypoglycemia-induced neuronal injury. Journal of Molecular Endocrinology,

32:129–144, 2004.

[149] D Urion, HJ Vreman, and MW Weiner. Effect of acetate on hypoglycemic

seizures in mice. Diabetes, 28:1022–1026, 1979.

[150] RC Vannucci and SJ Vannucci. Hypoglycemic brain injury. Semin Neonatol,

6:147–155, 2001.

[151] SJ Vannucci, F Maher, and IA Simpson. Glucose transporter proteins in brain:

delivery of glucose to neurons and glia. Glia, 21:2–21, 2997.

[152] L Velisek, J Veliskova, O Chudomel, KL Poon, K Robeson, B Marshall,

A Sharma, and S Moshe. Metabolic environment in substantia nigra reticu-

lata is critical for the expression and control of hypoglycemia-induced seizures.

The Journal of Neuroscience, 28:9349–9362, 2008.

Bibliography 84

[153] L Venance, D Piomelli, J Glowinski, and C Giaume. Inhibition by anandamide

of gap junctions and intercellular calcium signallin in striatal astrocytes. Nature,

376:590–594, 1995.

[154] JP Vessey, MR Lalonde, HA Mizan, NC Welch, ME Kelly, and S Barnes. Car-

benoxolone inhibition of voltage-gated Ca channels and synaptic transmission

in the retina. J Neurophysiol, 92:1252–1256, 2004.

[155] LJ Voss, G Jacobson, JW Sleigh, A Steyn-Ross, and M Steyn-Ross. Excitatory

effects of gap junction blockers on cerebral cortex seizure-like activity in rats

and mice. Epilepsia, 50:1971–1978, 2009.

[156] A Wallraff, R Kohling, U Heinemann, M Theis, K Willecke, and C Steinhauser.

The impact of astrocytic gap junctional coupling on potassium buffering in the

hippocampus. J Neurosci, 26:5438–5447, 2006.

[157] E Werder, HH Ehrat, R Morger, H Herzer, H Ludin, and R Illig. Hypoglycemic

attacks and convulsions due to an islet cell tumor in a 7-year-old-girl. Helv

Paediatr Acta, 26:131–143, 1971.

[158] T Wieloch. Hypoglycemia-induced neuronal damage prevented by an n-methyl-

d-aspartate antagonist. Science, 230:681–683, 1985.

[159] T Wieloch, RJ Harris, L Symon, and BK Siesjo. Influence of severe hypo-

glycemia on brain extracellular calcium and potassium activities, energy, and

phospholipid metabolism. J Neurochem, 43:160–168, 1984.

[160] C Wu, WP Luk, J Gillis, F Skinner, and L Zhang. Size does matter: generation

of intrinsic network rhythms in thick mouse hippocampal slices. J Neurophysiol,

93:2302–2317, 2005.

Bibliography 85

[161] A Yatani, M Wakamori, G Mikala, and A Bahinski. Block of transient outward-

type cloned cardiac K+ channel currents by quinidine. Circ Res, 73:351–359,

1993.

[162] JJ Yoon, CR Green, SJ O’Carroll, and LF Nicholson. Dose-dependent protec-

tive effect of connexin43 mimetic peptide against neurodegeneration in an ex

vivo model of epileptiform lesion. Epilepsy Res, 92:153–162, 2010.

[163] KR Zahs. Heterotypic coupling between glial cells of the mammalian central

nervous system. Glia, 24:85–96, 1998.

[164] ZJ Zhang, J Koifman, DS Shin, CM Florez, L Zhang, TA Valiante, and

PL Carlen. Transition to seizure: ictal discharge is preceded by exhausted

presynaptic GABA release in the hippocampal CA3 region. Neurobiology of

Disease, 32:2499–2512, 2012.

[165] J Ziburkus, JR Cressman, and SJ Schiff. Seizures as imbalanced up states: ex-

citatory and inhibitory conductances during seizure-like events. J Neurophysiol,

109:1296–1306, 2013.

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