Division of Physiology and Neuroscience
Department of Biosciences
Faculty of Biological and Environmental Sciences
University of Helsinki
Doctoral Programme in Brain & Mind
Animal models of early brain disorders: behavioural and
electrophysiological approaches
Alexey Y. Yukin
ACADEMIC DISSERTATION
To be presented for public examination, with the permission of the Faculty of
Biological and Environmental Sciences of the University of Helsinki
In the lecture hall 1041, Viikki Biocenter 2 (Viikinkaari 5),
On May 25th at 12 o’clock noon.
Helsinki 2016
Supervised by
Professor Kai Kaila
Department of Biosciences, and Neuroscience Center,
University of Helsinki, Finland
Professor Juha Voipio
Department of Biosciences, University of Helsinki,
Finland
Reviewed by
Professor Hannes Lohi
Research Program Unit, Molecular Neurology, Faculty of
Medicine
Department of Veterinary Biosciences, Faculty of
Veterinary Medicine
University of Helsinki, Finland
Folkhälsan Research Center, Helsinki, Finland
Professor Aarne Ylinen
Department of Neurological Sciences, University of
Helsinki, Finland
Department of Neurology, Helsinki University Central
Hospital, Finland
Opponent
Professor Heikki Tanila
A.I. Virtanen Institute, University of Eastern Finland,
Kuopio, Finland
Custos
Professor Juha Voipio
Department of Biosciences, University of Helsinki,
Finland
Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis
ISBN 978-951-51-2151-6 (paperback)
ISBN 978-951-51-2152-3 (PDF, http://ethesis.helsinki.fi)
To my loved ones
TABLE OF CONTENTS
ABBREVIATIONS.............................................................................................VII
ABSTRACT.......................................................................................................VIII
1 INTRODUCTION..............................................................................................1
2 REVIEW OF THE LITERATURE....................................................................3
2.1 THE SUBPLATE AND CORTICAL CIRCUIT DEVELOPMENT..............3
2.1.1 Role of subplate in cortex maturation...........................................................7
2.1.1.1 Formation of neocortical architectural hallmarks......................................7
2.1.1.2 Subplate and endogenous activity of the immature brain..........................8
2.1.2 Alterations in endogenous activity and functional architecture following
subplate ablation....................................................................................................9
2.1.3 Models of subplate disruption....................................................................11
2.2 FEBRILE SEIZURES...................................................................................12
2.2.1 Non-Genetic Mechanisms of Febrile Seizures...........................................13
2.2.1.1 Respiratory alkalosis...............................................................................13
Brain pH regulation............................................................................................14
GABAergic signalling and the role of carbonic anhydrase activity....................16
2.2.1.2 Inflammatory cytokines...........................................................................18
2.2.1.3 Role of brainstem in generation of febrile seizures.................................19
2.2.2 Animal models of febrile seizures..............................................................20
2.2.3 Antiepileptic therapy..................................................................................22
3. AIMS...............................................................................................................24
4. EXPERIMENTAL PROCEDURES...............................................................25
4.1 GENERATION OF THE TRANSGENIC MOUSE LINES AND IN
VITRO ELECTROPHYSIOLOGICAL RECORDINGS....................................25
4.2 ANIMAL SURGERY AND EEG RECORDINGS.......................................25
4.3 EXPERIMENTAL SEIZURES.....................................................................27
IV
4.4 BLOOD SAMPLING....................................................................................28
4.5 EEG ANALYSIS..........................................................................................29
4.6 STATISTICS.................................................................................................29
5 RESULTS........................................................................................................31
5.1 SUBPLATE NEURONS PROMOTE SPINDLE BURSTS AND
THALAMOCORTICAL PATTERNING IN THE NEONATAL RAT
SOMATOSENSORY CORTEX (I)....................................................................31
5.1.1 Local subplate ablation prevents spontaneous spindle bursts.....................31
5.1.2 Strengthening of the thalamocortical connection is prevented after
subplate ablation..................................................................................................32
5.1.3 Barrel patterning in S1 somatosensory area is dependent on subplate
function...............................................................................................................32
5.2 NEURONAL CARBONIC ANHYDRASE ISOFORM VII PROVIDES
GABAERGIC EXCITATORY DRIVE TO EXACERBATE FEBRILE
SEIZURES (II)....................................................................................................33
5.2.1 Sequential expression of CA VII and CA II in hippocampal neurons........33
5.2.2 Depolarizing GABA responses are promoted by CA VII and CA II..........34
5.2.3 Lack of electrographic febrile seizures in mice devoid of carbonic
anhydrase VII......................................................................................................35
5.3 FOREBRAIN-INDEPENDENT GENERATION OF HYPERTHERMIC
CONVULSIONS IN INFANT RATS.................................................................37
5.3.1 Disconnecting forebrain from the brainstem aggravates FS.......................37
6. DISCUSSION.................................................................................................39
6.1 STUDY I.......................................................................................................39
6.2 STUDY II......................................................................................................41
6.3 STUDY III....................................................................................................43
7 CONCLUSIONS AND FUTURE PROSPECTS.............................................44
8 ACKNOWLEDGEMENTS.............................................................................46
9 REFERENCES.................................................................................................48
V
LIST OF ORIGINAL PUBLICATIONS
This Thesis is based on the following publications referred to in the text by
their Roman numerals.
I.
Tolner EA*, Sheikh A*, Yukin AY, Kaila K, Kanold PO
(2012). Subplate neurons promote spindle bursts and thalamocortical
patterning in the neonatal rat somatosensory cortex. J Neurosci. 32:692-702.
II.
Ruusuvuori E*, Huebner AK*, Kirilkin I*, Yukin AY, Blaesse
P, Helmy M, Kang HJ, El Muayed M, Hennings JC, Voipio J, Šestan N,
Hübner CA, Kaila K (2013). Neuronal carbonic anhydrase VII provides
GABAergic excitatory drive to exacerbate febrile seizures. EMBO J. 32:
2275-86.
III.
Pospelov AS*, Yukin AY*, Blumberg MS, Puskarjov M, Kaila
K (2016). Forebrain-independent generation of hyperthermic convulsions in
infant rats. Epilepsia 57: e1-e6
These authors contributed equally to this work
The publications are referred to in the text by their roman numerals.
The doctoral candidate’s contribution:
In Study I the author performed surgery, histological staining and recorded
endogenous activity together with E.A.T., and contributed to data analysis.
In Study II the author contributed to designing the in vivo experiments and
febrile seizure paradigm, performed electrode implantations and EEG
recordings on CA VII KO and WT mice and contributed to data analysis.
In Study III the author participated in the design of the research, performed
all experimental work together with A.S.P., and wrote the manuscript
together with A.S.P. and K.K. with input from co-authors.
VI
ABBREVIATIONS
AED
BBB
CA
CNS
cRMS
CX
E
EEG
eFS
EPSC
Fb-EEG
FS
FSE
GABAAR
GD
GW
HFS
IL-1β
IL-1R1
IL-6
KO
LGN
LPS
MGN
ODC
P
pCO2
PCW
PFC
PVL
p75NTR
S1
SAT
SPN
TF
TNFα
V1
WT
antiepileptic drug
blood-brain barrier
carbonic anhydrase
central nervous system
cumulative root mean square
cortex
embryonic day
electroencephalography
experimental febrile seizures
excitatory post-synaptic current
full band EEG
febrile seizures
febrile status epilepticus
Ionotropic (type A) γ-aminobutyric acid receptor
gestation day
gestation week
high-frequency stimulation
interleukin-1β
interleukin type 1 receptor
interleukin-6
knock out
lateral geniculate nucleus
lipopolysaccharide
medial geniculate nucleus
ocular dominance column
postnatal day
partial pressure of CO2
post-conceptional week
prefrontal cortex
periventricular leukomalacia
p75 neurotrophin receptor
primary somatosensory cortex
spontaneous activity transient
subplate neuron
time frequency
tumour necrosis factor alpha
primary visual cortex
wild type
VII
ABSTRACT
This Thesis addresses fundamental mechanisms of brain disorders that occur
in preterm infants or during early childhood. To this end, we have developed
animal models to mimic disorder-specific pathological conditions in the
brain. During critical periods of development of the mammalian brain, cell
migration and synapse formation are crucially important to set up newly
developed neuronal circuits that will support complex network activity. A
particular set of cells, the subplate neurons, is the first to mature during the
earliest stages of cortical development. These neurons act as transient relay
and processing station for signals coming from subcortical structures to the
cortex. Their high dependence on oxygen supply makes them vulnerable to
injuries that take place during pregnancy. In the present work, conditions of
this kind were mimicked by specific toxin-based ablation of subplate neurons.
At a more advanced stage of brain development, a very common type of brain
disorder is febrile seizures (FS). They represent convulsive events in humans
that are promoted by respiratory alkalosis during febrile illness. The global
incidence of FS is estimated from 2 to 14 % (depending on ethnicity) of all
children in the age of 6 months to 5 years. Using an animal model based on
13-14 day old rats exposed to hyperthermia, we show that a pro-excitatory
action of GABA based on the neuron-specific carbonic anhydrase (CA)
isoform VII is crucially involved in the generation of these seizures. Finally,
while it is widely assumed that FS are limbic in origin, this Thesis provides
new evidence that the brainstem can have an independent and prominent role
in the generation of FS, and also of convulsions induced by kainic acid, which
so far, have been considered prototypical in studies of limbic seizures. As a
whole, this work demonstrates the high utility and heuristic value of customdesigned animal models in studies of basic mechanisms and consequences of
disorders in the developing brain.
VIII
1 INTRODUCTION
The developing brain undergoes global structural and functional changes. In
mammals a period in development known as the ‘brain growth spurt’ is
characterized by exponential increases not only in brain volume but in the
complexity of its wiring and functional organization. The timing of this
notable period relative to birth differs from one mammalian species to another
(Erecinska et al, 2004). In humans, the brain growth spurt commences
roughly by the end of the second trimester, peaking perinatally, and continues
into the first few years of life (Huttenlocher & Dabholkar, 1997; Erecinska et
al, 2004). In macaques, the timing of these events is shifted to take place
largely in utero and in mice and rats during the first three postnatal weeks
(Erecinska et al, 2004). Because of their exceptional plasticity, both adaptive
and maladaptive, developing networks are rendered highly vulnerable to
environmental perturbations (Volpe, 2001). Indeed, one of the most common
neurological manifestations of plastic changes in developing neural networks
during the brain growth spurt are seizures (Hauser et al, 1993). Occurrence of
seizures is highly age-dependent, with the highest rates of incidence in
neonates and infants, and peaking again at senescence (Hauser et al, 1993;
Volpe, 2008). The most common type of convulsive events in childhood are
FS, affecting children between 6 months to 5 years of age, with a peak
incidence at around 18 months (Hauser, 1994).
The clinical management of seizures in the paediatric population
predominantly relies on the use of GABAAR potentiating antiepileptic drugs
(AEDs), the rationale for use of which is rooted deep in a simplistic notion of
pharmacologically correcting the balance of excitation and inhibition
maintained respectively by a relationship of GABAergic and glutamatergic
1
synaptic inputs to cortical neurons (White et al, 2007; Fritschy, 2008; Kaila et
al, 2014). This rationale is further maintained by what is a widely held
assumption that the neocortical/limbic networks are the primary generators of
seizures, and thus are typically the targets of pharmacotherapy as well as
preclinical investigation.
2
2 REVIEW OF THE LITERATURE
2.1 THE SUBPLATE AND CORTICAL CIRCUIT DEVELOPMENT
The last trimester of gestation is considered to be one of the most critical
periods with regard to the development of the human cortex, especially for
the formation of the thalamo-cortical system (Zagha & McCormick, 2014),
which is thought to be critical for high frequency synchrony in the thalamocortico-thalamic network and underlie higher cognitive functions (Jones,
2002; Zhou et al, 2011). The developing mammalian neocortex is
characterized by the presence of the subplate, a transient relay and processing
station for thalamic neurons to establish their initial long-range connections
with cortex (Allendoerfer & Shatz, 1994; Kostović & Judas, 2006; Kanold &
Luhmann, 2010) (see Figure 1).
Birthdating studies in rodents report that the subplate is among the earliest
cortical structure to spawn and mature. In humans, the subplate is detectable
by the 14 - 15th postconceptional week (PCW), i.e. the start of the second
trimester. Subplate neurons (SPNs) are a temporary population of relatively
mature neurons of the immature neocortex, located in the developing white
matter underlying the cortical plate (Kostović & Rakic, 1980; Kanold &
Luhmann, 2010). They represent a distinct class of earliest born cells in the
developing cortex that form a transient layer between the cortical plate and
the intermediate zone of the fetal telencephalic wall (Kostović & Rakic, 1990;
Allendoerfer & Shatz, 1994; Price et al, 1997).
3
Figure 1. Schematic overview of the neuronal cell population in the developing human cortex,
including the subplate neurons (green) (modified from Hoerder-Suabedissen A. et al, 2015); VZ –
ventricular zone; SVZ – subventricular zone; IZ – intermediate zone; SP – subplate; CP – cortical
plate; MZ – marginal zone.
Instructed by the subplate, thalamocortical connections are established
primarily onto layer IV of the cortex during the third trimester of human
gestation (Kostović & Judas, 2002). While in rodents the subplate layer is at
most a relatively thin band of clearly defined neurons, in humans, the
subplate is about five times thicker than the cortical plate at PCW 18 to 22
when its maturity peaks (Meinecke & Rakic, 1992; Bystron et al, 2008; Tau
& Peterson, 2010). Towards birth, the thickness of the human subplate
decreases, disappearing during postnatal development (McConnell et al,
1989; Allendoerfer & Shatz, 1994). In rats subplate neurons are typically no
longer seen after the first postnatal month (see Table 1).
4
In humans, the end of the second trimester coincides with the peak of
developmental window and high vulnerability to damage (Bunney et al, 1997;
McQuillen & Ferriero, 2004). The relative maturity of the subplate is most
notably characterized by high metabolic demand rendering it particularly
susceptible to various perinatal insults, most notably hypoxia-ischemia in the
human preterm (Volpe, 2001; McQuillen et al, 2003).
Species
Mouse
Rat
Cat
Primate
Human
Gestation
19,5 GD
21 GD
65 GD
167 GD
40 GW
Birth of
E11-E13
E12-E15
E24-E30
E38-E43
GW5-6
SPN
(visual, somato-
(visual cx)
(somato-
sensory, auditory
sensory cx)
cx)
E43-E45
(visual cx)
Active
E14-P0
E16-E17
E36-E50
E78-E124
GW20-26
establish-
(2nd
ment of TC
trimester)
connections
Death of
E18-P21
E20-P30
P0-P28
E104-P7
GW34-41
SPN (about
E120-P7
2 years
80%)
(somato-
(PFC)
sensory cx)
Table 1. Development of subplate neurons in different species. (Modified from Kanold et al,
2010); CX – cortex; E – embryonic day; GD – gestation day; GW – gestation week; P – postnatal
day; PFC – prefrontal cortex; SPN – subplate neuron; TC – thalamocortical.
The most prominent feature of the subplate in cortical development is that it
serves as a relay and processing station for ingrowing thalamic afferent fibers
bringing glutamatergic input from thalamic nuclei into cortical layer IV
(Ghosh et al, 1990; Kanold & Luhmann, 2010; Kostović & Judas, 2010;
Judaš et al, 2013). Incoming thalamic axons arrive to the subplate long before
future layer IV neurons have completed their migration from the ventricular
5
zone (Shatz & Luskin, 1986). Work in the visual cortex has shown that
thalamic axons accumulate within the subplate for an extended period of time
in utero, from several days in rodents (Kanold & Luhmann, 2010) to several
weeks in cats (Shatz & Luskin, 1986), primates (Rakic, 1977; Kostović &
Rakic, 1984) and humans (Kostović & Rakic, 1984). Studies on the cat visual
system have demonstrated that SPN ablation results in arborization of axons
belonging to the thalamic lateral geniculate nucleus (LGN) below the visual
cortical plate instead of branching within layer IV. In effect, thalamic axons
grow past the visual cortex, forming an aberrant pathway within the white
matter (Ghosh et al, 1990; Ghosh & Shatz, 1992b; Ghosh & Shatz, 1993;
Kanold, 2009). Similarly, in studies on the cat auditory system the formation
of thalamocortical projections between medial geniculate nucleus (MGN)
axons and the primary auditory cortex is also compromised following SPN
ablation (Ghosh & Shatz, 1993).
After thalamic axons grow into the layer IV, subplate neurons continue to be
critically important for further synapse formation (Allendoerfer & Shatz,
1994; Kanold et al, 2003; Kanold & Shatz, 2006). During build-up of
thalamocortical projections, first forming thalamocortical synapses are weak
and functionally silent (Isaac et al, 1997), while strong inputs from subplate
neurons on to cortical postsynaptic terminals are already present (Friauf &
Shatz, 1991). Providing excitatory input to cortical neurons the subplate
converts, in an activity dependent manner (Finney et al, 1998), silent
synapses to functional ones (Kanold et al, 2003). Ablation of SPNs at the time
when thalamocortical projections have not yet formed, leads to prevention in
strengthening of synaptic input between the thalamus and cortex (Kanold et
al, 2003). Along with supporting maturation of glutamatergic thalamocortical
synapses, SPNs participate in the maturation of inhibitory circuitry and in
patterning of primary sensory areas (Hoerder-Suabedissen & Molnár, 2015).
6
2.1.1 Role of subplate in cortex maturation
2.1.1.1 Formation of neocortical architectural hallmarks
Interconnected neuronal circuits organized in columns that extend through
layers II - VI are the hallmark of neocortical cytoarchitecture. They represent
the functional elementary cortical unit of vertically grouped nets of neurons
activated by stimulation of the nearly same receptive fields (Mountcastle,
1957). The most investigated columnar organization is of the visual cortex,
where single orientation column and ocular dominance columns (ODC) are
defined as the elementary processing units of the primary visual cortex
(Hubel & Wiesel, 1979). Functional ODCs and orientation columns in the
primary visual cortex (V1) are the result of early activity-dependent
interactions driven by interplay of endogenous spontaneous activity and
sensory stimuli in the retina (Kanold & Shatz, 2006; Kanold & Luhmann,
2010). Formation of ocular dominance stripes results from subplatedependent thalamic segregation in layer IV. Due, in part, to gap-junction
driven activity that generates synchronized SPN oscillations, the subplate
provides the necessary prerequisites for cortical precolumn template
organization (Dupont et al, 2006). Additionally, by expressing molecular
guiding cues, SPNs direct thalamic axons (Kanold & Luhmann, 2010). In
particular, spatially graded expression of the neurotrophin receptor p75 and
the axon guiding cue ephrin-A5 in subplate guide thalamic axons to innervate
appropriate cortical area (Mackarehtschian et al, 1999). Notably, the
expression of the p75 receptor is enriched in subplate neurons compared to
neurons of the cortical plate (DeFreitas et al, 1991; Kordower & Mufson,
1992; Meinecke & Rakic, 1993; McQuillen et al, 2002) enabling its use in
7
ablation studies for specific SPN targeting (Kanold et al, 2003; Kanold &
Shatz, 2006).
2.1.1.2 Subplate and endogenous activity of the immature brain
In contrast to the adult electroencephalogram (EEG), neonatal EEG features
spatio-temporal distinct patterns of discontinuous activity (Vanhatalo &
Kaila, 2006). The fundamental EEG pattern of the immature mammalian
brain is characterized by intrinsic endogenous network events. In preterm
babies, the salient features of EEG consist of spontaneous activity transients
(SATs) and the intervals between them (Vanhatalo et al, 2005; Vanhatalo &
Kaila, 2006). Recordings from cortical structures during the period of early
cortical maturation showed similar EEG patterns for neuronal activity in rats
(Khazipov & Luhmann, 2006). EEG activity in the neonatal rat primary
somatosensory cortex is characterized by spindle-shaped bursts of fast
activity, defined as a slow wave with embedded high frequency oscillations
(Vanhatalo et al, 2002). They are thought to be homologous to human
premature delta brushes (Khazipov et al, 2004) and are essential for
generation of neuronal cortical circuits (Khazipov et al, 2004; Khazipov &
Luhmann, 2006). Spindle bursts typically last for around one second with
rhythmic activity at 5 - 25 Hz and occur approximately every 3 minutes
(Khazipov et al, 2004; Minlebaev et al, 2007). Similar events have been also
reported in the neonatal rat visual cortex (Hanganu et al, 2006; Khazipov &
Luhmann, 2006). Spindle bursts in somatosensory cortex are associated with
spontaneous muscle twitches that are evoked via motoneuronal bursts in the
spinal cord (Hamburger, 1975; Petersson et al, 2003). Triggered by direct
sensory feedback, spindle bursts result from spontaneous movements.
8
Experimental deafferentation of sensory inputs by severing the spinal cord
reduces the frequency of spindle bursts but does not abolish them (Khazipov
et al, 2004). These results suggest that spindle burst generation is not only
attributed to the activation of thalamocortical projections, but is also
associated with intrinsic oscillations of early cortical networks (Minlebaev et
al, 2007).
The subplate plays an important role in the generation of oscillatory activity
in the neocortex (Dupont et al, 2006). When the subplate gains its maximum
size and functionality at around 29 – 31 PCW, prominent network oscillations
in human preterm neonates are first observed (Milh et al, 2007). Work in vitro
has shown that rat SPNs are prominently coupled to each other and to cortical
plate neurons via gap junctions and upon electrical stimulation mediate
cortical oscillatory activity that synchronizes a columnar network 100-150
µm in diameter (Kanold & Luhmann, 2010). Functional cholinergic afferents
confined to subplate contribute to the oscillatory discharges in SPNs, and thus
can be a mechanism promoting intrinsic activity in neocortical circuits
(Dupont et al, 2006; Sun & Luhmann, 2007). Additionally, the excitatory
feedback from layer 4 neurons is involved in establishment of excitatoryexcitatory microcircuits that might take a part in generation of oscillatory
activity (Kanold & Luhmann, 2010). In addition to intracortical and
neuromodulatory cholinergic mechanisms, network oscillations in the form of
spindle bursts are generated in response to periphery sensory stimulation.
Subplate ablation has been shown to result in a loss of synchronized
oscillations in the cortical plate (Dupont et al, 2006).
2.1.2 Alterations in endogenous activity and functional architecture
following subplate ablation
9
Work by Shatz and colleagues (Ghosh & Shatz, 1992a; Kanold et al, 2003) on
visual cortex of cats demonstrated that SPN ablation disturbs the functional
development and organization of cortical networks. Microelectrode
recordings from layer IV in the visual cortex of subplate-ablated animals
revealed both decreased visual responses driven by the ipsilateral eye and
their impaired functional refinement. Furthermore, electric stimulation of
thalamic LGN neurons was shown to evoke qualitatively different field
potentials in subplate-ablated regions at retinotopically matching cortical
locations as compared with responses evoked in control regions. Cortical field
potentials in subplate-ablated regions were smaller in all cortical layers, most
prominently in layer IV. Notably, although LGN axons were shown to be
present in abundance in regions of layer IV overlying the ablated subplate, the
visual cortex at these sites remained uncoupled from the thalamus (Kanold et
al, 2003). Impairment in synaptic transmission of sensory inputs during the
first postnatal period has been suggested to result in an inability of thalamic
axons to segregate within the V1 region into ODCs and to establish the
functional architecture of the visual cortex (Kanold et al, 2003; Kanold,
2009). Indeed the result of SPN ablation during critical periods of the visual
system development is the impaired formation of visual cortical maps (Ghosh
& Shatz, 1992a; Kanold et al, 2003). The most prominent neurological
consequences of subplate dysfunction are impairments in cognitive, sensory,
behavioural, and motor domains (Kanold & Luhmann, 2010; HoerderSuabedissen et al, 2013). The end of the second trimester, when the subplate
has fully matured, represents a “window of vulnerability” for subplate
damage-related neurological consequences (Kostović & Rakic, 1990). Injury
of the developing periventricular white matter is one of the most common
neurological presentations diagnosed in preterm babies (McQuillen &
Ferriero, 2004). Consequent subcortical injury of developing white matter can
result in periventricular leukomalacia (PVL), one of the principal predictors
10
of cerebral palsy (McQuillen et al, 2003). Conversely, failure in programmed
cell death of subplate neurons may result in protracted retention of SPNs
within the subcortical white matter (Hoerder-Suabedissen & Molnár, 2015).
Interestingly, preservation of SPNs in form of interstitial white matter
neurons has been suggested to contribute to pacemaking properties and
development of epileptic foci (Kanold & Luhmann, 2010).
2.1.3 Models of subplate disruption
To shed light on mechanisms of cortical development and to further
understand the origin of early SPN death and its contribution to neurological
disorders, the two widely utilized methods of localized and selective SPN
ablation are: injection of kainic acid and immunolesioning by IgG-saporin
immunotoxin that binds to p75 neurotrophin receptor (p75NTR). A kainate
model of subplate ablation in cat on embryonic day (E) 42 was developed by
Shatz and colleagues to study the role of SPNs in the formation of the first
thalamocortical projections (Ghosh et al, 1990). A similar model has been
implemented to study the role of SPNs in the organization of functional
cortical columns during the first postnatal week (Ghosh & Shatz, 1992a). The
high selectivity of kainate towards SPNs has been attributed to early
maturation of SPNs and thus higher expression of glutamate receptors
compared to neurons of the cortical plate (Chun & Shatz, 1988).
A second approach that has been successfully used to eliminate SPNs
involves the use of p75NTR antibody coupled to a saporin immunotoxin
(Wiley, 1992; Moga, 1998). p75NTR is highly enriched in SPNs relative to
neurons of the cortical plate allowing its use as a target to rather specifically
ablate SPNs in the developing brain (Allendoerfer et al, 1990; Fine et al,
11
1997; Mrzljak et al, 1998; Kanold et al, 2003). Kainate acid injections create
larger regions of ablation within subplate while p75 antibody immunotoxin
method can provide pointed ablation of a specific region of interest.
2.2 FEBRILE SEIZURES
FS are the most common type of convulsive events in children (Hauser 1994).
Children between the ages of 6 months to 5 years are typically affected with a
peak of incidence at 16 - 18 months of age (Hauser, 1994; Shinnar & Glauser,
2002). Based on duration, recurrence patterns and behavioural phenotypes,
FS can be classified into two subgroups, simple and complex. Seizures that
are self-limited with generalized tonic-clonic convulsions lasting less than 10
- 15 minutes, with absence of repetitive clinical manifestation during the
same febrile illness, or that occur only once in a 24-hour period are often
classified as simple FS (Kliegman, 1996; Gordon et al, 2001; Waruiru &
Appleton, 2004). Complex FS are prolonged, lasting more than 15 minutes,
have a focal onset and persistent recurrence within 24 hours or within the
same febrile illness (Berg et al, 1990; Berg & Shinnar, 1996). In addition, FS
that last more than 30 minutes are classified as febrile status epilepticus
(FSE) (Lewis, 1999). Most FS incidences (~ 87 %) are clinically
characterized as benign events with low impact on the child’s development.
Minority of children (9 %) with FS have a prolonged form and 5 % of
children suffer from FSE (Sadleir & Scheffer, 2007). Both genetic and
environmental factors in the pre- and perinatal period have been implicated in
FS susceptibility (Kjeldsen et al, 2002; Baulac et al, 2004; Mulley et al,
2005). Family studies point to a considerable genetic factor in susceptibility
to FS (Tsuboi, 1987; Corey et al, 1991; Sadleir & Scheffer, 2007; Scantlebury
& Heida, 2010). Indeed, familial FS are thought to be due to channelopathies,
12
i.e. caused by mutations in ion channels, including certain types of voltagegated Na+ channels and GABAARs (Steinlein, 2002; Mulley et al, 2003) but
there is also evidence of genetic variation in gene encoding Cl- transporters
(Puskarjov et al, 2014). Non-genetic factors notably include overproduction
of inflammatory mediators e.g. cytokines (Dubé et al, 2005; Heida & Pittman,
2005; Heida et al, 2009; Vezzani et al, 2011) and hyperthermia-induced
respiratory alkalosis (Schuchmann et al, 2009).
2.2.1 Non-Genetic Mechanisms of Febrile Seizures
2.2.1.1 Respiratory alkalosis
Hyperthermia has been shown to result in a respiratory alkalosis that
precipitates seizures in rats (Schuchmann et al, 2006) and an analogous
mechanism has been proposed to account for generation of FS in humans
(Schuchmann et al, 2009). Notably, brain alkalosis is known to increase
neuronal excitability (Chesler, 2003; Ruusuvuori & Kaila, 2014) which
readily accounts for these observations. Homeothermic animals, such as rats
and humans, exhibit thermoregulation, keeping body temperature within
certain values regardless of perturbations in external ambient temperature.
Hyperthermia in rats induces an increase in the breathing rate (O'Dempsey et
al, 1993; Gadomski et al, 1994; Taylor et al, 1995; Schuchmann et al, 2006;
Schuchmann et al, 2009). It is thought that hyperthermia-induced increase in
breath rate serves a compensatory function by actively opposing further
temperature increase based on respiratory tract heat evacuation (Sutton, 1909;
Gadomski et al, 1994). In addition to thermoregulation, respiration also
13
controls and maintains the partial pressures of O2 and CO2 in the blood (Saiki
& Mortola, 1996; Mortola & Frappell, 2000; Putnam et al, 2004). The age
dependence of hyperthermia-induced seizures in rats confined to the first
weeks of life is thought to be due to immature systemic CO2 regulation by the
respiratory system of infant rats (Putnam et al, 2005). During this time, rats
exhibit a minimum threshold for experimental febrile seizures (eFS) to occur
(Holtzman et al, 1981; Bender et al, 2004). Analogously to the situation in rat
pups, in human infants hyperthermia is likely to trigger compensatory
respiratory tract heat evacuation resulting in hyperventilation with subsequent
net loss of CO2 and respiratory alkalosis (Schuchmann et al, 2009;
Schuchmann et al, 2011). Due to this, there is a tendency to FS precipitation
in children, however it is important to note that the net fever-induced
hyperventilation effect on brain pH will depend on the overall acid-base level,
but not solely on breath patterns of an individual (Schuchmann et al, 2011).
Brain pH regulation
Effective pH regulation is based on a combination of passive buffering and
active transport of acid-base equivalents that take place at different
organisational levels, from subcellular microdomains to the whole individual.
Alkalosis increases neuronal excitability and can be intense enough to trigger
epileptiform activity, while the opposite effect is observed with acidosis
(Balestrino & Somjen, 1988; Kaila & Ransom, 1998; Ruusuvuori et al, 2010).
An exception here are the chemosensitive neurons controlling breathing
(Putnam et al, 2004). The unique property of pH is to modulate protein
functions that are directly related to neuronal activity. To maintain tight pH
control, regulation thereof takes place at different systemic levels. At the
organ level two main regulators are involved: the lungs with their respiratory
14
function mediating acid excretion in the form of CO2 and kidneys that
actively regulate acid-base body balance through excretion of H+ in the form
of NH4 and H2PO4, or through reabsorption of HCO3-. To protect the brain
from possible blood plasma composition fluctuations, brain endothelial cells
line cerebral vasculature forming the blood-brain barrier (BBB), which
regulates interstitial content and mediates an effective pH regulation of the
brain parenchyma by its active transport mechanisms. At the cellular level,
the control of pH is maintained via buffering capacity and acid base transport
systems that actively move acid-base equivalents across the cell membrane of
neurons and glia and thereby regulate cytosolic pH. The total intracellular
buffering capacity can be divided into the non-bicarbonate or intrinsic
buffering capacity and CO2/HCO3--dependent component. Intracellular
buffering capacity arises from titratable imidazole residues of proteins and
from phosphates that are not able to cross the plasma membrane (Roos &
Boron, 1981). In rapid buffering, the capacity of the CO2/HCO3--buffering
system is rate limited by activity of the CA enzyme, which catalyses the CO2
hydration-dehydration reaction:
CA
CO2 + H 2 O ←⎯→
HCO3− + H +
In the absence of CA activity equilibration of the CO2 hydration reaction
takes tens of seconds, making the CO2/HCO3--buffering system ineffective
against rapid acid loads. On the other hand, transient shifts in CO2 or HCO3induce rapid pH responses only in the presence of CA activity (Maren, 1967;
Kaila et al, 1993; Supuran et al, 2004; Ruusuvuori & Kaila, 2014).
Among 13 identified catalytically active CA isoforms 11 of those are
expressed in the brain (Ruusuvuori & Kaila, 2014). With various localization
within brain cells, CA isoforms exert strong influence on the dynamics of pH
and CO2 homeostasis (Supuran et al, 2003; Ruusuvuori & Kaila, 2014). Of
15
particular interest in respect to neuronal excitability is the developmental
expression pattern and isoform identity underlying the neuronal carbonic
anhydrase activity.
GABAergic signalling and the role of carbonic anhydrase activity
The traditional concept of GABA and GABAergic system in brain physiology
is mostly based on its inhibitory properties and it is typically stated to be the
main mechanism to reduce neuronal excitability. Nevertheless, there are
evident exceptions to this paradigm. In immature neurons, intracellular
chloride concentration is maintained at an elevated level by active inward
transport of chloride ions (Kaila et al, 2014). Therefore GABAAR-mediated
responses are depolarizing or even excitatory (Rivera et al, 1999), and they
have been shown to be crucially involved in the generation of giant
depolarizing potentials (GDPs), a type of spontaneous network activity seen
in the hippocampus in vitro (Ben-Ari et al, 1989; Sipilä & Kaila, 2008).
During neuronal maturation a gradual shift from active net chloride uptake to
net chloride extrusion induces a negative shift in GABAAR responses (BenAri et al, 2007; Kaila et al, 2014). Following intense GABAergic synapse
activation in mature neurons, instead of neuronal suppression, direct
promotion of cell excitability takes place (Alger & Nicoll, 1982). Work
carried out on rat hippocampal slices, demonstrated that sustained GABAARmediated activation can paradoxically lead to a shift from hyperpolarizing to
depolarizing postsynaptic potentials. High-frequency stimulation (HFS) of
hippocampal interneurons in the absence of ionotropic glutamatergic
transmission was reported to produce massive and synchronous excitation of
hippocampal pyramidal neurons (Kaila et al, 1997; Smirnov et al, 1999;
Ruusuvuori et al, 2004). This type of GABAergic excitation represents a form
16
of ionic plasticity (Kaila et al, 2014) and it is strictly dependent on the
presence of intracellular HCO3- and GABAAR permeability to both Cl- and
HCO3- (Kaila & Voipio, 1987). During their channel-mediated efflux,
cytosolic HCO3- ions are continuously replenished via CA activity that
catalyses net hydration of CO2 that readily crosses the plasma membrane (see
pH regulation section).
The key finding of the study by Ruusuvuori et al, (2004) was the discovery
and characterization of a developmentally regulated CA isoform VII (CA
VII) that mediates rapid restoration of intracellular HCO3- levels in pyramidal
neurons. A steep increase in the expression of CA VII in hippocampal CA1
pyramidal neurons takes place approximately at the end of the second
postnatal week, whereas HFS-induced GABAergic depolarization is hardly
possible before this age (Ruusuvuori et al, 2004). CA II is another cytosolic
isoform that is ubiquitously expressed in a variety of tissues including the
brain (Ruusuvuori & Kaila, 2014). Study by Velíŝek et al, performed on CA
II-deficient mice reported significantly decreased susceptibility to flurothylinduced seizures as well as having a significantly decreased mortality rate
(Velíŝek et al, 1992) CA II-deficient mice displayed longer latencies to onset
of behavioural tonic-clonic seizures in both flurothyl and pentylenetetrazole
models compared to WT littermates (Velíŝek et al, 1993). Notably, mutations
in the human gene coding CA II are associated with chronic metabolic
acidosis (Pang et al, 2015).
Both application of GABAA agonists (Alger & Nicoll, 1979) and/or HFS
(Kaila et al, 1997; Viitanen et al, 2010) of CA1 GABAergic interneurons
evokes a biphasic membrane potential response in pyramidal neurons.
Immediately after GABAAR channel opening, the fast initial hyperpolarizing
step is followed by a gradual positive shift that is due to a conductive Cluptake driven by the depolarizing action of the outward bicarbonate flux
17
(Kaila & Voipio, 1987; Kaila et al, 1993). CA VII, using CO2 as a substrate,
efficiently buffers the intracellular HCO3- and promotes the electroneutral
uptake of Cl- and consequently shifts the EGABA to more positive values thus
potentiating depolarization effect once GABA channels are opened. The CAdependent anion redistribution during prolonged GABAergic stimulation
induces a net efflux of Cl- and K+ in a 1:1 stoichiometry thus leading to an
increase in interstitial [K+]o that ultimately results in a prolonged late nonsynaptic depolarization phase. The above mechanism clearly underscores the
potential for GABAergic signalling to change rapidly in a qualitative manner
(Kaila et al, 1997; Ruusuvuori et al, 2004; Viitanen et al, 2010; Kaila et al,
2014).
2.2.1.2 Inflammatory cytokines
Immune response to infection can be one of the factors that induce FS in
infants and children (Dubé et al, 2009; Heida et al, 2009). Clinical and
experimental data highlight the potential role of immune response in FS
aetiology. FS triggered by a fever are associated with enhanced release of
pro-inflammatory cytokines by both astrocytes and microglial cells (Heida et
al, 2009; Vezzani et al, 2013). Elevated levels of interleukin-1β (IL-1β),
tumour necrosis factor alpha (TNFα) and interleukin-6 (IL-6) are produced in
acute phase of developing fever and can ultimately contribute to seizure
generation (Heida et al, 2009). Particular interest represents IL-1β secretion,
which generates a pyrogenic effect and leads to the following massive
cytokine expression within the brain (Dubé et al, 2009). The potential
mechanism by which high levels of IL-1β provoke neuronal excitability has
been attributed to modulation of both glutamatergic and GABAergic
18
neurotransmission (Dubé et al, 2009). A seizure-promoting role of IL-1β
demonstrated in a series of in vivo experiments performed on knockout mice
lacking the IL-1β or its receptor (IL-1R1). This work showed that in the
absence of IL-1R1, much higher temperatures are needed to trigger
hyperthermic seizures (Dubé et al, 2005).
2.2.1.3 Role of brainstem in generation of febrile seizures
In rat models of FS the initial behavioural manifestations of seizures are
associated with freezing and oral automatisms (Baram et al, 1997). This and
other pertinent observations prompted the suggestion that the critical brain
areas responsible for generation of FS are those located in the limbic system
(Cendes et al, 1993). It has been proposed that later stages of eFS behavioural
manifestations can also involve midbrain structure activity (Dube & Baram,
2006). Possibility for the role of brainstem in generalized epilepsy in human
patients was reported by Kohsaka et al (Kohsaka et al, 1999). Widespread
enhanced brainstem activation preceded and triggered EEG seizure
discharges within the cortex (Kohsaka et al, 2002; Norden & Blumenfeld,
2002; Badawy et al, 2013). Evidence of brainstem participation has been
mainly obtained in adult rats and little information is available regarding
immature rodents (McCown & Breese, 1992). In rodents, behavioural seizure
manifestation induced by hyperthermia is characterized by typical limbic
seizures. Arrest of movement (freezing) followed by oral automatisms and
forelimb clonus (Gale, 1990) consequently evolve into more severe forms of
generalized tonic-clonic limb components. Several lines of evidence suggest
that tonic convulsions are the result of brainstem excitability. Experimental
animals with isolated brainstem subjected to electrical and chemical
stimulation exhibit the most severe form of stereotyped behavioural response
19
(Browning & Nelson, 1986). In addition, auditory stimulations can evoke
brainstem generalized tonic-clonic seizures independently from forebrain
structures (Browning et al, 1999). The above suggests that the brainstem
deploy powerful modulatory effect on cerebral cortex (Norden & Blumenfeld,
2002) and can act as a primary seizure generator (Velasco & Velasco, 1990).
2.2.2 Animal models of febrile seizures
Knowledge on the mechanisms and generators of FS has been gained through
studying animal models of febrile seizures (Dubé et al, 2009; Schuchmann et
al, 2009). To characterize and investigate FS, several animal models of
hyperthermia were developed with a focus on the role of body temperature
(Lennox et al, 1954; Holtzman et al, 1981; Baram et al, 1997). Rodents are
widely used to model FS. Hot water submersion (Jiang et al, 1999),
irradiation with infrared lamp (Holtzman et al, 1981), microwaves (Hjeresen
& Diaz, 1988) or the use of a hair dryer (Baram et al, 1997), have been the
commonly used for rapid achievement of critically high body temperatures to
generate seizures. To replicate infectious fever condition, including an
inflammatory response with endogenous pyrogen production as a result of
lipopolysaccharide (LPS) bacterial release (van Dam et al, 1998; Roth &
Blatteis, 2014), the model of LPS administration with subconvulsive dose of
kainate was developed in Pittman’s laboratory (Heida et al, 2004; Heida &
Pittman, 2005; Heida et al, 2005). In the hair dryer model, a very fast increase
in body temperature is achieved within 2 – 4 minutes (Dube & Baram, 2005;
Dube et al, 2007). Even before the eFS threshold is reached by the application
of a hot air stream, thermal nociceptors (Caterina et al, 1997) and
inflammatory responses in the ears and paws of animals will be activated
20
(Schuchmann et al, 2009). To minimize the possible effects of pain and
inflammatory responses, the original model of Holtzman (Holtzman et al,
1981) was modified by Schuchmann et al, (Schuchmann et al, 2006). Here,
the rise in core body temperature of the experimental animals was much
slower allowing induction of eFS with low mortality, enabling quantitative
tests of anticonvulsants to the study of the influence of genetic
background/mutations on eFS (Schuchmann et al, 2008).
Identification of eFS in animals relies on observation of behavioural seizure
patterns and/or on assessment of changes in EEG activity. The available
behavioural seizure scoring tests differentiate motor seizures with respect to
severity. In mice, sustained exogenous heating transforms normal explorative
animal behaviour patterns (stage 0) into hyperthermia-induced hyperactivity
and escape responses (stage 1). Thereafter sudden arrest and immobility
(stage 2) are seen and later turn into more severe forms of motor response.
Clonic seizures of one or more limbs and body trembling define stage 3.
Continuous tonic-clonic seizures with involvement of all extremities (stage 4)
can culminate with loss of postural control (van Gassen et al, 2008). Freezing
and consequent oral automatism with following involvement of extremities in
clonic shaking markedly indicates the onset of eFS. Work on mice may be
accompanied with difficulties in interpretation of behaviour seizure stages
(Schuchmann et al, 2008), thus epidural EEG recordings can be a reliable
way to perform eFS detection (Schuchmann et al, 2006). Ictal EEG activity
from hippocampus and cortex coincide with animal immobility and markedly
characterize the onset of eFS. Rhythmic trains of high amplitude spike-wave
activity with an interictal periods of relative silence are usually seen in rats
and mice (Dube & Baram, 2005).
21
2.2.3 Antiepileptic therapy
AEDs are the first step taken to control seizures in patients with various types
of epilepsy (Chong & Bazil, 2010). AEDs are directed to act on several
mechanisms of seizure regulation (Brodie et al, 2011). Among the most
commonly used AEDs are ones targeting the GABAergic signalling (Meisler
& Kearney, 2005), with the aim to enhance inhibitory activity by either
prolonging GABAAR open time or by inhibiting GABA transaminase (Kwan
et al, 2001). Treatment strategies in cases of complex FS or FSE are typically
directed to deal with acute event manifestations, and prophylaxis for
recurrence (Millichap, 1991; Fetveit, 2008; Bast & Carmant, 2013; Seinfeld
et al, 2014). The majority of FSE episodes do not stop spontaneously
(Hesdorffer et al, 2012). Fast acting benzodiazepines, notably diazepam,
remain to be the first-in-line anticonvulsant therapy from home till hospital
(Knudsen, 2000). Notably, in the light of neonatal seizures AEDs can cause
“electroclinical uncoupling” whereby electrographic seizure activity is either
unaffected or exacerbated through intensive GABAAR activation (Connell et
al, 1989).
In infants suffering from FS, benzodiazepines have been shown to suppress
the rate of breath (Orr et al, 1991; Tasker, 1998; Appleton et al, 2008) and
consequently they can be expected to reduce the respiratory alkalosis. It is
also worth to mention that a high rate of side effects has to be expected during
benzodiazepine treatment. In children younger than 2 years it is associated
with
adverse
effects
including
fatal
hepatotoxicity,
gastrointestinal
disturbances, pancreatitis, transient mild ataxia, hyperactive behaviour,
lethargy; respiratory depression, bradycardia, and hypotension (Fetveit, 2008;
Steering Committee on Quality Improvement and Management, 2008; Bast
& Carmant, 2013). Such prolonged treatment in case of continuous
22
prophylaxis of complex FS is associated with negative and long-lasting
effects on child memory formation (Seabrook et al, 1997), cognition,
behaviour and motor function (Daugbjerg et al, 1990; Offringa & Newton,
2013). Thus, there is a need for novel therapeutic approaches.
Based on the fact that acidosis provides attenuation of neuronal excitability, it
is worth to consider implementation of CO2 as a novel safe strategy in FS
treatment. It is well documented that CO2 shows a strong inhibitory effect on
neuronal activity. Already in 1928, in humans with absence seizures, a
suppressive action of carbogen mixture with 10 % CO2 was shown (Lennox,
1928; Lennox et al, 1936). Experiments on nonhuman primate, canine and
rodent model animals confirmed the anticonvulsant action of CO2 (Pollock,
1949; Pollock et al, 1949; Woodbury et al, 1958; Myer et al, 1961; Caspers &
Speckmann, 1972; Balestrino & Somjen, 1988; Ziemann et al, 2008).
Notably, CO2 in form of medical carbogen (5 % CO2 + 95 % O2) applied in
animal models of stimuli-induced myoclonic seizures, nonhuman primate
bicuculline-induced epilepsy, and in humans with drug-resistant partial
epilepsy, was shown to possess a potent fast-acting anticonvulsant effects
(Tolner et al, 2011). In the study by Schuchmann et al, (2006), hyperthermiaevoked electrographic activity in P10 rats along with associated behavioural
responses was rapidly and completely blocked by CO2 inhalation. Application
of 5 % CO2 prevented the development of electrographic ictal activity in the
neocortex and in the hippocampus. Additionally, 5 % CO2 also blocked
hyperthermia-induced long-lasting changes in neuronal signalling and
plasticity (Schuchmann et al, 2006). On the basis of the above data, it can be
concluded that CO2 is an effective therapeutic agent in the context of febrile
seizure intervention.
23
3. AIMS
Early developing patterned neuronal activity in the neonatal cortex promotes
network maturation that is organized by thalamacocrtical and intracortical
connectivity. On the other hand, since subplate neurons are the main
contributors to the establishment of thalamacortical connectivity during the
cortex maturation, we hypothesize that the SPNs are crucially involved in
generation of particular types of early network activity, which is an important
feature of cortical development. In the paradigm of febrile seizure generation
we hypothesize that carbonic anhydrase isoform VII is the key molecule in
age-dependent neuronal pH regulation that consequently affects febrile
seizure genesis. In literature, it is widely assumed that febrile seizures are
limbic in origin, with the ability to generalize due to invasion into the
brainstem in response to the initial forebrain seizure activity. Here we
hypothesize that brainstem could be capable of independently generating
tonic-clonic convulsions in febrile seizure model. To test the three
hypotheses, experimental work was designed and performed in order:
1. to examine the role of subplate neurons in endogenous patterned activity
and reveal the effects of subplate ablation on the formation of barrel patterns
in somatosensory area (I);
2. to investigate the involvement of intraneuronal carbonic anhydrase isoform
VII in the generation of febrile seizures (II);
3. to explore the role of brainstem activity in the mechanisms of febrile
seizure generation (III).
24
4. EXPERIMENTAL PROCEDURES
All material and methods used in this study are described in details in the
“Material and Methods” sections of the original papers. Methods used by the
author extensively during the study are discussed below. All experiments
were approved by the Ethics Committee for Animal Research at the
University of Helsinki.
4.1 GENERATION OF THE TRANSGENIC MOUSE LINES AND IN
VITRO ELECTROPHYSIOLOGICAL RECORDINGS
For detailed description of the materials and methods the reader is referred to
the original publications (I-II).
4.2 ANIMAL SURGERY AND EEG RECORDINGS
To register electrographic febrile seizures we used typically a pair of pups of
P14 age from the same litter. Ball-tipped silver wire electrodes (75 µm wire
diameter, Teflon insulated, ball tip diameter ca. 1 mm) were connected to
microconnectors and fixed with dental cement. Under isoflurane anesthesia
we implanted electrodes through small craniotomy made by drill with a 0.6
mm diameter carbide burr. Coordinates were measured by a stereotaxic
instrument (AP 2.5 mm; ML 1.3 mm). Reference electrode was placed above
cerebellum. After electrode implantation and its fixation we sutured the
incision and let animals recover for the next two hours in a warm
25
environment.
Before and during the eFS, we recorded continuous cortical EEG by a
custom-made amplifier. EEG signals were high-pass filtered (cut-off at 0.07
Hz), 1000x amplified, anti-alias filtered and sampled at 1kHz using a 16-bit
data acquisition interface and LabVIEW software (National Instruments,
Austin, Tx; custom-built functions courtesy of T. Maila).
To deliberately perform local subplate ablation in rat somatosensory cortex at
P0 - 1 age we anesthetized rats with isoflurane, drilled small craniotomies
overlying the S1 (limb region) cortex and injected 400 nl of anti-rat p75immunotoxin (192-IgG-saporin) with injector. To control immunotoxin
injections we used control toxin (anti-mouse p75-saporin “µ-toxin”, or IgGsaporin ”blank toxin”) or vehicle injections. Solutions were delivered into the
cortex by using a glass pipette (tip diameter 10 – 40 µm). Fluorescent
microspheres (Lumafluor Inc.) were coinjected for visualization of injection
sites.
EEG recordings in acute head-fixed rats were made between P7 and P10
under light isoflurane anesthesia (maintained at 1.5 – 2 %) using a miniature
stereotaxic in which an animal was kept warm at 35 °C. Through the window
above immunotoxin-injected area we inserted recording electrode arrays
consisting of 3 – 6 insulated tungsten wires (20 µm in diameter) at the depth
of 500 – 700 µm. Reference and ground electrodes (125 µm Teflon-insulated
silver wire) were placed over cerebellum. In parallel breath rate was
monitored by breath piezo sensor attached to the abdomen of the pup. To
register evoked spindle bursts in S1 area we inserted under the skin of the
dorsal surface of the left fore- and hindpaw bipolar stimulating electrodes (75
µm stainless steel). For chronic EEG recordings we used rats of P5 – 6 and
performed similar surgical and EEG recording approaches as described
above. To detect small movements such as sleep twitches and breathing
patterns during pup’s sleep we used a force transducer.
26
Rat pups assigned for the brainstem transection and sham operation at P13
underwent isoflurane anesthesia during which a small craniotomy was made 2
mm rostral from lambda. Brainstem transection was performed by insertion of
a blunt needle through the drilled hole until its tip touched the bottom of the
skull (Figure 2). By moving needle side-to-side we fully disrupted all
connections between midbrain and diencephalon.
Figure 2. Schematic representation of precollicular transection (Modified from Blumberg et al,
1995)
Sham-operated rats had the same surgical procedure apart from insertion of
the needle through the hole.
4.3 EXPERIMENTAL SEIZURES
To induce eFS pups were placed into a chamber with an ambient temperature
of 43 ± 1 °C for mice and 48 ± 1 °C for rats. Simultaneously with the
continuous cortical EEG and video recordings of the freely moving pups we
monitored their breath rate and core body temperature. Animals were exposed
27
to hyperthermia until their body temperature was critically high of 42.5 – 43
°C values (Dube & Baram, 2005).
Kainic acid-induced seizures were evoked by intraperitoneal injection of the
proconvulsant using the dose of 3 mg/kg (Pitkänen et al, 2006) in shamoperated and transected pups. Following injection, animals were kept at
thermoneutral conditions for 90 min to define the onset of seizures.
In experiments where diazepam was used (Stesolid Novum, 5 mg/ml) it was
intraperitoneally injected at 50 µg/kg, 150 µg/kg or 2.5 mg/kg dose 15
minutes before the onset of hyperthermia. Saline injection was repeated in
control animal group.
To test the effect of carbon dioxide on seizure generation, we applied a
preheated (43 – 44 °C) gas mixture of 5 % CO2, 19 % O2, and 76 % N2.
Behavioural seizures were quantified using the behavioural scale of mouse
eFS (see chapter 2.2.2).
4.4 BLOOD SAMPLING
Blood parameters were checked with a clinical blood gas and electrolyte
analyser device (GEM 4000 Instrumentation Laboratory) in wild type and CA
VII knockout animals. After collection of blood into plastic capillaries with
lyophilised lithium-heparin 100 I.U./ml, pH values were measured.
28
4.5 EEG ANALYSIS
In offline mode we analysed continuous EEG signals using Diadem,
LabVIEW and MATLAB. To detect spindle bursts during pup’s sleeping time
we filtered raw EEG between 5 and 40 Hz using a Butterworth 4th order filter.
Waking periods were excluded from analysis due to high signal
contamination by movement artefacts. Bursts lasting more than 100 ms with
at least 4 separate peaks with amplitude larger than 25 µV were considered
for further time-frequency (TF) and duration analysis. Neocortical sharp
potentials were defined as single sharp events of short duration (Bernard et al,
2005). Only sharp potentials of amplitude larger than 25 µV were included in
analysis. Cumulative root mean square (cRMS) values were quantified for the
frequency bands of 1 – 4 Hz, 4 – 10 Hz, or 5 – 45 Hz from 20 min EEG
signal. Fast Fourier transformation was used to calculate the power and peak
frequency over a 500 ms window from 5 – 40 Hz bandpass filtered (second
order Butterworth) local field potential signal. During offline EEG analysis of
mice subjected to eFS, electrographical seizure activity was recognised as a
burst of regular spikes with amplitude values around 150 – 600 µV, which is
at least 3x standard deviation of baseline EEG amplitude, and with durations
from 10 seconds up to one minute. Movement artefacts seen on the EEG
traces were routinely excluded.
4.6 STATISTICS
Statistical analysis between groups was performed using the Mann-Whitney
U test or Student’s t-test with two-tail distribution or Mantel-Cox test.
29
Average values of all numerical data presented are expressed as a mean ± SE.
p values <0.05 were considered as statistically significant.
30
5 RESULTS
5.1 SUBPLATE NEURONS PROMOTE SPINDLE BURSTS AND
THALAMOCORTICAL PATTERNING IN THE NEONATAL RAT
SOMATOSENSORY CORTEX (I)
5.1.1 Local subplate ablation prevents spontaneous spindle bursts
The main finding of this study was that endogenous activity in form of
spindle bursts are strongly influenced by subplate neurons. Wellcharacterized EEG patterns in somatosensory cortex (Khazipov et al, 2004;
Yang et al, 2009) were recorded by implementation of full band EEG (FbEEG). After subplate targeting at P0 – P1 age, electrodes were implanted into
P6 rats with further EEG registration during next consecutive days at P7 –
P10. In control conditions, non-injected rat pups along with µ-toxin, blank
toxin and vehicle-injected rats manifest regularly observed spindle bursts in
acute and chronic recordings. In contrast, animals subjected to SPN ablation
had significantly decreased spindle burst activity. Meanwhile, the peak
frequency of spindle bursts in ablated pups was slightly reduced and the
duration of rarely observed spindle bursts was comparable to the values of
control rats. Total EEG activity in specific frequency bands was evaluated by
cRMS of the EEG. A reduced cRMS in the frequency band that comprises
most spindle bursts (10 – 45 Hz) comparing with control pups was seen in
ablated pups. Sharp potentials as the second type of intrinsic activity in
neonatal rat cortex (Khazipov et al, 2004) were not affected in the parietal
and occipital regions in ablated and control pups.
31
5.1.2 Strengthening of the thalamocortical connection is prevented after
subplate ablation
In all our experimental animals subjected to subplate ablation there was a
marked reduction in spontaneous and sensory evoked activity in
somatosensory S1 limb area. Thalamocortical synaptic transmission can be
probed in vitro in the S1 barrel region (Katz & Shatz, 1996). After subplate
ablation at ~10th day whole cell patch-clamp recordings from layer IV were
performed. In slices of S1 we investigated how the absence of SPNs affect the
intrinsic properties of layer IV neurons. Stimulations of thalamocortical
projections with varying intensities while recording from layer IV were
performed. From both sets of layer IV neuron population, we observed short
latency excitatory post-synaptic currents (EPSCs). However, increasing the
stimulation level caused an increase in the amplitude of EPSCs only in slices
from non-ablated animals. Maximum levels of EPSCs in layer IV cells from
ablated pups were drastically smaller than in layer IV cells from non-injected
or control injected animals.
5.1.3 Barrel patterning in S1 somatosensory area is dependent on
subplate function
Barrel pattern in the area of somatosensory S1 cortex that represents whiskers
is an organizational hallmark of cortex formation (Erzurumlu & Kind, 2001).
Since endogenous patterned activity is instructive for functional and structural
development in barrel cortex (Minlebaev et al, 2007), we suggested that SPN
disruption would induce impairment in S1 organization. Ten days after
subplate ablation in rats, cytochrome oxidase staining of S1 whisker field
32
revealed large gaps or complete absence of stained barrels. Gaps in barrel
cortex had various sizes, which might be due to variability in the maturity of
barrel cortex at the time of toxin injections or because of the variability in
targeting procedures.
5.2 NEURONAL CARBONIC ANHYDRASE ISOFORM VII PROVIDES
GABAERGIC EXCITATORY DRIVE TO EXACERBATE FEBRILE
SEIZURES (II)
5.2.1 Sequential expression of CA VII and CA II in hippocampal
neurons
Multiple tissue Northern blot analysis of adult mouse tissues indicated
prominent expression of CA VII in the brain and spinal cord. A
developmental expression profile was obtained for CA VII using Western
blot analysis of protein lysates of whole hippocampi. The observed postnatal
increase in the expression level was due to CA VII since no signal was seen
in CA VII KO animals. Western blot analysis of glia/neuron cultures and pure
glia cultures revealed purely neuronal expression of CA VII.
Because of the possible contribution of other isoforms, CA VII specific
mRNA and protein expression data are not sufficient to find out the
developmental profile of carbonic anhydrase activity. Therefore we used an in
vitro functional assay (Ruusuvuori et al, 2004) in which the speed and
sensitivity to the CA inhibitor acetazolamide of intraneuronal pH responses
upon step changes in CO2 were used to indicate catalysis of CO2 hydrationdehydration. In WT neurons CA activity appeared at P10, whereas in CA VII
KOs CA activity was seen only starting at around P19. Thus a cytosolic
33
isoform other than CA VII is expressed in neurons at a somewhat later stage
of postnatal development, and it was identified as isoform II since no CA
activity was observed in CA II/VII double KO neurons even at the age of five
weeks or older.
5.2.2 Depolarizing GABA responses are promoted by CA VII and CA II
GABAergic responses were recorded from CA1 pyramidal neurons in P12 –
P16 WT and CA VII KO slices. In the presence of ionotropic glutamatergic
receptor and GABABR antagonists high-frequency stimulation applied at the
border of stratum radiatum and stratum lacunosum-moleculare evoked
GABAergic biphasic responses. WT and CA VII KO neurons responded with
an initial hyperpolarization followed by a prolonged depolarization that was
slower in KO, and the responses were abolished after picrotoxin application.
The prolonged GABAergic depolarization was able to evoke action potential
firing in WT neurons while in CA VII KO neurons action potential firing was
missing
in
every
recorded
neuron.
When
spiking
activity
was
pharmacologically blocked, microinjection of GABA to the border of stratum
radiatum and stratum lacunosum-moleculare resulted in a robust CO2/HCO3dependent depolarization that was larger in WT than in CA VII KO neurons.
On the other hand, when blockers of ionotropic glutamate receptors and
GABAB receptors but no tetrodotoxin were applied, GABA microinjections
evoked spiking activity as seen in field potential recordings, and this activity
was inhibited by the GABAA-receptor antagonist gabazine. Taken together,
these results indicate that expression of CA VII is crucial for the GABAergic
depolarization and excitation that is seen in hippocampal pyramidal neurons
at P12 – 16.
34
The very robust GABA injection-induced responses in pyramidal cell
membrane potential described above were used to address the functional role
of the two CA isoforms in slices from mature WT and KO mice (>P35). The
responses were indistinguishable in WT, CA VII KO and CA II KO neurons,
but much smaller in slices obtained from CA II/VII double KO animals.
These results indicate that mature neurons express two cytosolic CA isoforms
II and VII and either isoform alone is sufficient for rapid intraneuronal
bicarbonate replenishment during prolonged GABAAR activation.
5.2.3 Lack of electrographic febrile seizures in mice devoid of carbonic
anhydrase VII
The findings of study II showed that CA VII KO animals at P14 do not
develop experimental electrographic febrile seizures whereas WT mice of
similar age show pronounced electrographic seizures in response to induced
hyperthermia. Electrographic seizures in WT mice commenced in 24 ± 4 min
(n=9) after the onset of hyperthermia. Rectal temperature at this time point
was 40.9 ± 0.4 °C (n=9). Electrographic bursts of spikes (150-600 µV) with
frequency of about 2 – 4 Hz and duration of 10 - 60 s were recognized. Since
respiratory alkalosis is a main factor underlying eFS generation (Schuchmann
et al, 2006; Schuchmann et al, 2009; Schuchmann et al, 2011) we also
measured breath rate and blood pH in two animal groups. The breath rate in
WT mice increased to 177 ± 12 % (n=9) by the time of seizure onset, and a
similar increase in breath rate was seen in CA VII KO animals 24 min after
onset of hyperthermia. These increases in breath rate were paralleled by
respiratory alkalosis of the same amplitude in the blood of WT and CA VII
KO mice, and the body temperature increase in the KO (41.3 ± 0.4 °C, n=7)
35
was similar to that seen in the WT mice. In spite of these similarities, no
electrographic seizures were seen in CA VII KO animals, and the behavioural
activity in the KO mice was atypical of eFS. Periods of coordinated behaviour
patterns such as walking and exploratory activity were present, throughout the
hyperthermic period, which never happened in WT animals once seizures had
progressed to the clonic stage. In agreement with Shuchmann et al, (2006),
continuous application of 5 % CO2 in air prevented electrographic seizures
and all types of behavioural convulsions in WT animal group.
In order to gain further insight to the possible role of CA VII in to the
generation of eFS, we performed behavioural experiments on P14 rats
exposed to intraperitoneally applied low doses of diazepam that potentiate
GABAAR signalling (Eghbali et al, 1997). The lowest dose of 50 µg/kg
produced a non-significant decrease in the time to eFS onset from that seen in
saline-injected mice, whereas the dose of 150 µg/kg resulted in a significant
decrease in seizure latency. Importantly, these two low doses resulted in no
detectable breath changes under control conditions and gave similar increases
in breath rate during eFS induction. The much higher dose of 2.5 mg/kg
prevented seizure generation in all rats. Under control conditions 2.5 mg/kg
diazepam drastically suppressed breath rate and there was only a modest
increase in breathing rate in hyperthermic environment. These results point to
the role of CA VII dependent GABAergic HCO3- - dependent excitation in the
generation of eFS.
36
5.3 FOREBRAIN-INDEPENDENT GENERATION OF HYPERTHERMIC
CONVULSIONS IN INFANT RATS.
5.3.1 Disconnecting forebrain from the brainstem aggravates FS
Rats with precollicular brain transection subjected to hyperthermia were
compared with littermates that underwent sham surgery without no brain
damage. In response to elevated temperature, the onset of eFS was clearly
observed in all rats. Strikingly, rats with brain transection developed much
more intense hyperthermic convulsions with exclusive manifestation of tonicclonic behavioural pattern. In sham-operated pups, behavioural seizure onset
was characterized by severe clonic responses. Notably, body temperature of
transected animals at the moment of seizure manifestation was only 41.2 ±
0.4 °C (n=7) while sham-operated littermates had to be heated up to the
temperatures of 43.4 ± 0.2 °C (n=8). Additionally, latencies to seizure onset
were significantly shorter in the group of transected rats. It took 6.7 ± 1.1 min
for transected pups (n=7) to reach seizure threshold while sham-operated
pups had eFS attack only at 9.7 ± 0.9 min (n=8). Meanwhile, similar changes
in respiratory rate and blood pH were recorded in both groups. Hyperthermiainduced increase in breath rate at onset of convulsions in both animal groups
was of the same magnitude. After diazepam administration 15 min before
exposure to hyperthermia (2.5 mg/kg i.p.), hyperventilation in response to
hyperthermic conditions was prevented. Diazepam at lower doses (0.15
mg/kg) as well as vehicle injections in transected rats had no effect on the
latency to eFS and no significant differences were seen in respiratory rate at
the moment of eFS manifestation. The role of brainstem in seizure generation
was further investigated by application of kainic acid that is frequently used
to study seizures and mechanisms of epileptogenesis in limbic structures.
37
Similarly to hyperthermia, kainic acid evoked in transected rats only tonicclonic convulsions without preceding clonic pattern. As in eFS studies,
latency to seizures in transected pups (n=14) after kainic acid injections was
also significantly shorter than in sham-operated littermates (n=14), (11.8 ±
1.5 vs. 17.6 ± 1.5 min).
38
6. DISCUSSION
In this Thesis animal models were developed to mimic disorder-specific
pathological conditions in the brain, and the models were applied in order to
shed light on fundamental mechanisms of brain disorders that occur in
preterm infants or during early childhood. In the SPN ablation study the main
result is the evidence of the subplate influence on the intrinsic cortical
activity. Its removal in the S1 barrel cortical region abolishes the appearance
of the spindle bursts in this area and prevents the formation of the
characteristic barrel-like appearance. This absence of spindle bursts may be
the reason for further maturational deficits. Our second study demonstrates
the role of carbonic anhydrase isoform VII in pathophysiology of febrile
seizures. In the third study, we show that the brainstem can be an independent
seizure source with a prominent role in the generation of FS.
6.1 STUDY I
The results of this study show that subplate is a structure that participates in
the generation of a specific type of self-organized activity in the immature
cortex such as spindle bursts (Khazipov et al, 2004). Indisputable arguments
of subplate importance were the findings of impaired thalamocortical
transmission and reduction of endogenous activity in the cortex in subplate
ablated animals. In addition we observed a disruption in the development of
the patterning of the barrel field. While spindle bursts can be triggered by
peripheral sensory stimulation (Khazipov et al, 2004) they can also be
recorded in the absence of such inputs (Khazipov et al, 2004). During spindle
burst activity in vivo thalamus was shown to be active and was suggested to
be an active contributor to spindle bursts generation (Khazipov et al, 2004).
39
Since SPNs are the main target of thalamic axons and they massively project
to layer IV neurons they can relay incoming sensory information and mediate
spindle burst activity. SPNs are active during spindle bursts (Yang et al,
2009) and they oscillate in the spindle-frequency range (Hirsch & Luhmann,
2008), thus they might be directly related to the generation of these events.
Abolished spindle burst occurrence could be a direct consequence of the
absent subplate and/or of impaired thalamocortical transmission. Even though
thalamocortical synaptic transmission was impaired due to the subplate
absence, a large number of sharp potentials were observed. Thus, it is likely
that regularly seen sharp potentials are of intrinsic cortical circuit origin.
Remaining spindle bursts seen in ablated animals could originate from nearby
unaffected regions of S1. This coincides with previously shown data of slow
spindle burst spread within an area of at least a couple of mm across S1
(Khazipov et al, 2004; Sun & Luhmann, 2007).
The development of neocortical cytoarchitecture in visual cortex is thought to
be dependent on subplate dependent neuronal activity, experimental
destruction of which in cats lead to prevention of ocular dominance column
(ODC) formation (Kanold et al, 2003). Here we showed that subplate ablation
also prevents the formation of specific barrel pattern in somatosensory area.
Based on cytochrome oxidase staining we could conclude that SPN removal
affect S1 cytoarchitecture formation where clear gaps in the barrel filed were
present. This could partially reflect the decreased sensory driven activity
within layer IV since cytochrome oxidase levels are regulated by neuronal
activity.
40
6.2 STUDY II
CA isoforms are among the key molecules in neuronal pH and anion
regulation, and their involvement in the control of brain excitability is
evidenced by the antiepileptic actions of carbonic anhydrase inhibitors
although the underlying mechanisms of action have remained enigmatic
(Chesler, 2003; Casey et al, 2010). The cytosolic CA isoform VII has been
shown to play a key role in the postnatal upregulation of CO2/HCO3-dependent excitability of hippocampal networks (Ruusuvuori et al, 2004).
However, it was not known if other cytosolic CA isoforms are expressed in
CNS neurons in parallel with CA VII and if they exert similar effects on
neuronal excitability and on GABAergic signalling. In this study we showed
that hippocampal pyramidal neurons express cytosolic CA isoforms II and
VII. CA VII expression was found to be purely neuronal, whereas CA II is
known to be expressed also in glia (Kida et al, 2006; Ruusuvuori & Kaila,
2014). Both isoforms were found to be able to boost bicarbonate-dependent
biphasic GABAergic responses and consequent neuronal spiking activity
triggered by intense GABAAR activation. Emergence of this type of
GABAergic response was tightly associated with the onset of CA VII mRNA
expression at around P10 – 12, as shown before (Ruusuvuori et al, 2004).
Using our CA VII KO, CA II KO and double KO mouse models we found
that cytosolic CA activity in hippocampal pyramidal neurons is attributed to
the two isoforms II and VII. During P10 – 18 CA VII is the only cytosolic CA
isoform, but by P20 and onwards the two isoforms are expressed in parallel.
Somatic pHi recording and excitatory dendritic GABAAR responses revealed
no differences in the subcellular somatodendritic expression patterns of the
two isoforms. Consistent with the very high catalytic activity of CAs, we did
not detect any rate limiting effects when only one of the isoforms was absent.
41
Our present results clearly showed that CA VII activity alone is sufficient to
promote Cl- accumulation (Viitanen et al, 2010) by constant replenishment of
bicarbonate thus leading to the consequent GABAergic depolarization and
excitation (Kaila & Voipio, 1987; Viitanen et al, 2010). On the other hand,
CA VII expression in the human cortex and hippocampus already starts
perinatally, while FS are typically registered in children starting from 6
months of age. Therefore, the involvement of CA VII in generation of eFS in
the CA VII KO and WT mice was of particular interest to investigate. As it
was shown before, respiratory alkalosis is a trigger of FS in children
(Schuchmann et al, 2011). Data collected from blood measurements and
breath rate recordings showed that CA VII plays no role in hyperthermiainduced alkalosis in rodents and thus is not associated with systemic pH
changes that are triggered by breath rate changes. Nevertheless, exacerbation
of eFS in WT mice in comparison to CA VII KO pointed to a facilitatory role
of CA VII activity to promote FS with direct relationship to GABAergic
depolarization. Notably, low doses of diazepam potentiated GABAAR
opening and promoted eFS while high doses exerted opposite effect via
suppression of breathing. Especially in children, therapeutic doses interfere
with functions of respiratory system (Orr et al, 1991; Norris et al, 1999;
Appleton et al, 2008). Thus, our findings suggest that potentiation of
GABAergic responses in cortical neurons is not the prime mode of action of
diazepam in FS treatment.
42
6.3 STUDY III
The role of brainstem in seizure generation was proposed based on animal
models of circumscribed lesions and transections of the adult brainstem
(Browning & Nelson, 1986; Velasco & Velasco, 1990; Faingold, 2012), but
so far little is known about the role of brainstem in pathophysiological
activity of the immature brain (McCown & Breese, 1992; Coppola & Moshé,
2009). In response to hyperthermia first signs of seizure behaviour in
postnatal rodents are freezing and consecutive oral automatisms with clonic
forelimb development (van Gassen et al, 2008). It is postulated that these
initial events are of limbic origin (Dubé et al, 2009) while more severe forms
of seizure behaviour such as wild running and tonus-clonus have a brainstem
nature (Dube & Baram, 2006). These findings suggested us to use an animal
model of precollicular transection to investigate the role of brainstem in FS
generation. Strikingly, after isolation of the forebrain from the brainstem the
latency to seizure onset was significantly reduced in transected rats
comparing with WT mice. Not only the latency was shorter, but also the
magnitude of immediate behavioural response to eFS onset was much more
severe in transected pups. The above findings support the hypothesis that in
the intact brain, forebrain structures can actively suppress brainstem seizures
(Velasco & Velasco, 1990). With regard to respiratory alkalosis that promotes
FS (Schuchmann et al, 2006), we checked how low doses and high doses of
diazepam could contribute to breath rate changes. In agreement with previous
studies, high doses of diazepam effectively suppressed breathing rate whereas
low doses did not affect breathing rate. It is worth to mention that a similar
respiratory alkalosis was observed in all transected and sham-operated rats
subjected to hyperthermia only.
43
7 CONCLUSIONS AND FUTURE PROSPECTS
Study I demonstrates the crucial role of subplate neurons in morphofunctional maturation of the somatosensory cortex. This transient neuronal
population is necessary for the emergence of endogenous activity within
developing cortical networks and for promoting the organization of columntype cortical cytoarchitecture. In particular, experimental early subplate
ablation results in drastic reduction of somatosensory spindle bursts and
impairments in formation of cortical barrels, the hallmark architectonic
feature of the somatosensory cortex. These results expand on the previous
analogous findings made in the mammalian visual cortex, indicating a more
general instructive role of subplate networks in developmental patterning of
cortical maps.
Study II demonstrates that the most severe stages of electrographic
experimental febrile seizures are absent in mice lacking the neuron-specific
carbonic anhydrase isoform CA VII. Importantly, the results of this study
indicate that diazepines, such as diazepam, which are widely used in the
treatment of febrile seizures, may at low doses paradoxically exacerbate
febrile seizures by enhancing CA VII-dependent GABAergic depolarization
of neurons. Strikingly, the present work also shows that high doses of
diazepines, suppress febrile seizures most likely by blocking hyperthermiainduced respiratory alkalosis, a known trigger of febrile seizures.
Study III demonstrates for the first time that brainstem networks are alone
sufficient for the generation of severe tonic-cloning stages of febrile seizures.
The results of this work expand the conclusions of Study II by demonstrating
that diazepam administered at high doses prevents generation of febrile
44
seizures via blocking brainstem-dependent hyperthermia-induced respiratory
alkalosis.
The results obtained in this Thesis help us to better understand the possible
mechanisms that can cause seizures during febrile states. Defining carbonic
anhydrase isoform VII (CAVII) as a key molecule in pathological cascades in
febrile seizure generation, the design of novel isoform specific inhibitors of
CAVII can provide a novel approach in the treatment of FS. In addition, our
data suggests that previously recognized limbic area, as a primary locus of FS
generation should be re-evaluated and more attention should be given to
brainstem structures that so far can drastically contribute to manifestation of
seizure convulsions. Thus, the identification of brainstem as a locus of seizure
generation should aid future efforts aimed at prevention and treatment of FS
in children.
45
8 ACKNOWLEDGEMENTS
This work was carried out in the Laboratory of Neurobiology, Department of
Biosciences, University of Helsinki, Finland.
I would like to express my sincere gratitude to my supervisor Professor Kai
Kaila for his patience, wise mentoring and every day support throughout my
journey to pursue a PhD regardless of how difficult sometimes it was for me
to keep on going. His attitude and his devotion to science made me truly
believe that everything is possible and all you need is just to try one more
time especially when something goes wrong. I am also deeply grateful to my
supervisor Juha Voipio for his enthusiasm, kindness and willingness to give a
full-scale feedback no matter how busy he could be. His ability to clarify any
enigmatic phenomena is simply fantastic especially when it is related to
technical issues.
I am thankful to both my supervisors for their support and guidance.
I thank Else Tolner who taught me everything when I just came to the lab.
She was my mentor and friend during the first years of my work.
Thanks to Leonard Khirug for his great attitude and scientific help. His
constant support and trust in me cheered me up during these years.
I would like to thank Professor Heikki Tanila for kindly accepting the
invitation to be an opponent at the public defence of my Dissertation.
All comments and suggestion given to me by Professor Hannes Tapani Lohi
and Professor Aarne Ylinen how to improve my work are highly appreciated.
It would be absolutely impossible to complete studies and settle down my life
in Helsinki without outstanding work and personal dedication to the students
46
of Dr. Katri Wegelius. I am truly thankful for her help with numerous work
and life issues, for her kindness and friendship.
It’s a pleasure to acknowledge the members of the thesis advisory board
Docent Mikko Airavaara and Dr. Vootele Võikar for their helpful advices and
interest in my work at our meetings during these years.
My warmest regards to all my collaborators and friends who were beside me
over these years and helped me with experimental work: Ramil Afzalov,
Evgeny Pryazhnikov, Mikhail Yuriev, Svetlana Zobova and Dmitry
Molotkov. Furthermore I thank all current and former members of the lab:
Susanne Bäck, Henrike Hartung, Emma Prokic, Milla Summanen, Faraz
Ahmad, Peter Blaesse, Sudhir Sivakumaran, Henna-Kaisa Wigren, Alexander
Alafuzoff, Inkeri Hiironniemi, Albert Spoljaric, Jenna Lindfors, Martina
Mavrovic, Mari Virtanen, Britta Haas, Merle Kampura and Mairi Kuris. A
special thanks goes to: Eva Ruusuvuori, Alexey Pospelov and Anton
Tokarev. I would also like to express my gratitude to my office mates Martin
Puskarjov and Patricia Seja and to my close friend Stanislav Khirug for their
help, support, advices and friendship. Thank you, you guys are the best!
I was very lucky to meet my dearest and amazing Anya and I thank you for
your love and care. With you my final steps in book writing and preparations
for the Big D were much easy to tolerate J.
At last but at most, I acknowledge my family and relatives, especially my
wonderful mother Irina to whom I will be eternally grateful. For her
unwavering support in all what I choose to do.
47
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