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Functional role of presynaptic Kv3 channels on synaptic transmission at the basket cell - granule cell synapse
Inauguraldissertation zur
Erlangung der Doktorwürde
Fakultät für Biologie
Albert-Ludwigs-Universität
Freiburg im Breisgau
vorgelegt von
Marcus Illy
März 2012
Dekan: Prof. Dr. Gunther Neuhaus
Betreuer: Prof. Dr. Peter Jonas
Referent:
Koreferent:
Jahr der Promotion 2012
The research presented in this thesis was carried out at the Institut für Physiologie I of the
Albert-Ludwigs-Universität Freiburg between April 2007 and December 2011.
ContentZusammenfassung.................................................................................................................1
Abstract..................................................................................................................................2
Introduction.............................................................................................................................3
The role of the hippocampus in memory formation...........................................................3
Anatomy of the rat hippocampus.......................................................................................3
Interneurons of the DG......................................................................................................5
Feed-forward and feedback inhibition form the basis of complex network properties......7
The voltage-gated potassium channel family Kv3.............................................................9
Aim of this study...............................................................................................................11
Materials and Methods.........................................................................................................12
Electrophysiological recordings.......................................................................................12
Slice preparation..........................................................................................................12
Paired recordings........................................................................................................12
Data analysis and statistics.............................................................................................15
Morphology......................................................................................................................16
Solutions and substances................................................................................................17
Results.................................................................................................................................19
TEA prolongs AP half-duration of fast-spiking BCs.........................................................19
TEA-induced block of Kv3 channels increases synaptic transmission...........................21
TEA-induced block of Kv3 channels enhances multiple pulse depression.....................23
TEA-induced spike-broadening leads to recruitment of additional calcium channels.....25
Discussion............................................................................................................................29
Kv3 channels are involved in synaptic transmission at the BC-GC synapse..................29
TEA-induced block of Kv3 channels alters reliance of transmitter release on calcium
channel subtype...............................................................................................................32
Abbreviations.......................................................................................................................35
References...........................................................................................................................37
Acknowledgments................................................................................................................55
Curriculum Vitae...................................................................................................................56
Appendix A – Mathematica notebooks.................................................................................57
Calculation of multiple pulse relationships......................................................................57
Calculating skewness and CV.........................................................................................60
Appendix B – Selected parameters of individual cells.........................................................62
Zusammenfassung
Zusammenfassung
Spannungsgesteuerte Kaliumkanäle der Kv3-Unterfamilie spielen eine Schlüsselrolle bei
der Generierung des schnell feuernden Aktionspotentialsphänotyps in Korbzellen (basket
cells, BCs). Obwohl die elektrophysiologischen Eigenschaften dieser Kanäle vielseitig
untersucht wurden, ist nur wenig über ihre subzelluläre Verteilung bekannt. Eine
Expression von Kv3 konnte bereits in Somata und Dendriten gezeigt werden, während
dies für das Axon und die präsynaptische Terminale noch immer unklar ist. Es ist weiterhin
unbekannt, ob Kv3-Kanäle an der Regulation synaptischer Transmission beteiligt sind. Um
diese Fragen zu klären, führte ich simultane Paarableitungen zwischen präsynaptischen
BCs und post-synaptischen Körnerzellen (granule cells, GCs) in akuten hippocampalen
Hirnschnitten 19 bis 24 Tage alter Ratten im Gyrus dentatus (dentate gyrus, DG) durch.
Kombiniert mit der Applikation des Kv3-Antagonisten Tetraethylammonium (TEA) konnte
ich die Eigenschaften der Kv3-bedingten Transmitterausschüttung charakterisieren. Eine
Badapplikation von 1 mM TEA führte zu einer starken Verbreiterung des präsynaptischen
Aktionspotentials (APs) (0,91 ± 0,07 ms zu 1,15 ± 0,07 ms) und erhöhte dessen Spitzen-
amplitude. In der GC zeigten inhibitorische postsynaptische Ströme (IPSCs) einen Zu-
wachs der Amplitude (539,4 ± 76,7 pA zu 797,8 ± 112,4 pA). Durch Berechnung der
Schiefe der IPSC-Amplituden-Verteilung konnte gezeigt werden, dass dies auf eine Er-
höhung der Ausschüttungswahrscheinlichkeit zurückzuführen war. Zusätzlich führte 1 mM
TEA zu einer Erhöhung der multiplen Pulsdepression während gruppenevozierter APs. Es
ist bekannt, dass die synaptische Transmission an der BC-GC-Synapse unter Kontrollbe-
dingungen ausschließlich über spannungsgesteuerte Ca2+-Kanäle vom P/Q-Typ vermittelt
wird. Durch einen Block der P/Q-Typ Ca2+-Kanäle mit 1 µM ω-Agatoxin IVa in Anwesenheit
von TEA konnte ich eine kleine residuelle IPSC-Komponente aufzeigen. Das Integral
dieser Komponente betrug 7,59 ± 1,28% im Vergleich zur unter Kontrollbedingungen
aufgenommenen Komponente (100%). Durch zusätzliche Blockade von N-Typ Ca2+-
Kanälen durch 1 µM ω-Conotoxin GVIa, konnte ich ~54% der residuellen Kompontente
diesem Kanaltyp zuordnen. Zusammenfassend zeigen diese Ergebnisse, dass Kv3-
Kanäle in präsynaptischen Terminalen von DG-BCs an der Regulation der Transmitter-
ausschüttung beitragen. Weiterhin zeigen sie, dass die synaptische Transmission zu
einem kleinen Teil über N-Typ- und andere Ca2+-Kanäle vermittelt ist.
1
Abstract
Abstract
Voltage-gated potassium channels of the Kv3 subfamily play a key role in establishing the
fast-spiking action potential (AP) phenotype of basket cells (BCs). Even though their
electrophysiological properties are well understood, little is known about their subcellular
distribution. Kv3 has been shown to be expressed in somata and dendrites, but it is
unclear if they are expressed in axons and presynaptic terminals. Additionally it is unclear
if Kv3 channels are involved in the regulation of synaptic transmission. Adressing this
questions, I performed simultaneous paired recordings between presynaptic fast-spiking
BCs and postsynaptic granule cells (GCs) in acute hippocampal brain slices from 19- to
24-day-old rats in the dentate gyrus (DG). Combined with application of the Kv3 antagonist
tetraethylammonium (TEA) I characterized the properties of Kv3-related release. Bath
application of 1 mM TEA led to a strong broadening of the presynaptic AP (0.91 ± 0.07 ms
to 1.15 ± 0.07 ms) and increased its peak amplitude. In the GC, inhibitory postsynaptic
currents (IPSC) showed an increase in amplitude (539.4 ± 76.7 pA to 797.8 ± 112.4 pA).
Computing the skewness of the IPSC amplitude distribution showed that this was the
result of an increase in release probability. In parallel, 1 mM TEA increased the multiple
pulse depression during a train of APs. It is well known that under control conditions,
synaptic transmission at the BC-GC synapse is exclusively mediated by voltage-gated
Ca2+ channels of the P/Q-type. By blocking P/Q-type Ca2+ channels using 1 µM
ω-agatoxin IVa during simultaneous TEA application, I could reveal a small residual IPSC
component. The integral of this component was 7.59 ± 1.28% if compared to the
component recorded under control conditions (100%). With an additional block of N-type
Ca2+ channels by 1 µM ω-conotoxin GVIa, I could attribute ~54% of the residual
component to this channel type. However, a very small component still remained, which
could not been attributed to a certain voltage-gated Ca2+ channel-type. In conclusion these
results first suggest that Kv3 channels are also expressed in presynaptic terminals of DG-
BCs and contribute in the regulation of transmitter release. Second, this thesis suggests
that synaptic transmission can also rely to a small amount on N-type and other Ca 2+
channels if the AP waveform was broadened by TEA.
2
Introduction
Introduction
The role of the hippocampus in memory formation
Since the famous case of Henry G. Molaison (Scoville and Milner, 1957; Squire and
Wixted, 2011), mostly referred as H.M., the mammalian hippocampus is known as a
crucial part concerning memory storage and learning (Milner et al., 1998; Eichenbaum,
1999; Kennedy and Shapiro, 2004; Squire et al., 2004). In an attempt to cure H.M.'s
severe epileptic seizures, a bilateral temporal lobectomy was performed, including the
resection of parts of the anterolateral temporal cortex, the amygdala, and the anterior two-
thirds of his hippocampus. After surgery H.M. was affected from a permanent anterograde
amnesia. This gave strong evidence that the hippocampal formation is essential for
declarative memory formation (Zola-Morgan et al., 1986; Squire and Zola, 1996; Corkin,
2002; Squire, 2004). With hippocampal synaptic plasticity, few years later a molecular
mechanism was found capable to carry out memory formation on the subcellular level
which is still target of ongoing studies (Bliss and Lomo, 1973; Bliss and Collingridge, 1993;
Bennett, 2000; Pittenger and Kandel, 2003). Besides memory consolidation, the
hippocampus also contributes to other tasks, like as part of the hypothalamic-pituitary-
adrenal axis to stress response, and can be involved in mental illness, such as
schizophrenia (Tamminga et al., 2010; Stone and Hsi, 2011) and depression (Zhu et al.,
2011; Peng et al., 2012), if dysfunctional.
Anatomy of the rat hippocampus
The hippocampus belongs to the limbic system and is - as part of the allocortex - one the
phylogenetically oldest brain regions (Stephan, 1983; Lautin, 2001). Further parts of the
greater hippocampal formation are the presubiculum, subiculum, parasubiculum, and
entorhinal cortex (EC). Because of its strictly-layered anatomy, the hippocampus was an
early study-object for neuroanatomists in the 19 th century (Ramón y Cajal, 1893, 1909;
Golgi, 1906). Santiago Ramón y Cajal was one of the first scientists describing the
pyramidal cell (PC) layer (CA, from cornu ammonis) which was divided into three regions
(CA1, CA2, CA3) due to morphological differences in size and appearance of neurons
(Lorente de No, 1934; Slomianka et al., 2011).
3
Introduction
The main input region of the hippocampus is the dentate gyrus (DG) (Fig. 1). Seen in
medial to lateral order, the DG consists of the molecular layer (ML) which is typically
devoid of cell bodies, the principal cell-layer, which is mainly composed of granule cells
(GCs) and a subpopulation of mossy cells (MCs), and the hilar region, where a large
diversity of neurons can be found. Neurons located in EC layer II and III send axons via
the so-called perforant path (PP) to both GCs in the DG, CA1-PCs and CA3-PCs,
respectively (Fig. 1). In turn, the GCs send projections, called mossy fibers (MF), to PCs in
CA3. This PCs send axons in a bundle named Schaffer collateral (SC) terminating on PCs
in CA1. The pathway PP→ MF → SC is called the trisynaptic pathway of the hippocampus
(Fig. 1). There are two mutually connected recurrent networks in the hippocampus, given
rise by CA3-collaterals forming synapses on other CA3-PCs and DG-MCs. The latter
terminate on DG-GCs, forming the second part of the recurrent network. Finally, the axons
4
Figure 1: The hippocampal trisynaptic pathway.
Schematic drawing of a transversal hippocampal slice. The trisynaptic pathway is given rise from the perforant path (PP), forming synapses on dentate gyrus (DG) granule cells (GC). These send out mossy fibers (MF) to the dendrites of cornu ammonis layer 3 (CA3) pyramidal neurons (PC), which send in turn their axons in a bundle called Schaffer collaterals (SC) to the dendrites of PCs in CA2 and CA1. In the border area of DG GC layer, hilus, and CA PC layer, basket cells (BCs) are indicated as purple triangles. Output from the CA PC layer is managed by the associational commissural pathway (AC) and the axons of CA1 PCs (red).
Introduction
of CA1-PCs project back to subiculum, EC and neocortical areas. Thus EC serves as a
strongly associated input-output partner of the hippocampus (Van Hoesen and Pandya,
1975; Burwell, 2000; Suzuki and Amaral, 2004).
Interneurons of the DG
Beside the rather uniformly organized principal cell-layers there exists a large variation of
inhibitory interneurons throughout the hippocampus (Fig. 2) (Parra et al., 1998; McBain
and Fisahn, 2001; Somogyi and Klausberger, 2005). One of those is the parvalbumin (PV)-
expressing basket cell (BC), whose most obvious feature is high-frequency firing of action
potentials (AP). BCs exhibit a low input resistance of ~65 MΩ, (Aponte et al., 2006),
receive inputs by fast gated α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid
(AMPA)-receptors and release the neurotransmitter γ-aminobutyric acid (GABA) in a highly
synchronous manner (Geiger et al., 1997; Jonas et al., 2004). Since their resting
membrane potential (Vrest) is quite depolarized near firing threshold, BCs are easily
excitable (Fricker et al., 1999). It has been shown that synaptic transmission in BC
terminals is exclusively mediated by P/Q-type Ca2+ channels (Hefft and Jonas, 2005;
Bucurenciu et al., 2008, 2010), since blocking these channels leads to a complete loss of
synaptic transmission. At the BC-GC synapse, the synaptic transmission is characterized
by synaptic depression of ongoing IPSCs during repetitive firing of the BC (Bartos et al.,
2001). This is thought to be caused by incomplete refilling of synaptic vesicle pools (Liu
and Tsien, 1995a, 1995b; Weis et al., 1999; Kraushaar and Jonas, 2000). These
interneurons are crucial for the generation of network oscillations. Oscillatory activity
provides information about the cooperative activity of certain neuronal populations
(Buzsáki and Draguhn, 2004; Geisler et al., 2010) and are essential for higher cognitive
brain functions (Penttonen et al., 1998). BCs contribute to the generation of oscillations in
the gamma band range exhibited by fast feedforward and feedback inhibition, making
them essential for higher brain functions (Pouille and Scanziani, 2001; Bartos et al., 2007).
Interestingly, patients suffering from schizophrenia show deficits in the generation of
gamma-band oscillations (Light et al., 2006). In summary, BCs are highly optimized
interneurons with fast input and output characteristics for rapid information processing and
transmission.
5
Introduction
Concerning their morphology, BCs in the DG have their axon highly restricted to the
granule cell layer (GCL) with their soma located at the GCL-hilar border (Jonas et al.,
2004; Hefft and Jonas, 2005; Nörenberg et al., 2010). Because of the extensive axonal
arborization they project to a high number of GCs in the DG, making them ideal candidates
to study synaptic transmission. Like the vast majority of inhibitory interneurons, BCs are
devoid of dendritic spines (Freund and Buzsáki, 1996; Martínez et al., 1999), but can be
rather easily identified by their fusiform- or triangular-shaped somata (Freund and Buzsáki,
1996). In addition to hippocampus, BCs are abundant in several brain regions such as
neocortex (Blazquez-Llorca et al., 2010; Fazzari et al., 2010), thalamus (Klostermann and
6
Figure 2: Domain-specific innervations of interneurons in the DG
Schematic drawing of five different types of GABAergic interneurons in the DG, illustrating dendritic and axonal arborization and somatic location, respectively. Red circles represent interneuron somata, green cirles GC somata and green lines the corresponding dendritic arborization. Red, solid lines indicate the predominant orientation of the dendritic tree. Blue boxes show the main target region within the DG laminae where the axon of each interneuron typically arborizes. The shown laminae are as followed: outer molecular layer (OML), inner molecular layer (IML), granule cell layer (GCL), and hilus. The pictured interneuron types are the parvalbumin-expressing basket cell (PV-BC), CCK expressing interneuron (CCK-IN), molecular layer perforant path-associated cells (MOPP) and hilar perforant path-associated cells (HIPP).
Modified from Freund and Buszaki, 1996.
Introduction
Wahle, 1999; Traub et al., 2005), and cerebellum (Caillard et al., 2000; Collin et al., 2005;
Wierzba-Bobrowicz et al., 2011).
Beside the PV-BC, there is another type of BC, in literature mostly named the
cholecystokinin-expressing interneuron (CCK-IN) (Kawaguchi and Kubota, 1997; Hefft and
Jonas, 2005). In contrast to PV-positive BCs, these cells do not show sustained fast-
spiking of APs and release GABA in an asynchronous manner (Hefft and Jonas, 2005; Ali
and Todorova, 2010). Actually their neurotransmitter release relies on the expression of
N-type Ca2+ channels. Interestingly, in the DG their axonal arborizations do not overlap
with those of BCs since CCK-IN axons are confined to the inner molecular layer (IML).
Contrariwise, in CA1 and CA3, both BCs and CCK-INs, terminate on the perisomatic
domain of PCs (Pawelzik et al., 2002; Freund, 2003; Morozov and Freund, 2003).In the
current view CCK-INs are thought to work as plastic fine-tuning devices providing
subcortical inputs into the hippocampus, which is in contrast to the oscillation-modulatory
function of PV-BCs (Freund and Katona, 2007).
The DG also contains further types of interneurons which are classified by their dendridic
and axonal arborization (Freund and Buzsáki, 1996). Axo-axonic or chandelier cells are
interneurons terminating at the axon initial segment of a large number of principal cells and
are ideal candidates for creating synchronized synaptic output within large cell populations
(Somogyi et al., 1983; Freund and Buzsáki, 1996; Klausberger and Somogyi, 2008).
Molecular layer perforant path-associated (MOPP) cells form synapses restricted to the
dendritic shaft of GCs (Halasy and Somogyi, 1993) and help to maintain firing frequency
within physiological levels by feedforward inhibition and therefore protect GCs from noisy
input from EC (Ascoli and Atkeson, 2005; Ferrante et al., 2009). Hilar perforant path-
associated (HIPP) cells project to GC dendrites in the outer molecular layer (OML) (Sik et
al., 1997) and play together with MCs a key role in pattern separation (Myers and
Scharfman, 2009).
Feed-forward and feedback inhibition form the basis of complex network properties
Hypothesizing a simple network of neurons only connected by excitatory synapses (Fig. 3)
makes it easyly comprehensible that stimulation of a single cell would result in a feedback
loop exciting all the neurons in the population (Fig. 3A). Inhibition is therefore crucial to
maintain complex spiking patterns in neural networks (for review see Jonas and Buzsaki,
7
Introduction
2007). Feedback inhibition establishes a form of self-regulation withing the network. This
can be illustrated by a simple two-cell model, consisting of an excitatory principal cell and
an inhibitory interneuron with mutual connections (Fig. 3A). Driving the excitatory neuron
by external input would rise the discharge rate of the postsynaptic interneuron, which in
turn would hyperpolarize the principal cell and thus preventing further increase in firing
rates.
In a second model, an inhibitory interneuron projects on an excitatory principal cell, both
driven by the same input, thus creating feed-forward inhibition (Fig. 3B). In this case, the
driven interneuron will decrease the activity of the principal cell, acting like a filter of
synaptic input. This simple pairings are effective enough to increase temporal precision of
synaptic transmission (Buzsáki, 1984; Ferrante et al., 2009) by narrowing time-windows
(Pouille and Scanziani, 2001), which is an essential feature in coincidence detection, for
instance (Yang et al., 1999; Couchman et al., 2010; Pavlov et al., 2011; Tang et al., 2011).
Coincidence detection plays a key role in the integration of spatial and temporal context
during memory consolidation (Katz et al., 2007), one of the main tasks of the
hippocampus.
Adding more cells to our simple two-cell model will dramatically increase complex firing
behavior which is hard to predict and requires more complex mathematical models. During
8
Figure 3: Different types of inhibiting circuits
A. Feedback inhibition performed by an interneuron (red) stabilizes principal cell (green) activity. “-” represents inhibitory connections, “+” shows excitatory connections, respectively. Blue arrow show the site of synaptic input to the model. Black arrows in the somata indicate changes in activity.
B. Feed-forward inhibition filters and thereby decreases principal cell activity.
C. Lateral inhibition segregates the activity of the middle principal cell by suppressing the discharge rate of neighboring principal cells.
Modified from Jonas and Buzsaki, 2007.
Introduction
lateral inhibition (Dizon and Khodakhah, 2011), a special case of feed-forward inhibition, a
single active principal cell is able to suppress surrounding principal cells by recruiting
nearby inhibitory interneurons (Fig. 3C).
Thus, small differences in the input will create large differences in the output and enable
pattern separation of electric activity within a subset of the network. In the retina, lateral
inhibition enhances the contrast by of visual signals (Eggers et al., 2007; Vigh et al., 2011).
Because PV-BCs in the DG project on a high number of GCs and receive inputs from GCs
and other interneurons, they are known to be involved in feed-forward and feedback
inhibion (Jedlicka et al., 2010).
The voltage-gated potassium channel family Kv3
For establishing the fast-spiking phenotype, the voltage-gated potassium channel Kv3
subfamily is attributed to a key role. As all vertebrate Kv channels, Kv3 channels are
tetramers and formed by four identical α-subunits (Gutman et al., 2005). Each subunit
consists of six transmembrane domains (S), with its N- and C-termini facing the
cytoplasmic domain (see Fig. 4B). S4 contains the voltage-sensor, while S5 and S6 form
the P-loop which creates the conducting pore. Furthermore, Kv channels can be
modulated in their electrophysiological aspects, like permeability, as well as in their surface
expression level by additional non-conducting β-subunits (Pongs et al., 1999; Li et al.,
2006). It is remarkable that the S4 - S5 sequence of Kv3 protein is completely preserved in
a large number in species including humans suggesting a strong evolutionary pressure for
it (Rudy and McBain, 2001; Choe, 2002).
The Shaw-related Kv3 subfamily is encoded by the KCNC gene family (Rettig et al., 1992)
and holds two members in the A-type category (Kv3.3, Kv3.4) as well as two delayed
rectifiers (Kv3.1, Kv3.2). All of them are high-threshold activated channels showing
conductance at depolarizations around -20 mV (Coetzee et al., 1999). All Kv3 subtypes
also exhibit extremely fast activation and deactivation kinetics (Lien and Jonas, 2003). In
BCs these features enable a fast membrane repolarization and by this means a fast
reactivation of sodium channels capable for repetitive spiking at high frequencies (Martina
and Jonas, 1997). Most remarkably, this is achieved without affecting threshold and timing
of the AP and prolonging the refractory period (Rudy and McBain, 2001). Antagonizing Kv3
channels leads to a loss of fast-spiking phenotype in hippocampal oriens alveus
9
Introduction
interneurons (Lien and Jonas, 2003), a GABAergic interneuron type with similar AP firing
properties like BCs. Contrariwise, dynamic clamp recordings were capable to rescue fast-
spiking in this neurons when artificial Kv3 conductances were mimicked (Lien and Jonas,
2003). This suggests the expression of Kv3 conductances is a main requirement for
establishing fast-spiking phenotypes in hippocampal interneurons.
In the thalamic reticular nucleus, ablation of Kv3.1 and Kv3.3 results in an abolishment of
network oscillations and disturbed slow-wave sleep in mice (Espinosa et al., 2008). Since
the role of GABAergic interneurons for the maintenance of hippocampal oscillations is
thought to be essential for higher brain functions such as temporal encoding of information
(Singer, 1999; McBain and Fisahn, 2001; Bartos et al., 2002), this suggests an
involvement of Kv3 channels in the maintenance of hippocampal network oscillations.
Beside the electrophysiological properties of the Kv3 family, little is known about the
subcellular localization of Kv3 channels. An expression of Kv3 channels in somata
(Martina et al., 1998) and dendrites (Hu et al., 2010) of BCs has been previously
10
Figure 4: Genetic origin and transmembrane structure of voltage-gated potassium
channels.
A. Phylogenetic tree of the Kv family. The Kv3 subfamily is highlighted red. Modified from Coetzee et al., 1999.
B. Transmembrane structure of a single Kv channel α-subunit. Transmembrane segments are represented as orange rectangles. S4 contains the voltage sensor (“++” indicates the positive charges within this segment), while S5 and S6 form the P(pore-forming)-loop. For a functional conducting pore, four P-loops are required.
Modified from Choe, 2002.
Introduction
demonstrated, but it is still unclear whether this also holds true for axon and presynaptic
terminals. A presence of Kv3 channels in synaptic boutons would suggest a pivotal role in
shaping the presynaptic spike and subsequently the recruitment of voltage-gated Ca2+
channels. It has been shown that during a block of Kv3 channels in slices of layer II/III
barrel cortex, AP-evoked Ca2+ transients increased in amplitude and duration (Goldberg et
al., 2005). However, the molecular identity and number of the channels mediating this
effect still remains unknown. In the current view a small number of Ca 2+ channels is
sufficient to release transmitter with high temporal precision at the BC-GC synapse, due to
a tight-coupling of Ca2+ channels and sensors (Bucurenciu et al., 2008, 2010). The
molecular identity of the Ca2+ sensor in BC terminals remains unresolved, however former
works suggest synaptotagmin 2 (Sheng et al., 1997; Südhof, 2002; Bucurenciu et al.,
2008).
Aim of this study
This thesis addressed four questions by performing paired recordings at the BC-GC
synapse in the rat hippocampus:
(1) Are Kv3 channels functionally expressed in BC axons and/or terminals?
(2) Do Kv3 channels control the release of GABA?
(3) Are other Ca2+ channels additionally to P/Q-type recruited if Kv3 channels are
antagonized?
(4) Can the block of Kv3 channels be used to get new information about the coupling of
voltage-activated Ca2+ channels and Ca2+ sensors?
The results first suggest a novel view of Kv3 channels contributing to rapid signaling in BC
terminals by stabilizing the transmitter release and avoiding synaptic depression. Second,
these results argue for a model of synaptic transmission with tight-coupling of few, but
highly sensitive voltage-gated Ca2+ channels to Ca2+ sensors. Third, a contributing role of
N-Type Ca2+ channels to synaptic release could be uncovered.
11
Materials and Methods
Materials and Methods
Electrophysiological recordings
Slice preparation
19- to 25-day-old Wistar rats were rapidly decapitated without anesthesia in accordance
with national and institutional guidelines. Experiments were approved by the Animal Care
Committee Freiburg according to national regulation (Tierschutzgesetz, §15; registry T-
04/10 and X-07/05A). The head was immediately submerged in iced (~3°C) artificial
cerebrospinal fluid (ACSF). Fur and skin were removed and the skull opened from the
caudal side. The brain was cut at the bulbus olfactorius and cerebellum, removed, and
carefully placed in fresh ice-cold ACSF. The hemispheres were separated by a sagittal cut.
The right hemisphere was preferably used for experiments, the left one was discarded. A
part of the dorsal cortex was removed by a cut performed parallel to the bisecting line of
the angle formed by the edges of the hemisphere. Afterwards, the hemisphere was glued
at the cutting area and placed in cooled slicing chamber using a cyanoacrylate-based glue.
The brain was placed with bulbus olfactorius left and cortex right to minimize the distance
the blade has to enter the tissue during slicing. Transversal hippocampal slices (300 μm
and 350 µm thickness) were cut with either a vibratome (custom-made) (Geiger et al.,
2002) or alternatively a Leica VT1200 (Leica Microsystems GmbH, Wetzlar, Germany).
During slicing, the chamber was continuously bubbled with carbogen (95% O2 / 5% CO2)
to maintain a stable pH and provide oxygen-support. After cutting, the slices were
transferred to a custom-made maintenance chamber (Edwards et al., 1989) with
carbogen-bubbled ACSF at 33°C for 20 min to recover channel kinetics and other
temperature-depended cell parameters. Finally, the maintenance chamber was transferred
to room temperature (21 - 24°C, checked by a digital thermometer (custom-made)) for
recordings.
Paired recordings
All experiments were done at room temperature (21 - 24°C). In order to study synaptic
transmission, paired recordings of BCs and GCs in the DG were performed (Kraushaar
and Jonas, 2000) under visual control using an infrared differential interference contrast
12
Materials and Methods
(IR-DIC) video-microscope system (Edwards et al., 1989; Stuart et al., 1993; Koh et al.,
1995). Patch pipettes were pulled from thick-walled borosilicate glass (2 mm outer
diameter, 1 mm inner diameter). Filled with intracellular solution, the open-tip resistance
was 2 - 5 MΩ. To prevent dust particles being attached at the pipette, a positive pressure
of 80 - 110 mbar was applied depending on tip-size. Putative BCs were selected by their
soma-shape and location at the border of the GC-hilus-layer or deeper in the GC-layer.
The highest chance of getting a BC was achieved if fusiform-shaped cells were selected
(Fig. 7A). Cells embedded deeper in the slices (~ 20 - 70 µm) were preferentially used
since synaptic transmission is highly dependent on intact axon arborization. First, the
pressure was released and a tight-seal (> 3 GΩ) cell-attached configuration was
established. Second, a whole-cell-configuration was achieved by suction-induced rupture
of the cell membrane.
The series resistance (Rs) was calculated and Vrest was checked. Only BCs with a
Vrest < -54 mV were used for experiments. Selected cells showed a fast-spiking firing
pattern (> 50 Hz) upon injection of a depolarizing current (1 s at 900 pA) (Martina et al.,
1998). Subsequently, a whole-cell recording of a GC at the GC-layer/IML-border was
obtained using the same procedures as described before. Only GCs with a V rest < -60 mV
were used. In 20 - 50% of all experiments BCs and GCs were synaptically connected as
revealed by IPSCs in response to AP evokation in the BC.
During paired recordings, first a single AP in the presynaptic BC was elicited by a brief
depolarizing current injection (2 nA, 1 ms). In the subsequent recorded sweep, a repetitive
(“train”) stimulation was used by injection depolarizing current pulses (2 nA, 1 ms) with a
interstimulation interval of 19 ms to evoke APs at a frequency of 50 Hz. The intersweep
interval was set to 10 s to prevent synaptic depression.
For recordings, a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA)
was used. To eliminate intercellular variations in Vrest, all BCs were held at -70 mV by
injecting a hyperpolarizing current (> -160 pA). For the same reason, postsynaptic GCs
were clamped to -80 mV. Only recordings with a Rs < 15 MΩ were used for analysis, since
recorded inhibitory postsynaptic current (IPSC) amplitudes decrease with increasing Rs
(Numberger and Draguhn, 1996). Recordings with a change in Rs by more than 20% were
discontinued to ensure electric stability during the recording. In current-clamp
configuration, Rs was compensated by the build-in function of the patch-clamp amplifier.
13
Materials and Methods
14
Figure 5: Calculation of waveform parameters in BC-GC paired recordings
A1. Representation of depolarizing current pulse, injected via the patch clamp pipette (A2) into a BC (B2).
B1. Representation of an AP waveform. The examined parameters were as followed: the amplitude (AAP) from baseline to the peak of the AP, the half-duration (HD), measured as the time from 50% of the amplitudes upward to the 50% downward deflection and the rise-time (RT), measured as time-interval from 20% to 80% of the AP amplitude.
C1. Representation of an IPSC recorded in a GC (C2). The amplitude (AIPSC) was measured from baseline to peak. Furthermore, the integral under the curve was computed as a 400 ms time-interval starting with the begin of the current-injection.
Materials and Methods
Contrariwise, Rs was monitored, but not compensated in voltage-clamp configuration in
order to avoid manipulations of the recorded currents by the compensatory build-in
workflow of the amplifier. Membrane potentials are given without correction for liquid
junction potentials. Signals were low-pass filtered at 10 kHz (4-pole low-pass Bessel) and
sampled at 50 kHz.
Pulse generation and data acquisition was done by a 1401Power interface (CED,
Cambridge, UK) and a PC. The program Igor 5.057 (WaveMetrics, Lake Oswego, OR,
USA) with the plugin Fpulse 3.18 (Igor-plugin by Ulrich Fröbe, Physiologisches Institut I,
Freiburg, Germany) was used for data acquisition.
After the recordings, pipettes were removed carefully to establish outside-out patches to
preserve the cell. Finally, the outside-out patch was destroyed by applying pressure.
Subsequently, the offset-potential was checked for stability during recording in order to
exclude baseline-drift as a possible case of defect.
All traces shown in this work recorded under current-clamp are single traces. All
postsynaptic traces recorded under voltage-clamp configuration represent average traces
calculated from single traces during the steady-state phase of the experiments.
Data analysis and statistics
Firing frequency of BCs was determined under current-clamp as the inverse of the mean
interspike interval during a 1 s depolarizing current injection (0 pA to 900 pA in steps of
100 pA). AP amplitude, AP rise time (time from 20% to 80% of the AP amplitude) and half-
duration (time from 50% rising phase to 50% descending phase of AP waveform) were
measured from the baseline preceding current injection. The maximal rate of rise was
calculated from the first derivative of the AP waveform. If the area under the curve was
determined, the integral in a 400 ms interval starting at the beginning of the current
injection was calculated.
In order to examine multiple pulse relationships during train stimulations, the single
amplitudes of every IPSC were required. (1) Therefore, the amplitude of the first IPSC was
measured from baseline. (2) Then, by exponentially fitting this first IPSC and subtracting
the fit from the IPSC pattern (see Fig. 9B) a new baseline was calculated. (3) This baseline
was used to evaluate the amplitude of the subsequent IPSC. Then, the steps (2) and (3)
15
Materials and Methods
were repeated until all train IPSC amplitudes were computed. Because of the extremely
small IPSC amplitudes after application of peptide-toxins, an exponential fit did not reach
the required precision of the program. In these cases, the values were evaluated by hand.
For every single IPSC withing the train, the baseline was set just before the beginning of
the IPSC. Then the amplitude of this IPSC was measured in accordance to the hand-set
baseline.
Analysis was done in Mathematica 5.2 and 8.0 (Wolfram Research, Champaign, IL, USA,
see Appendix A for Mathematica notebooks), Excel 2003 (Microsoft Corporation,
Redmond, WA, USA), Origin 8.5G (Origin Lab Corporation, Northampton, MA, USA) and
stimfit 0.9 (custom-made by C. Schmidt-Hieber and P. Jonas, http://www.stimfit.org).
Statistics were done in GraphPad Prism 3 (GraphPad Software, La Jolla, CA, USA). All
numeric values and graphic representations (points an error bars) in this work represent
means ± standard error of the mean (SEM). All data samples showed a parametric
distribution. Due to the small numbers of experiments, a Wilcoxon signed-rank test for
nonparametric distributions was performed to check for significant differences between the
data samples obtained from paired observations. In order to analyze data from non-paired
observations, a Mann-Whitney-Test was performed. Significance of differences was
assessed at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). If data sets showed extremely
strong differences, GraphPad Prism 3 was not able to compute an exact P-value. In these
cases, the P-values are indicated by P < 0.0001.
Morphology
To confirm cell identity, potential BCs were filled with 0.2% biocytin (Horikawa and
Armstrong, 1988; McDonald, 1992) during recording and fixed over night using 2.5%
paraformaldehyde, 1.25% glutaraldehyde and 15% picric acid in 100 mM phosphate buffer
(PB) at pH 7.3 and a temperature of 4°C. After fixation slices were rinsed and incubated in
1% hydrogen peroxide followed by a treatment with 10% sucrose PB and 20% sucrose PB
for cryoprotection. Accordingly, slices were shock-frozen in liquid nitrogen and incubated
overnight in PB including 1% avidin-biotinylated horseradish peroxidase complex (ABC;
Vector Laboratories, Peterborough, UK) at a temperature of 4°C. Slices were heavily
rinsed in PB in order to remove excessive ABC and subsequently treated with 0.05% 3,3'-
diaminobenzidine tetrahydrochloride and 0.1% hydrogen peroxide. Finally, slices were
16
Materials and Methods
extensively rinsed in PB and embedded in aqueous mounting medium (Mowiol, Höchst or
Roth, Germany). After processing, cells were compared with IR-DIC-images taken during
recording ensuring morphology was not altered during the fixation process.
Solutions and substances
All substances were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck
(Darmstadt, Germany) and Honeywell (Seelze, Germany), unless specified differently. For
BCs, a potassium-gluconate-rich internal solution (KGluc) was used, containing 135 mM
KGluc, 20 mM KCl, 100 nM EGTA, 2 mM MgCl2, 2 mM Na2ATP, 10 mM HEPES, and 0.2%
biocytin (Mobitec, Göttingen, Germany). To boost IPSC amplitudes in GCs, an internal
potassium-rich solution (KCl1) was used, containing 140 mM KCl, 10 mM EGTA, 2 mM
MgCl2, 2 mM Na2ATP, and 10 mM HEPES. APs were suppressed by adding 3 mM of N-
(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) to the KCl1-
solution. Both internal solutions were adjusted to pH 7.28 with KOH and tested for an
osmolarity ~ 302 mOsm. A maximal offset of ± 10 mOsm was accepted.
ACSF contained 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM
glucose, 2 mM CaCl2, and 1 mM MgCl2. The osmolarity was ~320 mM after equilibration
with carbogen at ~ 7.4 pH.
To block glutamatergic transmission, 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)
(Tocris Bioscience, Bristol, UK) and 20 µM D-2-amino-5-phosphonopentanoic acid
(D-AP-V) (Tocris Bioscience) were added to the ACSF. Kv3 channels were blocked with
1 mM tetraethylammonium (TEA). All drugs were applied via bath-superfusion.
Ca2+ channels were antagonized by peptide toxins as followed: P/Q-type with 0.5 - 1 µM
ω-agatoxin IVa (Tocris Bioscience) and N-type by 1 µM ω-conotoxin GVIa (Tocris
Bioscience). Since proteins form strong, non-specific interactions with surfaces
(Sagvolden, 1999), adhesion of the peptides to glass materials and tubings was prevented
by adding 1 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich) to the solution. Peptide
toxins were applied with a solution-recycling peristaltic pump (Ismatec, Wertheim-
Mondfeld, Germany) with a total volume of ~ 5 ml. The solution was equilibrated with
carbogen (95% O2 / 5% CO2) to ~ 7.4 pH. In every case, peptide-toxins were applied in
addition to 1 mM TEA.
17
Materials and Methods
In two experiments only ω-agatoxin IVa was applied after TEA (Fig. 6). In another two
cases TEA followed an application of ω-agatoxin IVa, again followed by an application of
ω-conotoxin GVIa. Finally, in two other experiments, ω-agatoxin IVa and ω-conotoxin GVIa
were co-applied after TEA. The proportions of toxin-sensitive currents from co-application
of ω-agatoxin IVa and ω-conotoxin GVIa as well as subsequent application of ω-conotoxin
GVIa after ω-agatoxin IVa were similar. Therefore results of these experiments were
pooled. For the same reason, the data derived from 0.5 µM and 1 µM ω-agatoxin IVa,
respectively, were pooled as well.
18
Figure 6: Paradigms for isolating Ca2+ dependend currents after TEA application
A. Paradigm shows bath application of 1 mM TEA followed by subsequent bath application of 0.5 or 1 µM ω-agatoxin IVa, followed by 1 µM ω-conotoxin GVIa.
B. Alternative paradigm, showing bath application of 1 mM TEA with subsequent co-application of 1 µM ω-agatoxin IVa and 1 µM ω-conotoxin GVIa.
Results
Results
The results of this work indicate that Kv3 channels are expressed in presynaptic terminals
of fast-spiking BCs of the hippocampal DG and that they play a crucial role in synaptic
transmission. First, a block of Kv3 channels enhanced synaptic transmission by prolonging
the AP duration. Second, this augmented transmission did not exclusively rely on the
known P/Q-type Ca2+-channel activation, but N-type and probably R-type and/or L-type
Ca2+ channels. The N-type mediated transmission showed different characteristics as
known from other cells, suggesting a novel role of N-type Ca2+ channels in synaptic
transmission. Finally these results suggest a tight-coupling of few, but easily activatable
voltage-gated Ca2+ channels to the Ca2+ sensor.
TEA prolongs AP half-duration of fast-spiking BCs
Although 4-aminopyridine (4-AP) is a well-known blocker for Kv3.1 and Kv3.2 subunits
(Henderson et al., 2010; Pedroarena, 2011; Wykes et al., 2011), a bath application of
100 µM resulted in an dramatically increased spontaneous spiking activity of the
presynaptic BC (not shown). This increase was most likely due to a block of voltage-gated
potassium channels from the Kv1 subfamily, where several members (Kv1.1, 1.2, 1.3, 1.5
and 1.6), show a sensitivity to 4-AP in the micromolar range (Coetzee et al., 1999; Guan et
al., 2007; Yang et al., 2007; Higgs and Spain, 2011). Under control conditions, Kv1
channels activate at low thresholds and repolarize membrane potential before reaching
firing threshold. If antagonized, as presumably appeared in here, an increase in
spontaneous spiking is likely. As a consequence, 1 mM TEA was used as Kv3 channel
antagonist in all experiments of this study.
To examine presynaptic effects of TEA, cells were recorded in current clamp configuration.
In order to eliminate intercellular variations in BCs Vrest, all BCs were held at -70 mV
throughout the whole experiment. APs were elicited by application of a brief depolarizing
current pulse (2 nA, 1 ms).
Two to five minutes after starting the bath application of 1 mM TEA a highly significant
broadening of the APs was observed, indicated by an increase of the half-duration from
0.91 ± 0.07 ms to 1.15 ± 0.07 ms (P < 0.0001; n = 21; Fig. 7C, Appendix B, Tab. 1). This
was most likely due to a block of Kv3 channels by TEA application. Because the AP
19
Results
20
Figure 7: TEA-induced block of Kv3 channels increases half-duration and amplitude
of somatic APs in fast-spiking BCs
A. Upper panel: IR-DIC image of a hippocampal BC. The fusiform-shaped cell is located at the GCL-hilus border. For better identification, the BC soma is labeled by transparent red color done by digital post-processing of the image. Lower panel: Fast-spiking firing pattern (72 Hz) of a BC upon injection of a depolarizing current pulse (900 pA, 1 s).
B. Typical voltage traces showing a single AP elicited by injecting a short depolarizing current pulse of 20 pA for 1 ms before (black), during (red) and after (green) application of 1 mM TEA. Note the broadening of the AP waveform and the increased peak amplitude during application of 1 mM TEA. The washout partially recovered the AP waveform recorded under control conditions.
C - F. Summary graphs of the effect of TEA on half-duration (C), rise-time 20% - 80% (D), peak amplitude (E) and the maximal rate of rise (F). Black open boxes show date from individual experiments, red boxes the corresponding mean values. TEA significantly increased half duration (P < 0.0001) and peak amplitude (P < 0.01). All data from n = 21 cells, washout performed in n = 4 cases.
Results
broadening was easily recognizable during the recordings, this effect was also used as a
positive control in all experiments. The TEA-induced Kv3 antagonization significantly
increased peak amplitude of APs from 116.5 ± 2.1 mV to 120.3 ± 2.4 mV (P = 0.007,
n = 21, Fig. 7E, Appendix B, Tab. 2). Furthermore, more hyperpolarizing current was
needed being injected to maintain membrane potential at -70 mV during the recording (not
shown). This finding suggests a slight depolarization of the BCs V rest caused by
antagonization of Kv3 channels by TEA.
TEA-application did not significantly affect rise time (change from 0.64 ± 0.07 ms to
0.61 ± 0.06 ms, n = 21, Fig. 7D) and maximal rate of rise (change from 314.3 ± 22.2 V/s to
321.4 ± 22.3 V/s, not significant, n = 21, Fig. 7F) of the AP. This indicates TEA did not
affect voltage-gated sodium channels as expected. In four cases a washout could be
performed. During washout-phase, the half-duration showed a decrease in 3 of 4 cases
which was on average 0.93 ± 0.03 ms (not significant, n = 4). This suggests a partial
recovery.
In summary, these results indicate that Kv3 channels are likely being expressed at the
soma of BCs and are capable to shape the somatic AP waveform in half-duration and peak
amplitude. Kv3 channels in the presynaptic terminal were expected to have an measurable
influence on synaptic transmission.
TEA-induced block of Kv3 channels increases synaptic transmission
In the next step, the contribution of Kv3 channels in the regulation of synaptic transmission
was examined. Paired recordings from presynaptic fast-spiking BCs and postsynaptic GCs
were performed. In order to record postsynaptic currents, GCs were recorded in voltage-
clamp configuration. Postsynaptic GCs were clamped to -80 mV to eliminate intercellular
variations in Vrest.
Under control conditions the average IPSC amplitude recorded at the postsynaptic GC
was 539.4 ± 76.7 pA (including failures, n = 21) and showed a moderate increase to
797.8 ± 112.4 pA (P < 0.0001, n = 21, Fig. 8C, Appendix B, Tab. 3) when TEA was applied.
As already known, TEA can be removed from slices by washout (Ishikawa et al., 2003).
Therefore, approximately 15 min after beginning of the washout-phase a recovery of IPSC
amplitude back to a mean of 526.1 ± 240.6 pA could be monitored (not significant if tested
against values recorded under TEA conditions, n = 5, Fig. 8B and 8C).
21
Results
To distinguish between an increase of vesicle release probability or an increase in the
number of synaptic release sites, the skewness of the IPSC amplitude distribution was
examined (Kerr et al., 2008). Righ-skewed (v > 0) amplitude distributions indicate low
release probabilities, whereas left-skewed (v < 0) amplitude distributions are suggesting
higher release rates. The distribution changed from right-skewed (0.20 ± 0.17) under
control conditions to a left-skewed (-0.32 ± 0.10) after TEA application. This suggests a
significant increase of the release probability (P = 0.007, n =18, Fig. 8D).
22
Figure 8: TEA-induced block of Kv3 channels increases unitary IPSC amplitude by
raising release probability
A. Representative traces of a BC-GC paired recording (black: control, red: 1 mM TEA, green: washout). Upper traces show GC recording, lower traces the corresponding IPSCs recorded at the postsynaptic BC. The dashed horizontal line shows 100% of the IPSC amplitude under control conditions.
B. Amplitude-time relationship of a single paired recording. The time of application of 1 mM TEA is denoted by a black line. Few minutes after begin of TEA application a strong increase of the IPSC peak amplitude was notable. This effect was almost completely reversible during washout.
C - E. Summary graphs of the effect of TEA on peak amplitude (C), skewness (D) and coefficient of variation (CV, E). Black open boxes show date from individual experiments, red boxes the corresponding mean values. TEA significantly changed peak amplitude (P < 0.0001, n = 21), as well as skewness (P = 0.007, n = 18), and CV (P = 0.0003, n = 18) of the amplitude distritubtion.
Results
This is supported by the finding that the coefficient of variation (CV, for further information
on computation of CV see Appendix A), which indicates the dispersion of the IPSC
amplitude distribution, was changed. A low CV results from a narrow distribution and would
indicate a high synaptic release, since the maximal number of synaptic vesicle quanta are
released in most cases. Indeed, the CV was significantly reduced (P = 0.0003, n = 18, Fig.
8E) during TEA treatment, further suggesting that the presynaptic terminal reached its
maximal release probability.
During washout a partial recovery of skewness (0.07 ± 0.12, not significant, n = 5, Fig. 8D)
and full recovery of CV (0.35 ± 0.12, not significant, n = 5, Fig. 8E) were observed.
Taken together these results indicate that Kv3 channels are present at presynaptic
terminals. Furthermore, they are capable to regulate synaptic transmission by modulating
the release probability of synaptic vesicles.
TEA-induced block of Kv3 channels enhances multiple pulse depression
In order to examine dynamic changes of synaptic release, a train stimulation paradigm
(see methods section, “paired recordings”) was used which evoked 10 single APs at a rate
of 50 Hz in the BC. Again, the corresponding IPSCs were recorded at the postsynaptic
GC.
Under control conditions, the first evoked IPSCs showed a similar amplitude as reported in
the last paragraph (Fig. 8), demonstrating the intersweep interval of 10 s was long enough
to allow full recovery from synaptic depression (Fig. 9A). Looking at the IPSC train in a
single recorded trace, an increasing depression of IPSC amplitude from IPSC1 to IPSC10
was observed. This depression is typical for the BC-GC synapse and has been reported
previously (Kraushaar and Jonas, 2000). After TEA-application, the amplitude of the first
evoked IPSC was again augmented in the similar way as described for single evoked
IPSCs (see last paragraph), but the subsequent IPSCs showed only little increases.
To quantify this depression, an exponential fit was derived from IPSC1 (as described in the
methods section, “data analysis and statistics”, Appendix A and Kerr et al., 2008) and
subsequently subtracted from the entire trace (Fig. 9B). As a result, the amplitude of
IPSC2 could be evaluated. This procedure was repeated in order to assess all the single
IPSC amplitudes within train.
23
Results
24
Figure 9: TEA-induced block of Kv3 channels enhances multiple pulse
depression of IPSC amplitudes
A. Representative traces of a BC-GC paired recording upon presynaptic train stimulation. (black: control, red: 1 mM TEA, green: washout). Upper traces show a single GC recording, lower traces the corresponding average IPSCs recorded at the postsynaptic BC. GCs were stimulated by 10 single depolarizing current injections at a frequency of 50 Hz in order to evoke IPSC trains in the BC. The dashed horizontal line shows 100% of the IPSC amplitude under control conditions.
B. Representation of peak amplitude measurement of second and subsequent IPSCs after iterative subtraction of fitted decay phases of preceding IPSCs. An exponential fit of the preceding IPSC (e.g. IPSC1) was calculated and subtracted from the whole recorded waveform. By this means the amplitude of the IPSC (e.g. IPSC2) could be determined. This process was repeated until all IPSC amplitudes in the train were evaluated.
C. Summary plot showing multiple pulse depression (MPD) of IPSCs during a 50 Hz train of 10 APs (black: control, red: 1 mM TEA, green: washout). TEA increased MPD strongly significant (*** indicates P < 0.0001, n = 21).
Results
On average, the ratio of peak amplitudes for IPSC10 / IPSC1 was 0.28 ± 0.02 under
control conditions (Fig. 9C). The multiple pulse depression (MPD) of IPSC amplitudes
during TEA application was strongly enhanced with a heavily declining component
followed by a more sustained component. If TEA was applied, the ratio of peak amplitudes
for IPSC10 / IPSC1 significantly decreased to 0.13 ± 0.01 (P < 0.001, n = 21). This effect
was almost completely reversed during washout of TEA (0.23 ± 0.1, not significant, n = 2).
Thus, 1 mM TEA reversibly increased fast depression at the BC-GC synapse during trains
of APs, indicating Kv3 channels play a role in stabilizing synaptic release during high
frequency trains of somatic APs.
TEA-induced spike-broadening leads to recruitment of additional calcium channels
In the current view, evoked synaptic transmission at the BC-GC synapse is exclusively
mediated by P/Q-type Ca2+ channels (Hefft and Jonas, 2005), as in most fast-spiking
GABAergic interneurons (Poncer et al., 1997; Wilson et al., 2001; Zaitsev et al., 2007). Yet,
it is unclear whether other Ca2+ channels are expressed at low levels at the presynaptic
terminal and if they contribute to synaptic transmission. The brief presynaptic AP of BCs
might only selectively activate P/Q-type Ca2+ channels, because of their faster gating
(Wheeler et al., 1996; Li et al., 2007). Since the TEA-induced block of Kv3 channels leads
to a massive broadening of the AP, it was hypothesized that this stronger depolarization
could be effective enough to activate less sensitive Ca2+ channels than of P/Q-type.
In order to produce large depolarizations in the BCs presynaptic terminal and thus a strong
activation of voltage-gated Ca2+ channels, a train stimulation to evoke 10 APs at 50 Hz was
used. After TEA application, the peptide blockers ω-agatoxin IVa (0.5 µM and 1 µM), a
specific P/Q-type Ca2+ channels blocker, and 1 µM ω-conotoxin GVIa, also a highly
specific blocker of N-type Ca2+ channels, were subsequently added to the bath. Because
peptide-toxins can not be removed by washout (Sagvolden, 1999), no washout was
performed.
Because the train stimulation paradigm was the same as in the last paragraph, the
recordings under control conditions and in the presence of 1 mM TEA were similar
(Fig. 10A). Again, a clear multiple pulse depression within the IPSC train was recorded
under control conditions that increased after application of 1 mM TEA. The integral under
the IPSC train was evaluated and normalized to the control recording, since it is an
25
Results
indicator for total charge flow in current-time relationships. As expected, during application
of TEA, the integral calculated under the IPSC train increased significantly to
139.55 ± 14.80% (P = 0.008, n = 6, Fig. 10C). This indicates a higher total charge flow due
to the stronger depolarization caused by TEA-induced block of Kv3 channels.
After application of ω-agatoxin IVa, the IPSC amplitudes showed a fast decline to almost
baseline within four to eight minutes (Fig. 7B). Interestingly, the application of
ω-agatoxin IVa did not block all of the unitary IPSCs, but revealed a small residual
component (Fig. 10A). The average amplitude of the initial first IPSC within this component
was 7.6 ± 3.1 pA and differed significantly from that recorded under TEA conditions
(P < 0.001, n = 4). Likewise, the integral significantly decreased to 7.59 ± 1.28% (P = 0.01,
Mann-Whitney-Test, n = 5). Because the failure rate of triggering IPSCs increased
dramatically, at least 30 traces were necessary to calculate a representative average and
determine the IPSC characteristics.
With TEA and ω-agatoxin IVa continuously applied to the bath, 1 µM ω-conotoxin GVIa
was added. Again, a residual current component remained. The average amplitude of
IPSC1 was not significantly changed (9.4 ± 3.4 pA), whereas the integral was further, but
not significantly reduced (3.53 ± 2.67%). Notably, the failure rate of triggering IPSCs in
response to presynaptic APs increased further since to the recordings acquired during
ω-agatoxin IVa application (Fig.10A, lower panel right).
Because the IPSC train pattern differed strongly from that recorded under control
conditions and in the presence of TEA, the multiple pulse relationships were calculated.
The block of P/Q-type Ca2+ channels by ω-agatoxin IVa diminished the heavily declining
component and, more surprisingly, revealed a new slowly rising component in the IPSC
train (Fig. 10D). It emerged after IPSC6 and showed a strong facilitation. The strongest
mean facilitation (2.66 ± 0.55, P = 0.002, Mann-Whitney-Test, n = 4) was evaluated during
IPSC8/IPSC1. With co-application of ω-conotoxin GVIa, the facilitation was attenuated to
values around 1 but was not significantly different if compared to those calculated from the
experiments where only ω-agatoxin IVa was used. Only during the first and last single
IPSCs within the train a slight facilitation could still be monitored (Fig. 10D), e.g.
IPSC2/IPSC1 was 1.51 ± 0.46 (not significant, n = 4).
These results suggest that synaptic transmission at the BC-GC synapse is not only
26
Results
27
Figure 10: Release at the BC-GC synapse is not exclusively mediated by P/Q-type
Ca2+ channels
A. Typical traces of a BC-GC paired recording (black: control, red: 1 mM TEA, blue: 1 µM ω-agatoxin IVa, green: 1 µM ω-agatoxin IVa and 1 µM ω-conotoxin GVIa; see Fig. 6 for detailed paradigm). Upper traces show GC recording, lower traces the corresponding IPSCs recorded at the postsynaptic BC. In case of the ω-agatoxin IVa and ω-conotoxin GVIa recordings, the traces in grey boxes show a magnification of the corresponding recordings.
B. Amplitude-time relationship of a single paired recording (color coding equals Fig. 10A). Few minutes after application of 1 mM TEA a strong increase of the IPSC peak amplitude was notable. Additional treatment with 1 µM ω-agatoxin IVa reduced most of the IPSC amplitudes near baseline-values, but still some single events were notable. The same was observed if 1 µM ω-conotoxin GVIa was additionally added to the experiment.
C: Summary graph of the effect of TEA, ω-agatoxin IVa and ω-conotoxin GVIa on the integral of IPSCs (color coding equals Fig. 10A; empty boxes show single experiments, filled boxes the corresponding average integral). While the integral increased significantly under TEA conditions, additional treatment with ω-agatoxin IVa strongly reduced it (** represents P = 0.01, Mann-Whitney-Test). Subsequent addition of ω-conotoxin GVIa reduced it further, but not significantly.
D: Summary plot showing multiple pulse relationships of IPSCs during a 50 Hz train of 10 APs (black: control, red: 1 mM TEA, blue: 0.5 µM / 1 µM ω-agatoxin IVa green: ω-conotoxin GVIa). Application of TEA led to a depression whereas treatment with ω-agatoxin IVa and ω-conotoxin GVIa induced a facilitation.
All data were taken from two experiments treated with TEA, ω-agatoxin IVa and ω-agatoxin IVa / ω-conotoxin GVIa, two experiments treated with only TEA and ω-agatoxin IVa and two experiments treated with TEA and ω-agatoxin IVa / ω-conotoxin GVIa.
Results
restricted to P/Q-type Ca2+ channels, but also to N-type and probably R-type and/or L-type
Ca2+ channels. The alterations of the multiple pulse relationships during IPSC trains
suggest that different quantitative recruitment of these channels is capable to strongly
modify transmitter release. By that, the shape of a IPSC train can be changed from
depression to a facilitation, when additional Ca2+ channels are activated by long-lasting
depolarizations. These strong depolarizations can be achieved by a block of Kv3 channels,
suggesting these channels might contribute in the recruitment of different voltage-gated
Ca2+ channels.
28
Discussion
Discussion
In this study, the role of presynaptic Kv3 channels in synaptic transmission at the BG-GC
synapse was investigated. Simultaneous recordings of synaptically connected BCs and
GCs combined with pharmacological treatment gave new insights in the contribution of
presynaptic voltage-gated potassium channels and voltage-gated Ca2+ channels to
synaptic transmission. The results indicate first that Kv3 channels allow BCs to keep APs
short, which is essential for the fast-spiking phenotype of BCs. Second, block of Kv3
channels by TEA suggests that these channels help to stabilize synaptic transmitter
release during high-frequency trains of APs by lowering the release probability and thus
preventing synaptic depression. Finally, it was shown that synaptic transmission not only
relies on P/Q-type Ca2+ channels, but also N-type and probably R-type and/or L-type when
the presynaptic terminal is strongly depolarized. This suggests an expression of these
Ca2+ channels in the presynaptic terminal at low quantities.
Kv3 channels are involved in synaptic transmission at the BC-GC synapse
In cerebellar BCs, presynaptic Kv3 channels have been previously reported (Southan and
Robertson, 2000), but did not show an involvement in synaptic release (Tan and Llano,
1999; Southan and Robertson, 2000). In other synapses, such as the calyx of Held, a
high-fidelity synapse in the the auditory brainstem, featuring fast-spiking and rapid signal
transmission, the expression of presynaptic Kv3 channels is well-known (Ishikawa et al.,
2003; Nakamura and Takahashi, 2007), but their role in synaptic transmission remains
unresolved. This works shows that TEA broadens the somatic AP waveform by blocking
Kv3 channels (Laerum and Storm, 1994; Wang and Kaczmarek, 1998; Hlubek and
Cobbett, 2000; Ishikawa et al., 2003). This stronger depolarization might induce a
prolonged Ca2+-influx in presynaptic terminals of BCs and thus results in a higher rate of
transmitter release. Taken together, this work suggests a contribution of Kv3 channels on
synaptic transmission, whereas it remains still unclear if Kv3 channels are expressed in
axon and/or presynaptic terminals of hippocampal DG-BCs. Future studies can address
this question by focal application of TEA to different segments of the BCs axonal
arborization, beginning at the axon initial segment and ending up at the BC terminal.
Additional morphological information about the subcellular distribution of Kv3 channels can
be gathered by immunogold labeling of Kv3 channels as already done for Kv3.1 and Kv3.3
29
Discussion
in the rat cerebellum (Alonso-Espinaco et al., 2008; Puente et al., 2010).
Still there remain some caveats, especially concerning the antagonist TEA used during this
work. TEA is known as a reliable blocker for channels in the Kv3 subfamily (Rettig et al.,
1992; Grissmer et al., 1994; Martina et al., 1998; Erisir et al., 1999; Lien et al., 2002) , but
lacks some specificity. In the millimolar range, TEA antagonizes the low-threshold
potassium channel subunits Kv1.3 and Kv1.6 (Al-Sabi et al., 2010) whereas the
homomeric Kv1.1 channel is sensitive to TEA in even submillimolar concentrations
(Hopkins, 1998). Kv1 channels show a low activation threshold and thus prevent cells from
firing APs at low depolarizations. Antagonizing these channels results in a broadening of
the AP waveform in the axons, but not the soma (Kole et al., 2007). By this, the recorded
broadening of the somatic AP in BCs and thus the effects on synaptic release can be
addressed to some point to a block of Kv1 channels. However, it is unclear if members of
the Kv1 subfamily are expressed in DG-BCs. Kv1 channels have been shown being
present in the axon initial segment of neocortical fast-spiking interneurons, where they
selectively filter out small somatic depolarizations and thus only allow AP generation in
response to large inputs (Goldberg et al., 2008). Comparably, in PCs, expression is also
highest at the axon initial segment (Wang et al., 2004; Kole et al., 2007). The performed
somatic recordings in this work require further investigation, since no information is
gathered about the true axonal and presynaptic AP waveform, which can be heavily
modified in amplitude and duration while traveling towards the terminal (Kole et al., 2007).
Upon the application of TEA a significant increase of the somatic AP peak amplitude was
observed, suggesting the activation of Kv3 channels is fast enough to attenuate the
overshoot of presynaptic APs. This has been already observed in calyceal terminals of rats
(Ishikawa et al., 2003) but not mice (Wang and Kaczmarek, 1998).
It is also known that the repolarization phase of the AP in various cell-types is assisted by
large-conductance Ca2+-activated K+ (BK) channels (Storm, 1990; Johnston et al., 2000;
Hu et al., 2001). These channels also contribute to fast-spiking of APs in CA1 PCs (Ghatta
et al., 2006; Guan et al., 2007). BK channels are sensitive to TEA in the submillimolar
range (Barrett et al., 1982; Kang et al., 1996; Poolos and Johnston, 1999), but since now,
their expression pattern in BCs has not been investigated (Erisir et al., 1999). Furthermore,
a recent work suggested a different role of Kv3 and BK channels in deep cerebellar
nuclear neurons. Although BK and Kv3 channels share quite similar electrophysiological
30
Discussion
properties, it has been shown that BK channels were mainly responsible for the regulation
of spontaneous firing of APs, whereas Kv3 channels only controlled firing frequency
(Pedroarena, 2011).
Taken together, it is therefore unlikely, but can not be ruled out that at least a part of the
effects induced by the application of TEA can also be explained by a partial involvement of
Kv1 and BK channels. In order to eliminate the uncertainty similar experiments could be
repeated with knock-out (KO) mice. A suitable double-KO mouseline for Kv3.1 / Kv3.2 has
been reported previously (Ozaita et al., 2004).
Rapid synaptic signaling is a hallmark feature of fast-spiking PV-positive interneurons. In
BC-GC synapses the typical latencies (measured from steepest slope of the AP till the
beginning of the IPSC) are about 1 ms at near physiological properties (Kraushaar and
Jonas, 2000). Proximity of voltage-gated Ca2+ channels to the Ca2+ sensor and thus the
release machinery is critical for rapid and efficient signal transmission at high temporal
precision (Llinás et al., 1976; Stanley, 1993; Neher, 1998; Cao and Tsien, 2010). In the BC-
GC synapse, a small number of voltage-gated Ca2+ channels, which show a thight-
coupling to the Ca2+-binding sensors in the nanometer range, have been shown to be
effective enough in reliably triggering synaptic release (Bucurenciu et al., 2008; Müller et
al., 2010). In the current point of view this can be explained by two scenarios: First, few but
strongly conducting Ca2+ channels are activated or, second, a large number of Ca2+
channels are activated, which exhibit smaller individual conductances. If scenario two
would be right, the longer and stronger presynaptic depolarization induced by the block of
Kv3 channels would have been expected to recruit more of the hypothesized less
conducting channels. In turn, this would result in a higher total rate of transmitter
exocytosis and thus a stronger augmentation of the IPSC amplitudes. Since the
augmenting effect of TEA on IPSC amplitudes was quite moderate (Fig. 8), the first
scenario is more likely.
A block of Kv3 channels by bath application of TEA augmented the multiple pulse
depression (Fig. 9) by increasing release probability (Fig. 8) within a train of 10 single APs.
Most likely, this is due to a faster depletion of the releasable pool. In an alternative
explanation the longer presynaptic depolarization may produce a stronger inactivation of
voltage-gated Ca2+ channels, leading to a decrease in exocytosis rates in response to
following APs (Xu and Wu, 2005). Since transmitter recycling during endocytosis also
31
Discussion
depends on intracellular Ca2+ (Wu et al., 2005), it is possible that reduced Ca2+ transients
may also further promote multiple pulse depression. By this means, these results suggest
that presynaptic Kv3 channels prevent depression in high-frequency trains and therefore
stabilize synaptic transmission. On top of this positive effect, Kv3 channels are also useful
to minimize energy consumption of the brain caused by synaptic transmission. In
particular, this is important for fast-spiking interneurons like BCs since the vertebrate brain
uses a wide proportion of his energetic resources for the generation and transmission of
APs (Sengupta et al., 2010). Kv3 channels are extremely applicable for fast membrane
repolarization at a comparable low energetic cost (Alle et al., 2011). Thus, these channels
contribute to an energy-efficient but yet stable synaptic transmission at high presynaptic
firing rates.
TEA-induced block of Kv3 channels alters reliance of transmitter release on calcium
channel subtype
In DG-BCs, vesicle exocytosis is exclusively triggered by Ca2+ influx through P/Q-type Ca2+
channels (Hefft and Jonas, 2005). Antagonizing these channels with the selective blocker
ω-agatoxin IVa (0.5 µM or 1 µM) resulted in an almost complete loss of synaptic
transmission, with an integral computed from under the IPSC of 3.9 ± 11.6% (n = 3, from
Hefft and Jonas, 2005). Now, this work showed an integral of 7.59 ± 1.28% (n = 4) after
blocking P/Q-type Ca2+ channels by ω-agatoxin IVa, which was applied in the presence of
1 mM TEA. Furthermore, in 2 out of 4 cases, the concentration of ω-agatoxin IVa was
higher (1 µM) than the concentration used in Hefft and Jonas, 2005. By this, a further
reduction of the integral was expected due to the selectivity of ω-agatoxin IVa. In
conclusion the increased presynaptic depolarization caused by TEA was efficient enough
to activate a voltage-gated Ca2+ channel with slower gating kinetics. To identify the
responsible Ca2+ channels, 1 µM ω-conotoxin GVIa was additionally applied and revealed
that ~54% of the residual component was mediated by voltage-gated N-type Ca2+
channels. These channels show a decreased efficacy in being activated by APs in
comparison to P/Q-type Ca2+ channels (Li et al., 2007) which might explain the strongly
increased IPSC failure rate (Fig. 10A).
However, a further reduced residual current component remained and the responsible
channels were not identified during this work. It is possible that the residual current is
32
Discussion
mediated by R-type Ca2+ channels. These channels are known to be expressed in
terminals of CA1-PCs (Giessel and Sabatini, 2011) as well as in the calyx of Held (Wu et
al., 1998), respectively. They are known to play an essential role in fast glutamatergic
transmission in hippocampal CA3-PCs (Gasparini et al., 2001). R-type Ca2+ channels show
a lower efficacy in triggering IPSCs than N-type Ca2+ channels (Wu et al., 1998), which
could explain the further increase in the IPSC failure rate. Future studies could address
this possible contribution of R-type Ca2+ channels by additional application of SNX-482, a
selective antagonist of these channels (Li et al., 2007). Furthermore it is also thinkable that
L-type Ca2+ channels are involved, which usually play a key role in pacemaker activity e.g.
in dopaminergic neurons of the substancia nigra (Putzier et al., 2009; Guzman et al., 2010;
Li and Baccei, 2011), gene expression in hippocampal neurons (Bading et al., 1993;
Lipscombe et al., 2004; Tuckwell, 2012), and several pathological states, like bipolar
disorder (Casamassima et al., 2010). The role of N-type and probably R-type and/or L-type
Ca2+ channels in synaptic transmission at the BC-GC synapse therefore still remains to be
investigated.
Up to now there was no evidence that synaptic transmission was mediated by other
channels than of P/Q-type in fast-spiking GABAergic interneurons (Hefft and Jonas, 2005;
Zaitsev et al., 2007). P/Q-type Ca2+ channels can be activated by short APs, as has been
shown in BCs, preserving synaptic transmission even at high firing rates (Li et al., 2007).
Longer lasting presynaptic depolarizations, in this work induced by the application of TEA,
were capable to activate further types of Ca2+ channels. Having a closer look to the
residual current after co-application of TEA, ω-agatoxin IVa and ω-conotoxin GVIa, a
mayor difference was the change from a depressing to a facilitating multiple pulse
relationship. It was was more profound after application of ω-agatoxin IVa, but still
observed when ω-conotoxin GVIa was additionally applied (Fig. 10). Interestingly, N-type-
mediated synaptic transmission is know to resemble a depressing multiple pulse
relationship (Giugovaz-Tropper et al., 2011). This further supports the hypothesis that
N-type Ca2+channels play a role in synaptic transmission at the BC-GC synapse. Previous
findings suggested that most or all synaptic terminals expressing P/Q-type Ca2+ channels
may also express N-type Ca2+ channels, as has been shown in cultured mouse
hippocampal neurons (Cao and Tsien, 2010). In P/Q-type Ca2+ channel KO mice, N-type
Ca2+ channels take the dominant role in triggering transmitter release in the calyx of Held
33
Discussion
(Jun et al., 1999; Ishikawa et al., 2005; Giugovaz-Tropper et al., 2011), hippocampal
neurons (Jun et al., 1999), and neuromuscular junctions (Urbano et al., 2003). lt is also
known that the expression of P/Q-type Ca2+ channels dramatically increases during
development (e.g. for the rat calyx of Held between P8 and P13), while synaptic
transmission before this time-point mainly relies on N-type Ca2+ channels (Iwasaki and
Takahashi, 1998; Rosato Siri and Uchitel, 1999; Iwasaki et al., 2000; Urbano et al., 2002;
Fedchyshyn and Wang, 2005). In contrast, these channels play no or only a minor role in
synaptic transmission in adult animals. It is possible that after treatment with the peptide-
toxins some of the Ca2+ channels preserved during ontogenesis in BCs were uncovered.
During pre- and early postnatal development, the multiple pulse facilitation of IPSCs
caused by these channels may assist synaptic maturation, strengthening and outgrowth
since GABAergic transmission is excitatory before P12 in rats (Ben-Ari, 2002; Akerman
and Cline, 2007; Rheims et al., 2008). Excitatory transmission is known to be essential for
synaptic maturation during ontogenesis (Sutor and Luhmann, 1995). A recent paper further
demonstrated that PV-expressing fast-spiking interneurons in the juvenile neocortex of
mice are able to form interconnected networks at the end of the first postnatal week
(Pangratz-Fuehrer and Hestrin, 2011). This suggests that the expression of Ca2+ channels
other than of P/Q-type in hippocampal BCs may support synapse formation in the DG.
Contrariwise, it can be hypothesized that the presence of these Ca2+ channels at the BC-
GC synapse may serve another reason in the mature brain. Since the IPSC waveform
showed strong differences between P/Q-type and N-type mediation it can be assumed that
this an unknown mechanism of BCs switching the output between a depression to a
facilitation. A stronger presynaptic depolarization could be induced by neuromodulators,
such as hormones (Mora et al., 2012), substances like histamine that are released in
response to inflammations (Ellender et al., 2011) or modulators applied via ingestion, like
caffeine (Simons et al., 2012). Because the N-type mediated transmission recorded in this
work contributed only by few percent to the total synaptic transmission, the P/Q-type Ca2+
channels would remain to be silenced. Up to now there is no hint such a synaptic
mechanism exists, rendering this hypothesis highly unlikely.
So if N-type, and R-type or L-type Ca2+ channels are dormant or able being activated
without artificial manipulations is still an ongoing question. Its answer could give new
insights in the mechanisms of synaptic transmission and information processing.
34
Abbreviations
Abbreviations
ABC: avidin-biotinylated horseradish peroxidase complex
AC: associational commissural pathway
ACSF: artificial cerebrospinal fluid
4-AP: 4-aminopyridine
AMPA: α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid
AP: action potential
BC: basket cell
BK channel: large-conductance Ca2+-activated K+ channels
BSA: bovine serum albumin
CA: cornu ammonis
CCK: cholecystokinin
CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione
CV: coefficient of variation
D-AP V: D-2-amino-5-phosphonopentanoic acid
DG: dentate gyrus
EC: entorhinal cortex
GABA: γ-aminobutyric acid
GC: granule cell
HIPP cell: Hilar perforant path-associated cell
IML: inner molecular layer
IPSC: inhibitory postsynaptic current
IR-DIC: infrared differential interference contrast videomicroscopy
KCl1: potassium-rich solution
KGluc: potassium-gluconate-rich solution
35
Abbreviations
KO: knock-out
MC: mossy cell
ML: molecular layer
MOPP cell: molecular layer perforant path-associated
MPD: multiple pulse depression
OML: outer molecular layer
PB: phosphate buffer
PC: pyramidal cell
PP: perforant path
PV: parvalbumin
QX-314: N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide
Rs: series resistance
SC: Schaffer collateral
SEM: standard error of the mean
TEA: tetraethylammonium
Vrest: resting membrane potential
36
References
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Acknowledgments
Acknowledgments
First of all I would like to express my gratitude to Prof. Dr. Peter Jonas for giving me the
opportunity to do my PhD in his group, his patience and great encouragement in my thesis
and all the countless hours providing me help with experimental designs and data analysis
even after the institute moved to the IST in Klosterneuburg / Austria. I learned more about
electrophysiology I could ever imagine and a lot of skills my future career will benefit from.
I would also like to thank Prof. Dr. Josef Bischofberger and Prof. Dr. Marlene Bartos for the
discussions and helpful suggestions during our lab seminars.
I thank Dr. Klaus Haverkampf for his assistance if anything on the microscopy or setup
was broken or needed adjustment. I would also like to thank Hans-Joachim Weber and the
people from the workshop for immediate repairs of broken recording chambers etc.
I thank Dr. Yexica Aponte for her friendship and for introducing me into the depths of patch-
clamp recordings and BC identification.
Furthermore, I would like to thank Selma Becherer, Margit Northemann, Ulrike Thirimanna
and Karin Winterhalter and especially Lilia Dumont and Ina Koeva-Slancheva for preparing
the solutions, helping out with ordering chemicals and all the other helpful things they
provided.
I also would like to thank Dr. Anja Matthiä for her friendship and helping me out with some
tricky Mathematica files.
Therefore I also thank my friends in the lab for the great working atmosphere and all the
help they provided when anything was necessary.
I thank the people from the Neuroscience Graduate School in Freiburg for organizing all
the interesting talks and social events.
I would like to thank my family and friends especially for their love and support beside the
scientific parts of life.
Finally, I want to thank my beloved partner Alexandra for her help even in scientific
questions, but mostly for all the love and the good time we had together here in Freiburg.
I'm looking forward to our new life in Ulm!
55
Curriculum Vitae
Curriculum Vitae
Name: Marcus Illy
Nationality: German
Date of birth: April 23, 1980 in Speyer am Rhein / Germany
Education
2007 – present: PhD-student in the laboratory of Prof. Dr. Peter Jonas, Institute of
Physiology I at the University of Freiburg, Germany.
Supervisor: Prof. Dr. Peter Jonas.
2007 – 2010: Member of the Graduiertenkolleg 843 at the University of Freiburg,
Germany
2006 – 2007: Diploma thesis in the laboratory of Prof. Dr. Eckhard Friauf,
Department of animalphysiology at the TU Kaiserslautern, Germany,
entitled Optical imaging with voltage-sensitive dyes reveals delay
lines in the chick nucleus laminaris.
Supervisor: Dr. Stefan Löhrke
2001 – 2006: Studies in Biology at the TU Kaiserslautern
2000: Abitur at the Käthe-Kollwitz-Gymnasium in Neustadt a. d. Weinstraße,
Germany
56
Appendix A – Mathematica notebooks
Appendix A – Mathematica notebooks
Calculation of multiple pulse relationships
Evaluation MPRFitting;
modified after Angharad Kerr Evaluation MPDFitting
Analysis of depression by fitting and subtraction of exponentialsVersion 2File: PPDFittingPJ and AKmodified by AN and MI
Manual:1.) Put following information into red marked parts:- Import data: pathname, filename, sample interval- Sort data: last sweep of control-phase, last sweep of drug-phase, last sweep of washout-phase, sweeps to delete, testrange (e.g. Control ->controlBegin, ControlEnd)
Choose from the blue fields the corresponding delete-range and put them into the grey code- Build average: put delete-ranges into grey code- Analysis: check fittings; if necessary, change fitrange, amplitude and/or tau
Initialise
Clear[filename, sweepAPs, sweepPSC]<< Statistics`NonlinearFit`;(* for nonlinear least squares fit*) Statistics`ContinuousDistributions`;(* for mean*)<< Graphics`Graphics`Off[General::spell]Off[General::spell1]
ink1 = Install["C:/UserIgor/UserC/read3new/read3.exe"];
(* link2 = Install["d:/user/C/sincha/debug/sincha"] *)
f[t_] := a Exp[-t/ \[Τ]];...fitAndSubtract[av_, totalIPSC_, varForFit_] := Module[{}, ampIPSCs = Table[0, {totalIPSC}]; tPeaks = Table[0, {totalIPSC}]; tStart = (startTrain)/sampleInt; tEnd = (startTrain + trainInt)/sampleInt; ampIPSCs[[1]] = Min[Take[av, {tStart, tEnd}]]; tPeaks[[1]] = First[First[Position[av, ampIPSCs[[1]]]]]; avCut = Drop[av, tPeaks[[1]]]; For[i = 2 , i <= totalIPSC, i = i + 1, fitResult = BestFitParameters /.NonlinearRegress[Take[avCut, Round[varForFit[[i]][[1]]/sampleInt]], …..f[x], x, {{a, varForFit[[i]][[2]]}, {\[Tau], varForFit[[i]][[3]]}}, RegressionReport ->BestFitParameters];Print["IPSC", i - 1, ": ", fitResult];gr1 = ListPlot[avCut, PlotStyle -> PointSize[0.008], PlotRange -> All, PlotLabel -> "IPSC " <>
ToString[i – 1], DisplayFunction -> Identity];gr2 = Plot[f[x] /. fitResult, {x, 0, 2000}, PlotStyle -> RGBColor[1, 0, 0], PlotRange -> All, PlotLabel -> "IPSC " <> ToString[i - 1], DisplayFunction -> Identity];
57
Appendix A – Mathematica notebooks
Show[gr1, gr2, PlotRange -> {{-50, (startTrain + totalIPSC*trainInt) / sampleInt(*1100*) }, {ampIPSCs[[i - 1]] - 100, 50}}, PlotLabel -> "IPSC " <> ToString[i - 1], DisplayFunction -> …..$DisplayFunction];
g[x_] := f[x] /. fitResult;avCut = avCut - g /@ Table[j, {j, 1, Length[avCut]}];ampIPSCs[[i]] = Min[Take[avCut, trainInt/sampleInt + 10/sampleInt]];Print[ampIPSCs[[i]]];Show[ListPlot[Take[avCut, trainInt/sampleInt + 10/sampleInt], DisplayFunction -> Identity], Graphics[ {RGBColor[0, 1, 0], Line[{{trainInt/sampleInt - 10/sampleInt, ampIPSCs[[i]]}, {trainInt/sampleInt + 500, ampIPSCs[[i]]}}]}], PlotRange -> All, DisplayFunction -> $DisplayFunction];tPeaks[[i]] = First[First[Position[avCut, ampIPSCs[[i]]]]];avCut = Drop[avCut, tPeaks[[i]]]; (*tPeaks = train interval + transmission latency*)];Print[tPeaks ];ampIPSCs];
Import data
pathName = "D:\\path\\to\\file\\";fileToAnalyze = "recording";fileName = pathName <> fileToAnalyze <> ".dat";
info = ReadCfsInfo[pathName <> fileToAnalyze <> ".dat", -1];
sampleInt = 0.02; (*in ms*)trainInt = 20 (*in ms*);startTrain = 100;(*in ms*)
voltageTrace[x_] := ReadCfsSections[fileName, x, x, 0, 0, Range[0, ReadCfsSectionPoints[fileName, x, x]]];currentTrace[x_] := ReadCfsSections[fileName, x, x, 0, 1, Range[0, ReadCfsSectionPoints[fileName, x, x]]];
ListPlot[voltageTrace[1], PlotRange -> All];ListPlot[currentTrace[1], PlotRange -> All];
posFirst = StringPosition[info, "Last sct: "];posLast = StringPosition[info, "Total pts: "];
nrSectionStr = StringTake[info, {Flatten[posFirst][[2]] + 1, Flatten[posLast][[1]] - 1}];
totalSweeps = ToExpression[nrSectionStr]
Sort data
controlEnd = 70; (* put last trace of control phase *)
teaEnd = 139; (* put last trace of TEA phase *)
deleteRangeControl = {};deleteRangeTea = {};deleteRangeWashout = {};toDeleteControl = deleteRangeControl/2;toDeleteTea = deleteRangeTea - controlEnd/2;toDeleteWashout = deleteRangeWashout - teaEnd/2;controlBegin = 2; (*"1" for normal protocol, "2" for alternating protocol*)teaBegin = controlEnd + 1;
58
Appendix A – Mathematica notebooks
washoutBegin = teaEnd + 1;washoutEnd = totalSweeps;testRange = Range[controlBegin, controlEnd, 2]; (*put "2" for every second sweep, if alternating protocol was used*)
Length[Delete[testRange, toDeleteControl]]ListPlot[currentTrace[#], PlotRange -> All, PlotLabel -> #] & /@Delete[testRange, toDeleteControl];
Build average
av1original = Mean[currentTrace[#] & /@Delete[testRange, toDeleteControl]];(*all traces*)av1 = Drop[av1original - Mean[Take[av1original, 2000]], -2000];
av2original = Mean[currentTrace[#] & /@Take[Delete[testRange, toDeleteControl], -10]];(*last 10 traces*)av2 = Drop[av2original - Mean[Take[av2original, 2000]], -2000];
av3original = Mean[currentTrace[#] & /@Take[Delete[testRange, toDeleteControl], -5]];(*last 5 traces*)
av3 = Drop[av3original - Mean[Take[av3original, 2000]], -2000];
ListPlot[av1, PlotRange -> All];grTest = ListPlot[av2, PlotRange -> All];ListPlot[av3, PlotJoined -> True, PlotRange -> All];Show[Graphics[{RGBColor[0, 1, 0], Line[{{0, -800}, {500, -800}}]}], PlotRange -> All]
Analysis
Fitting of traces(*fitrange [ms], amplitude [pA], tau [ms]*)variables = {{15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}, {15, -900, 200}};resultAv2 = fitAndSubtract[av2, 10, variables]
Summary
{resultAv2}#[[10]]/#[[1]] & /@ {resultAv2}
grAv2 = ListPlot [Transpose[{Range[10], resultAv2}], PlotStyle -> {RGBColor[1, 0, 0], PointSize[0.02]}, Axes -> True, AxesOrigin -> {0, 0}, TextStyle -> {FontFamily -> "Arial", FontSize -> 12}, PlotRange -> {All, All}];
Show[grAv1, grAv2, grAv3, Axes -> True, AxesOrigin -> {0, 0}, TextStyle -> {FontFamily -> "Arial", FontSize -> 12}, PlotRange -> {All, All}];
result = Transpose[{Flatten[{fileToAnalyze, Range[10]}], Flatten[{"last_10", resultAv2 }] // MatrixForm
SetDirectory["D:\\path\\to\\export\\directory"];
Export["output.txt", result, "TSV"]
59
Appendix A – Mathematica notebooks
Calculating skewness and CV
Initialise
Clear[filename, sweepAPs, sweepPSC]
<<Statistics`NonlinearFit`;(* for nonlinear least squares fit*)<<Statistics`DescriptiveStatistics`; (* for skewness *)<<Statistics`ContinuousDistributions`;(* for mean*)<<Graphics`Graphics`Off[General::spell]Off[General::spell1]
link1=Install["c:/UserIgor/UserC/read3new/read3.exe"];
dummy = Table[0, {40000}];
Import data
path = "D:\\path\\to\\file\\\"; file = "recording ";
info=ReadCfsInfo[path<>file<>".dat",-1]
sampleInt=ReadCfsSmpInt[path<>file<>".dat"]*1000;
trainInt=20 (* in ms *);startTrain=400 (* in ms *);
voltageTrace[x_]:=ReadCfsSections[path<>file<>".dat",x,x,0,0,Range[0,ReadCfsSectionPoints[path<>file<>".dat",x,x]]];
currentTrace[x_]:=ReadCfsSections[path<>file<>".dat",x,x,0,1,Range[0,ReadCfsSectionPoints[path<>file<>".dat",x,x]]];
ListPlot[voltageTrace[1],PlotRange->All];ListPlot[currentTrace[1],PlotRange->All];
posFirst=StringPosition[info,"Last sct: "];posLast=StringPosition[info,"Total pts: "];
nrSectionStr=StringTake[info,{Flatten[posFirst][[2]]+1,Flatten[posLast][[1]]-1}];
totalSweeps=ToExpression[nrSectionStr]
Data Sorting
rawDeleteRange=Sort[Flatten[{Range[61,82],Range[131,220]}]]; (* sweep number *)controlEnd=60; (* sweep number *)teaEnd=130; (* sweep number; must be even - see Peak Detection *)controlBegin=1; (* sweep number *)teaBegin=controlEnd+1; (* sweep number *)washoutBegin=controlEnd+1; (* sweep number *)washoutEnd=totalSweeps; (* sweep number *)
allSweeps=Range[controlBegin,totalSweeps];toAnalyze=Delete[allSweeps,Flatten[Position[allSweeps,#]&/@rawDeleteRange,1]];
60
Appendix A – Mathematica notebooks
deleteControl=Length[Select[rawDeleteRange,#<=controlEnd&]];deleteTea=Length[Select[rawDeleteRange,controlEnd<#<=teaEnd&]];deleteWashout=Length[Select[rawDeleteRange,teaEnd<#<=washoutEnd&]];
Length[rawDeleteRange]==deleteControl+deleteTea (* must be TRUE if NO WASHOUT was performed! *)
Length[rawDeleteRange]==deleteControl+deleteTea+deleteWashout (* must be TRUE if washout was performed, OTHERWISE remove deleteWashout at Peak Detection! *)
Analysis
Subtract baseline
base=Mean[Take[currentTrace[#], 2000]]&/@toAnalyze
subBaseCurrTrace[x_]:=currentTrace[x] - Mean[Take[currentTrace[x], 2000]];ListPlot[subBaseCurrTrace[#], PlotRange -> All, PlotLabel -> #] &/@ toAnalyze;
Peak Detection
peaks=Min[Take[subBaseCurrTrace[#],{5000,5500}]]&/@toAnalyze;
peaksInverted=peaks*(-1);
peakControl=Take[peaksInverted,{1,controlEnd-deleteControl}];peakTEA=Take[peaksInverted,{controlEnd+1-deleteControl,teaEnd-deleteControl-deleteTea}];peakWashout=Take[peaksInverted,{teaEnd+1-deleteControl-deleteTea,IntegerPart[washoutEnd- deleteControl-deleteTea-deleteWashout]}];
Length[peaksInverted]==Length[peakControl]+Length[peakTEA]+Length[peakWashout]
SkewnessskewControl=Skewness[peakControl]skewTEA=Skewness[peakTEA]skewWashout=Skewness[peakWashout]
Coefficient of Variation
CoefficientOfVariation[peakControl]CoefficientOfVariation[peakTEA]CoefficientOfVariation[peakWashout]
61
Appendix B – Selected parameters of individual cells
Appendix B – Selected parameters of individual cells
62
Table 1: Effect of bath application of 1 mM TEA on half-duration and rise-time (20%-80%).
Parameter n = 21control washout
single mean SEM single mean SEM single mean SEMBC 01 0,71
0,91 0,07
1,10
1,15 0,07
0,93
1,03 0,08
BC 02 1,21 1,39BC 03 1,14 1,28 1,30BC 04 0,87 1,24BC 05 1,29 1,44 1,38BC 06 0,52 0,93BC 07 0,51 0,64 0,53BC 08 0,46 0,58BC 09 0,66 0,91BC 10 0,75 1,18BC 11 0,90 1,01BC 12 0,74 1,22BC 13 0,70 0,91BC 14 0,79 0,87BC 15 1,44 1,73BC 16 1,24 1,47BC 17 1,62 1,69BC 18 0,88 1,25BC 19 1,12 1,39BC 20 0,85 1,07BC 21 0,63 0,83
P < 0,0001BC 01 0,26
0,64 0,07
0,31
0,61 0,06
0,50
0,64 0,07
BC 02 0,96 0,95BC 03 0,92 0,97 0,98BC 04 0,73 0,95BC 05 0,80 0,78 0,80BC 06 0,42 0,55BC 07 0,27 0,30 0,27BC 08 0,20 0,18BC 09 0,27 0,25BC 10 0,48 0,40BC 11 0,70 0,48BC 12 0,39 0,44BC 13 0,33 0,29BC 14 0,72 0,51BC 15 0,60 0,67BC 16 0,95 0,93BC 17 1,30 0,96BC 18 1,24 1,21BC 19 0,65 0,57BC 20 0,83 0,59BC 21 0,48 0,47
P = 0,3438
1 mM TEA
half-duration
(ms)
rise-time 20%-80%
(ms)
Appendix B – Selected parameters of individual cells
63
Table 2: Effect of bath application of 1 mM TEA on peak amplitude and max. rate of rise.
Parameter n = 21control 1 mM TEA washout
single mean SEM single mean SEM single mean SEMBC 01 110,6
116,4 2,1
114,5
120,3 2,4
123,2
127,2 2,6
BC 02 130,1 139,0BC 03 132,6 136,3 137,9BC 04 116,2 131,5BC 05 125,5 133,7 135,8BC 06 105,9 115,2BC 07 111,9 114,0 111,8BC 08 111,5 108,7BC 09 116,3 117,2BC 10 116,0 116,8BC 11 115,5 109,6BC 12 109,5 115,0BC 13 110,9 113,2BC 14 107,1 107,0BC 15 113,7 127,8BC 16 129,8 134,1BC 17 139,7 139,1BC 18 113,7 121,5BC 19 115,5 118,0BC 20 112,7 108,4BC 21 100,7 105,4
P = 0,0066BC 01 411,7
314,3 22,2
398,1
321,4 22,3
346,7
310,2 25,0
BC 02 211,6 196,9BC 03 241,2 235,0 232,7BC 04 302,9 323,5BC 05 204,7 200,1 205,4BC 06 434,7 390,4BC 07 446,2 451,4 455,8BC 08 530,4 524,0BC 09 440,0 457,4BC 10 348,3 362,8BC 11 306,5 333,4BC 12 355,6 339,0BC 13 359,4 378,8BC 14 286,6 375,7BC 15 186,4 186,0BC 16 200,9 192,9BC 17 149,1 183,8BC 18 238,6 209,0BC 19 268,2 281,2BC 20 300,5 317,9BC 21 376,8 412,7
P = 0,2777
peak amplitude
(mV)
max. rate of rise (V/s)
Appendix B – Selected parameters of individual cells
64
Table 3: Effect of bath application of 1 mM TEA on IPSC1 amplitude and skewness.
Parameter Cellcontrol 1 mM TEA washout
single mean SEM single mean SEM single mean SEMBC 01 1237,4
539,4 76,7
1840,3
797,8 112,4
1077,6
526,1 117,4
BC 02 295,4 477,9BC 03 831,8 1399,6 1150,0BC 04 283,7 340,3BC 05 422,1 542,5 104,94BC 06 601,1 752,5BC 07 151,3 341,1 187,0BC 08 534,8 751,0BC 09 621,7 971,9BC 10 1240,0 1431,5BC 11 501,7 621,8BC 12 479,9 792,2BC 13 388,0 470,1BC 14 1062,5 1539,4BC 15 173,5 322,5BC 16 251,5 465,4BC 17 385,1 529,7BC 18 835,7 1185,8BC 19 836,0 1667,9BC 20 85,2 133,2BC 22 109,0 176,9 110,97
n = 21 P < 0,0001
skewness
BC 01 -0,35
0,20 0,16
-0,55
-0,32 0,09
0,01
0,07 0,06
BC 02 0,73 -0,16BC 03 0,44 -0,24 0,35BC 04 -0,46 -0,10BC 05 0,26 -0,19 0,29BC 06 0,10 -0,54BC 07 -0,08 0,08 0,00BC 08 0,19 -0,75BC 09 0,10 -0,07BC 10 -0,14 -0,17BC 11 -0,29 0,48BC 12 -0,36 -0,47BC 13 -0,21 -0,63BC 14 -0,25 -0,73BC 15 0,24 0,00BC 16 -0,04 -1,46BC 17 1,07 -0,42BC 22 2,65 0,12 -0,29
n = 18 P = 0,0068
amplitude IPSC1 (pA)