The calyx of Held

Abstract

The calyx of Held is a large glutamatergic synapse in the mammalian auditory brainstem. By using brain slice preparations, direct patch-clamp recordings can be made from the nerve terminal and its postsynaptic target (principal neurons of the medial nucleus of the trapezoid body). Over the last decade, this preparation has been increasingly employed to investigate basic presynaptic mechanisms of transmission in the central nervous system. We review here the background to this preparation and summarise key findings concerning voltage-gated ion channels of the nerve terminal and the ionic mechanisms involved in exocytosis and modulation of transmitter release. The accessibility of this giant terminal has also permitted Ca(2+)-imaging and -uncaging studies combined with electrophysiological recording and capacitance measurements of exocytosis. Together, these studies convey the panopoly of presynaptic regulatory processes underlying the regulation of transmitter release, its modulatory control and short-term plasticity within one identified synaptic terminal.

Full text

REVIEW
The calyx of Held
Ralf Schneggenburger & Ian D. Forsythe
Received: 6 April 2006 /Accepted: 7 June 2006 / Published online: 8 August 2006
#
Springer-Verlag 2006
Abstract The calyx of Held is a large glutamatergic
synapse in the mammalian auditory brainstem. By using
brain slice preparations, direct patch-clamp recordings can
be made from the nerve terminal and its postsynaptic target
(principal neurons of the medial nucleus of the trapezoid
body). Over the last decade, this preparation has been
increasingly employed to investigate basic presynaptic
mechanisms of transmission in the central nervous system.
We review here the background to this preparation and
summarise key findings conc erning voltage-gated io n
channels of the nerve terminal and the ionic mechanisms
involved in exocytosis and modulation of transmitter
release. The accessibility of this giant terminal has also
permitted Ca
2+
-imaging and -uncaging studies combined
with electrophysiological recording and capacitance mea-
surements of exocytosis. Together, these studies convey the
panopoly of presynaptic regulatory processes underlying
the regulation of transmitter release, its modulatory control
and short-term plasticity within one identified synaptic
terminal.
Keywords Calyx of Held
.
Synaptic transmission
.
Plasticity
.
Synaptic vesicle
.
Exocytosis
.
Voltage-gated
channel
.
Modulation
.
Metabotropic receptor
A short history of the calyx of Held
The size of a synapse is a significant technical constraint for
electrophysiological recording. The large dimensions of
some invertebrate synapses have been exploited to provide
considerable insight into presynaptic function (Llinas et al.
1972; Augustine et al. 1985; Young and Keynes 2005).
However, the progress of similar studies in vertebrates was
long hampered by the technical difficulty of presynaptic
recording from small nerve terminals. Over the last 50 years,
a range of preparations have contributed to our understand-
ing of presynaptic mechanisms, from the neuromuscular
junction (Katz 1969) to chromaffin cells (Neher and Marty
1982), chick ciliary ganglion (Martin and Pilar 1963;
Stanley and Goping 1991), neurohypophysial nerve termi-
nals (Lemos and Nordmann 1986; Jackson et al. 1991) and
synaptosomal preparations (Nicholls and Sihra 1986), to
name just a few. Given the predominance of fast gluta-
matergic synapses in the mammalian central nervous
system (CNS) and their pivotal role in in formation
processing, the development of a preparation at which
direct presynaptic patch-clamp recordings were feasible and
at which glutamate was the neurotransmitter was desirable.
One candidate was the hippocampal mossy fibre terminal,
from which direct recordings have indeed been achieved
(Geiger and Jonas 2000). Another approach has made use
of anatomical evidence for two giant synapses in the
auditory pathway, the endbulbs of Hel d and calyces of
Held, respectively.
Both synapses are glutamatergic and form part of the
relay pathway subserving sound-source localisation in
the aud itory brains te m (Fig. 1a). They owe their name to
the German anatomist Hans Held, who working in Leipzig
in the late 19th century, studied the central auditory
pathways by the Golgi staining technique in cats (Held
Cell Tissue Res (2006) 326:311–337
DOI 10.1007/s00441-006-0272-7
R. Schneggenburger (*)
Laboratory of Synaptic Mechanisms,
École Polytechnique Fédérale de Lausanne (EPFL),
Brain Mind Institute,
Bâtiment AAB, Station 15,
CH-1015 Lausanne, Switzerland
e-mail: ralf.schneggenburger@epfl.ch
I. D. Forsythe
Cellular & Molecular Neuroscience Group,
MRC Toxicology Unit, University of Leicester,
Leicester LE1 9HN, UK

1893). Ramón y Cajal intensively studied these giant
synapses by using Golgi material from several species
(Ramón y Cajal 1972) and rendered the first high-
resolution images of calyces of Held at the light-
microscop ic leve l. Held also g ave his na me to an oth er
auditory giant synapse called the endbulb of Held, which
is the primary afferent synapse onto the bushy cells of the
anterior ventral cochlear nucleus (aVCN; Lorente de No
1981). Physiological characterisation and intra-axonal
recording from axons in the trapezoid body (Fig. 1)of
cats showed bushy cell projections to several ip si lat er al
and contralateral nuclei, with all axons giving rise to one
or rarely two calyces in the contralateral medial nucleus of
the trape zoid body (M NTB; Spirou et a l. 1990;Kuwabara
et al. 1991; Smith et al. 1991). Immunohistochemical
evidence first suggested that the calyx of Held could be
glutamatergic (Grandes and Streit 1989) and this was
confirmed by in vitro current-clamp recordings in brain slices
showing block of excitatory inputs by the glutamate
receptor antagonist DNQX (Banks and Smith 1 992).
Whole-cell voltage-clamp from MNTB neurons showed dual
component excitatory postsynaptic currents (EPSCs) with a
fast time-course component mediated by α-amino-3-5-meth-
yl-4-isoxazolepropionic acid (AMPA) receptors and a slower
time-course N-methyl-D-aspartate (NMDA) receptor-mediat-
ed current (Forsythe and Barnes-Davies 1993).
Direct recordings from the calyx of Held presynaptic
terminal were subsequently achieved by using patch-clamp
methods in an in vitro slice preparatio n of the rat brainstem
(Forsythe 1994; Borst et al. 1995). Presynaptic recording
from the endbulb of Held synapses in the aVCN has proven
technically more difficult and, so far, has only been
achieved in the chick (Sivaramak ri shna n a nd Laurent
1995). The access ibility of the calyx of Held has since
been used to investigate presynaptic ion channels, Ca
2+
influx, transmitter release and its short-term modulation
under direct voltage-clamp control of the presynaptic
terminal. The calyx of Held has also become a model
Fig. 1 The calyx of Held synapse in the auditory brainstem circuit.
a Representation in the coronal plane of the brainstem auditory
pathway and the calyx of Held synapse, which forms part of the
auditory circuit at the level of the superior olivary complex (SOC).
The calyx of Held is an excitatory glutamatergic synapse arising
from globular bushy cells in the anterior ventral cochlear nucleus
(aVCN) onto a principal cell in the medial nucleus of the trapezoid
body (MNTB). The principal cells provide an inhibitory projection
to other nuclei of the SOC such as the lateral superior olive (LSO).
The bushy cells in the aVCN receive excitatory input from the
auditory nerve fibres. The calyx of Held is thus a tertiary auditory
synapse that rapidly relays information, providing the LSO and
other nuclei with (inhibitory) information with regard to sound
arriving at the contralateral ear. b Representation of a single calyx of
Held synapse onto a given single MNTB principal cell (modified,
with permission from Elsevier, from Walmsley et al. 1998). The
MNTB principal c ells receive additional inhibitory and excitatory
input through small bouton-like synapses but, in most c ases, a given
MNTB principal cell is thought to receive input from only one large
calyx of Held. Thus, a one-to-one synapt ic relati onsh ip exists
between a given globular bushy cell and an MNTB principal cell. c
Development of afferent fibres originating from the aVCN (r eprin-
ted, with permission of Wiley-Liss, from Kandler and Friauf 1993).
The first fibres cross the midline approximately by embryonic day
15 (E15) and, at postnatal day 3 (P3), large calyceal synapses are
formed and the one-to-one synaptic relationship is established. Later
in development (P14), characteristic changes occur in the morphol-
ogy of the calyx. (Data in this and subsequent figures are f rom rat)
312 Cell Tissue Res (2006) 326:311–337

system for studying developmental changes of presynaptic
function prior to hearing “onset” when the auditory canal
opens (this occurs at around postnatal day 11 (P11) or P12
in rats and mice; Blatchley et al. 1987; Geal-Dor et al.
1993) and on towa rds ma turation at around 20 days
postnatally. The direct access of the nerve terminal to
patch-clamp recording allows the manipulation of the
intracellular biochemical environment, the introduction of
Ca
2+
indicators and light-sensitive Ca
2+
chelators and the
manipulation of the presynaptic Ca
2+
concentration. In this
review, we will briefly introduce the structure of the calyx
of Held and then describe those research fields of synaptic
transmission in which work on the calyx of Held has made
important advances. Finally, we will discuss the advan-
tages, limitations and future potential of the calyx of Held
as a model presynaptic preparation.
Function of the calyx of Held in the auditory brainstem
circuitry
The calyx of Held is thought to aris e from globular bushy
cells in the aVCN, which project onto principal neurons of
the contralateral MNTB (see Fig. 1; Harrison and Irving
1966; Friauf and Ostwald 1988 ;Spirouetal.1990;
Kuwabara et al. 1991; Smith et al. 1991). It therefore
forms a tertiary synapse in the auditory pathway. The
MNTB principal cells provide inhibitory glycinergic pro-
jections to neighbouring nuclei in the superior olivary
complex, including the lateral superior olive (LSO; see
Fig. 1 ; Tollin 2003) and the medial superior olive (MSO;
Banks and Smith 1992; Joris et al. 1998; Brand et al. 2002).
The LSO and MSO are the first nuclei in which binaural
information conv erges, with the calyx of Held/MNTB
synapse forming a fast “inverting” relay, at which excitation
originating from the contralateral cochlea is converted into
inhibition to the ipsilateral auditory brainstem. The large
size of the calyx of Held allows it to harbour hundreds of
active zones (see below) and thus a single presynaptic
action potential (AP) releases hundreds of quanta , generat-
ing a large EPSC that rapidly depolarises the MNTB
neuron to threshold. Hence, the large size of the presynaptic
terminal guarantees rapid signalling, preserving the timing
information of the acoustic signal for processing by the
binaural circuits underpinning sound localisation (Oertel
1999; Trussell 1999).
A single MNTB principal neuron receives input from
only one calyx-type synapse, although multiple calyceal
inputs are occasionally observed in ~5% of principal
neuron recordings in mice (Bergsman et al. 2004) and
~20% of the afferent fibres give rise to two calyces on
separate MNTB principal neurons (Kuwabara et al. 1991;
Smith et al. 1991; Rodríguez-Contreras et al. 2006). In
addition to the calyceal input, principal cells receive conven-
tional excitatory synapses (Forsythe and Barnes-Davies 1993;
Hamann et al. 2003) and inhibitory inputs (Banks and Smith
1992;Awatramietal.2004). The calyx of Held develops
early, with trapezoid axons growing from the cochlear
nucleus, crossing the midline by E15 and forming large pre-
calyceal nerve endings by P3 (Fig. 1c; Kandler and Friauf
1993; see also Morest 1968; Hoffpauir et al. 2006).
Morphology and ultrastructure of the calyx of Held
A number of electron-microscopic (EM) studies have been
conducted on calyces of Held (Lenn and Reese 1966 ;
Nakajima 1971; Jean-Baptiste and Morest 1975
; Sät zler
et al. 2002; Taschenberger et al. 2002) and endbul bs of
Held (Lenn and Reese 1966; Ryugo et al. 1996;Nicoland
Walmsley 2002). They show that despite the large size of
the nerve terminal (Fig. 2a), individual active zones of
calyceal nerve endin gs in the MNTB and VCN are
morphological ly simila r to those of conventional sm all
nerve terminals (Fig. 2b). Calyceal t erminals aris e from a
myelinated axon, which can be thick in cats (5 –10 μm;
Rowland et al. 2000) but is thinner in rats (<2 μm;
Rodríguez-Contreras et al. 2006). Small synap tic vesicles
(SSVs) accumulate at electron-dense contac t sites, the
active zones (Fig. 2b). Interes tingly, calyceal SSVs are of
slightly larger diamete r (~45 nm) than those co ntained in
many s mall bouton-like h ippocamp al and cerebellar syn-
apses,whichare~35nmindiameter(Schikorskiand
Stevens 1997; Xu-Friedman et al. 2001). An EM
reconstruction of an entire calyx of Held from a P9 rat,
by using serial ultrathin sections (Sätzler et al. 2002 ), has
shown the pres ence of ~550 in dividua l active zon es, with
an average nearest-neighbour separation of ~0.6 μm.
Numerous non-synaptic contact sites, named p uncta
adherentia (also observed by Jean-Baptiste and Morest
1975), have also been found. Synaptic contact sites
(activezones)haveanaveragesurfaceareaof0.1μm
2
,
similar to estimates for hippoc ampal and cerebellar
excitatory synapses (Schikorski and Stevens 1997;Xu-
Friedman et al. 2001) an d contain an average o f two
morphologically docked vesicles (Sätzler et al. 2002).
Another study has estimated th at the extrapolated number
of act ive zones increas es from ~300 at P5 to ~680 at P14
(Taschenberger et al. 2002).Thenumberofactivezones
corresponds well to the estimated number of functional
active zones based on EPSC fluctuation analysis (Meyer
et al. 2001;andseeFig.5b). Thus, the calyx of Held can
be seen as a vast “parallel” arrangement of hundreds of
active zones, all activated by a single presynaptic AP.
Although the calyx of Held contains “conventional” active
zones, there is some evidence for structural specialisations
Cell Tissue Res (2006) 326:311–337 313

beyond mere size. Investigations of mature calyces by Row-
land et al. (2000) in the cat have revealed that the non-
synaptic contact sites (puncta adherentia) are associated with
tethered mitochondria within 200 nm of the membrane, a
complexthattheyhavecalledthe“mitochondria-associated
adherens complex” (MAC). Recently, Wimmer et al. (2006)
have found, by using confocal fluorescence microscopy after
virus-mediated over-expression of synaptic vesicle proteins
(Wimmer et al. 2004), that vesicle clusters and active zones
are organised in “donut”-like assemblies of ~1 μmin
diameter. Electron microscopy has revealed that the “donuts”
are comprised of ~5–9 active zones clustered around the
same number of mitochondria. Interestingly, “donuts” only
appear during maturation of the calyx of Held after the
opening of the auditory canal (at P11/12; Blatchley et al.
1987;Geal-Doretal.1993). The intricat e arrangement of
the release apparatus (vesicle clusters and active zones) and
mitochondria might represent an optimal spatial arrange-
ment for fast re-supply of ATP and for local Ca
2+
sequestration into mitochondria (Billups and Forsythe
2002). This arrangement might be advantageous for
sustaining high rates of transmitter release during the high
frequency firing that occurs physiologically at the calyx of
Held (Kopp-Scheinpflug et al. 2003).
Presynaptic AP and ion channels of the calyx of Held
Direct recording of presynaptic conductances at synaptic
terminals in the mammalian CNS has so far only been
achieved at three sites: the excitatory calyx of Held, the
mossy fibre terminals (Geiger and Jonas 2000) and the
cerebellar inhibitory pinceau (Southan and Robertson
1998). An example of a calyx of Held (filled with Lucifer
yellow) viewed by differential interference contrast optics
and then under fluorescence illumination is shown in
Fig. 3a. Because of the continuity of the calyceal nerve
terminal and its axon, whole-cell recording from the calyx
will include currents arising from proximal parts of the
axon, with the current and voltage respon ses of a terminal
being influenced by the length of the intact axon. For
instance, terminals with short axons fire a single AP on
sustained depolarisation, whereas terminals with intact
axons in excess of 150 μm exhibit a sustained repetitive
AP firing throughout the depolarisation (Dodson et al.
2003). Our objective here is to summarise the ionic
conductances regulating excitability and AP generation at
the calyx of Held.
Presynaptic resting membrane potentials (RMPs) at the
calyx are around −75 mV (Forsythe 1994; Borst et al. 1995;
Fig. 2 Morphology and ultra-
structure of the calyx of Held.
a Electron micrograph of the
calyx of Held from a P9 rat
(yellow presynaptic calyx, blue
postsynaptic MNTB principal
neuron, red its nucleus, boxed
area active zone). Bar 5 μm.
Taken, with permission, from
Sätzler et al. (2002); copyright
2002 by the Society for Neuro-
science. b High resolution EM
images of active zones within
calyces of Held (green vesicles
identified as morphologically
docked). Left Two neighbouring
active zones from a P5 rat. Right
Single active zone in a P14 rat.
Bars 200 nm. Reprinted, with
permission from Elsevier, from
Taschenberger et al. (2002)
314 Cell Tissue Res (2006) 326:311–337

Borst and Sakmann 1996), which is slightly more negative
than the RMPs of the postsynaptic MNTB neuron at around
−65 mV. Upon afferent fibre stimulation, the calyx shows
overshooting orthodromic presynaptic APs (Fig. 3b) reach-
ing +30 mV, with half-widths of 0.41 ms at 25°C (Dodson
et al. 2003) and 0.26 ms at 36°C (Borst and Sakmann
1998b; Kushmerick et al. 2006). A developmental acceler-
ation in the presynaptic AP time-course is also apparent,
with halfwidths at P14 being less than half of those at P7
(Fig. 3c; Tasch enberger and von Gersdorff 2000). As
previously observed from intra-axonal recording of mye-
linated axons (Barrett and Barrett 1982 ), the calyx APs are
accompanied by d epolaris ing after-potentials (DAP) of
around 10 mV in amplitude (Borst et al. 1995; Dodson et
al. 2003; see Fig. 3b). DAPs peak with late ncies of around
5 ms, last between 20 and 100 ms and are unaffected by
blocking Ca
2+
channels, by transmitter release or by
increasing Ca
2+
buffering, consistent with a postulated
origin involving passive discharge of internodal capacitance
(Barrett and Barrett 1982), although they may be further
enhanced by the capacitive load of the terminal itself.
Presynaptic APs are blocked by tetrodotoxin (TTX;
Forsythe 1994; Borst et al. 1995;Leaoetal.2005). In
contrast to the ciliary ganglion (Martin and Pilar 1963), no
evidence has been found for direct electrical transmission
across gap junctions. The presynaptic axon is myelinated and
hence AP propagation is via saltatory conduction with
voltage-gated Na
+
channels located at nodes of Ranvier
and K
+
channels at juxtaparanodal regions (Rasband and
Shrager 2000). Voltage-gated Na
+
channel structure is similar
to that of Ca
2+
channels (Catterall et al. 2005). In the CNS, a
developmental transition from expression of Nav1.2 to
Nav1.6 occurs at maturation (Caldwell et al. 2000; Rios et
al. 2003) in many areas of the brain. Immunohistochemical
labelling shows that Na
+
channel density is low at the calyx,
supporting the idea of passive AP propagation into the
terminal but, by P12, Nav1.6 is located at a high density in
the last segment of the axon (heminode, which is unmyelin-
ated; Leao et al. 2005). This pattern of localisation differs
from the situation in en-passant terminals, such as mossy-
fibre boutons, where a high density of voltage-gated Na
+
-
current has been found in outside-out patches from the
terminal (Engel and Jonas 2005). With maturation up to P15,
calyceal Na
+
currents increase in magnitude and show
accelerating inactivation and recovery from inactivation
(tau: 0.5 ms at 35°C), contributing to increased afferent fibre
excitability (Leao et al. 2005).
K
+
channels Most alpha (α) subunits of K
+
channels have a
structure analogous to one domain of voltage-gated Na
+
channels; hence, four subunits must assemble to form a
functional channel. Channels may be heterologous but are
usually composed of subunits from within the same family,
plus beta subunits and/or accessory proteins. With around
100 K
+
-channel subunit genes in more than 10 families,
there is potential for huge diversity (Coetzee et al. 1999)
and thus relating native channels to their recombinant
counterparts is difficult, and it is worth providing a brief
overview by way of introduction to presynaptic K
+
currents.
There are four families of mammalian voltage-gated K
+
channels which can generate the classic “delayed rectifier”
characteristics. The shaker-related Kv1 subunits (of which
there are seven members) form channels activated by small
depolarisations from RMP (10–40 mV, hence low voltage-
activated) and are involved in regulating excitability and
the threshold for AP firing. Kv1 channel s may exhibit
voltage-depend ent inactivation through an N-terminal
(N-type, ball and chain) mechanism (Aldrich 2001) but
this depends on the subunit composition and/or presence of
beta subunits. The two shab-related Kv2 subunits associate
with many accessory subunits and generate a broad range
of conductances. Kv2.1 channels are widely expressed but,
as yet, little evidence exists for their immunolocalisation in
the MNTB or synaptic terminals (R.E.W. Fyffe, personal
communication). The four shaw-related Kv3 subunits are
activated by larger depolarisations (>50 mV, hence high
voltage-activated). These voltages are only achieved during
APs and Kv3 channels participate in repolarisation,
particularly in fast-spiking interneurons (Rudy and McBain
2001). Finally, the three shal-related Kv4 subunits that
generate transient inactivating subunits underlie A-type (I
A
)
currents (Jerng et al. 2004). Like Kv1 channels, they are
activated by small depolarisations but at resting potentials
require membrane hyperpolarisation to remove steady-state
inactivation. Other related K
+
channels, such as Kv7
(KCNQ; Delmas and Brown 2005) and twin-pore K
+
channels (KCNK; Goldstein et al. 2001) are expressed in
the auditory brainstem (Karschin et al. 2001; J. Johnston,
A. Skrzypiec, M. Postlethwaite and I.D. Forsythe, in
preparation) but will not be considered here.
The postsynaptic MNTB neuron expresses both low
voltage-activated (I
K,LV
) and high voltage-activated (I
K,HV
)
K
+
currents, which regulate firing threshold and AP re-
polarisation, respectively (Brew and Forsythe 1995; Dodson
et al. 2002). I
K,LV
is blocked by the black mamba snake
toxin, dendrotoxin-I (DTx-I), confirming mediation by Kv1
channels. Kv1 channels are located in cell bodies,
dendrites, synaptic terminals (Wang et al. 1994) and
juxtaparanodal regions of myelinated axons (Rasband and
Shrager 2000). I
K,HV
is mediated by Kv3 channels, which are
generally associated with high frequency AP firing and
mediate rapid AP repolarisation in many areas of the CNS
(Rudy and McBain 2001), including the auditory brainstem,
and are blocked by low (mM) concentrations of tetraethylam-
monium (Brew and Forsythe 1995; Wang and Kaczmarek
1998; for a review, see Kaczmarek et al. 2005). Activity-
Cell Tissue Res (2006) 326:311–337 315

dependent changes in Kv3 channel activity are mediated by
channel phosphorylation by casein kinase II (Macica and
Kaczmarek 2001) and protein kinase C (PKC; Macica et al.
2003). In the MNTB, there is high basal phosphorylation at
ser503 of Kv3.1b and this decreases the postsynaptic K
+
-
current amplitude. Exposure to moderate sound levels causes
dephosphorylation and increases I
K,HV
in the MNTB neuron
(Song et al. 2005), thus improving the ability of MNTB
principal cells to follow AP firing at high frequencies.
Characterisation of the presynaptic K
+
currents is not
complete but several studies have shown that, like the
postsynaptic bushy cell and MNTB principal cell bodies,
both low and high voltage-activated outward currents play a
major role in regulating presynaptic AP firing.
I
K,LV
currents mediated by Kv1 channels Voltage clamp
recordings have shown that calyces of Held possess a
current activating over a low voltage range (−60 to
−30 mV, Fig. 3e,f, closed symbols). These currents were
blocked by DTx-I confirming mediation by Kv1 channels,
and by tityustoxin Kα, which is specific for channels
containing Kv1.2 subunits, but were less sensitive to
dendrotoxin-K, which blocks Kv1.1-containing channels
(Dodson et al. 2002, 2003 ). Immunohistochemistry has
confirmed that Kv1.2 subunits are located in the axon (see
Fig. 3g) immediately adjacent to, but excluded from, the
terminal itself. Thus, Kv1.2 localisation seems to overlap
with the Nav1.6 location (Leao et al. 2005), compatible
with a role in regulating threshold excitability.
Blockade of presynaptic Kv1 channels has no effect on
AP halfwidth (Dodson et al. 2003 ) or on evoked transmitter
release (Brew and Forsythe 1995) from a single AP but
blockade increases DAP amplitude, which then elicits
additional aberrant APs during the DAP. These results
suggest that presynaptic Kv1 functions to shunt and to
suppress terminal hyperexcitability and so minimises AP
“reflection”. Reflection arises because the duration of the
DAP outlasts the Na
+
-channel refractory period, thus
generating an antidromic AP under certain conditions
(Dodson et al. 2003). Of note, Kv1 currents of the calyx
of Held show little inactivation (Forsythe 1994; Dodson et
al. 2003) and the calyx exhibits no short-term plasticity
attributable to Kv1 channels. However, inactivating K
+
currents do contribute to AP repolarisation at mossy fibre
terminals (Geiger and Jonas 2000) and neurohypophysial
terminals (Jackson et al. 1991; Tho rn et al. 1991), where
accumulation of inactivation during AP trains increases AP
duration and Ca
2+
influx and causes short-term facilitation
of transmitter release.
I
K,HV
currents mediated by Kv3 channels Early recordings
from the calyx have demonstrated that calyceal high
voltage-activated outward K
+
currents are blocked by
micromolar concentrations of 4-aminopyridine (Forsythe
1994). Low millimolar (1–3 mM) concentrations of
tetraethylammonium (which blocks Kv3 currents) increase
AP duration (Wang and Kacz marek 1998) and transmitter
release (Ishikawa et al. 2003; Fig. 3d). Immunohistochem-
ical studies at the light-microscopic level show that Kv3
subunits are absent from the heminode and so do not
overlap with Kv1 or Nav1.6 channels (Dodson et al. 2003;
Fig. 3g). Intriguingly, in EM studies, little or no Kv3.1b
immunostaining has been observed on the release face but
is concentrated on the non-release face of the synapse
(Elezgarai et al. 2003). The reasons for this are unknown
since, from a biophysical perspective, their location on one
synaptic face or another would have little impact on their
ability to repolarise presynaptic APs. One rationale, given
that the calyx can occupy over 60% of the postsynaptic
soma surface, is that accumulation of K
+
in the small
volume of the synaptic cleft would have a dramatic impact
on pre- and postsynaptic membrane potential and so, by
locating these channels on the non-release face, secondary
depolarisation of the pre- and postsynaptic membrane
potentials is probably minimised; however, the mechanism
of this localisation or exclusion from the release face is
unknown. Given the broad distribution of phosphorylated-
Kv3.1b in both postsynaptic and presynaptic compartments
Fig. 3 Presynaptic patch-clamp recordings from the calyx of Held:
the nerve terminal action potential (AP) and voltage-gated K
+
currents. a Nomarski image (differential interference contrast) of a
single MNTB neuron with surrounding calyx (arrows). Presynaptic
recording from this terminal was confirmed by labelling with Lucifer
yellow from the patch pipette. Scale Neuronal diameter: 18 μm. b An
orthodromic presynaptic AP followed by a depolarising after-potential
(DAP). Modified, with permission, from Borst et al. (1995). c Normalised
presynaptic APs at three different postnatal developmental stages. The
presynaptic AP is brief at P7 (half-width: ~0.5 ms) but becomes even
briefer with further postnatal development. Modified, with permission,
from Taschenberger and von Gersdorff (2000); copyright 2000 by the
Society for Neuroscience. d Paired pre- and postsynaptic recording.
Application of 1 mM tetraethylammonium (TEA; blocks the high-
voltage-activated K
+
current) increases AP duration (Pre)andincreases
transmitter release (EPSC). Taken, with permission, from Ishikawa et al.
(2003 ); copyright 2003 by the Society for Neuroscience. e Outwar d K
+
-
currents of the calyx of Held generated on depolarisations from a holding
potential of −70 mV. Current traces are shown for voltage steps from −70
to −5 mV, under control conditions in the presence of tetrodotoxin (top)
and following application of tityustoxin-Kα (100 nM, TsTx-Kα,Kv1.2
antagonist). The current amplitudes observed with steps to −40 mV
(which largely correspond to the low-voltage-activated K
+
current, I
K,LV
)
are indicated by filled black bar (left). f Current-voltage relationship of
outward K
+
currents plotted at the time indicated by filled and open
symbols in e.NotethatTsTX-Kα blocks all outward current at voltages
up to −30 mV. Taken, with permission, from Dodson et al. (2003 ).
g Immunolocalisation of Kv3.1b and Kv1 .2 subu nits in the calyx
(left, red)andthelast2μm of the axon (arrow). Kv1.2 subunits are
not located in the calyx but are present in the last portion of the
axon (centre, green). The overlay (ri ght) shows that Kv3 and Kv1
channels are located in distinct compartments (stars, daggers). Bar
10 μm. Taken, with permission, from Dodson et al. (2003)
b
316 Cell Tissue Res (2006) 326:311–337

of the MNTB (Song et al. 2005), Kv3.1 modulation
probably also occurs in the presyn aptic terminal, but this
has yet to be directly demonstrated.
I
H
currents As yet, little information has been obtained
regarding K
+
channels responsible for setting RMPs in the
MNTB, but there is good evidence for the participation of
an I
K,LV
and I
H
in octopus cells (Bal and Oertel 2001).
Hyperpolarisation-activated non-specific cation currents,
known as I
H
, are mediated by HCN subunits and are
permeable to K
+
and Na
+
(their reversal potential is around
−30 mV). They have relatively slow kinetics but are active
Cell Tissue Res (2006) 326:311–337 317

at RMPs and are broadly expressed in the CNS (Santoro et
al. 2000), being associated with oscillat ory rhythm (Hu et
al. 2002) and the control of dendrite excitability (Day et al.
2005). I
H
currents are present in the calyx of Held and the
postsynaptic MNTB neuron (Banks et al. 1993; Cuttl e et al.
2001). HCN1 and HCN2 subunits are expressed in specific
neuronal patte rns in various auditory brainstem nuclei
(Koch et al. 2004). I
H
localised in presynaptic terminals
can influence exocytosis at the crustacean NMJ (Beaumont
and Zucker 2000) but, although initial studies have
suggested similar effects in the hippocampus (Mellor et al.
2002), this has not bee n co nfirmed (Chevaleyre and
Castillo 2002) and, at the calyx of Held, the blocking of
I
H
does not modulate transmitter release (Cuttle et al.
2001). I
H
is also present at inhibitory synaptic terminals of
cerebellar basket cells (Southan et al. 2000) where the
blocking of I
H
(with ZD7288) increases the frequency of
spontaneous inhibitory postsynaptic currents. I
H
is modu-
lated by intracellular cAMP at both the postsynaptic MNTB
neuron (Banks et al. 1993) and the presynaptic terminal
(Cuttle et al. 2001) and so could contribute to activity-
dependent modulation of the presyn aptic RMP, in concert
with other low voltage-activated curren ts and leak channels.
Ca
2+
-activated K
+
channels There is good evidence that
large conductance (BK) Ca
2+
-activated K
+
channels
(generated by slo1 subunits) are widely expressed and
influence transmitter release at the amphibian (Robitaille
et al. 1993) and mammalian (Katz et al. 1995)neuromus-
cular junctions. Recent immunohistoch emical studies
have shown that slo1 is present in axons and terminals
associated with glutamatergic synapses (Misonou et al.
2006). Pharmacological studies have shown that the BK
antagonist iberiotoxin blocks a slow current in the calyx
of Held (Ishikawa et al. 2003) and another study h as noted
aK
+
current activated by Ca
2+
uncaging in the calyx of
Held; this cu rrent is suppressed by tetraethylammonium
(Wölfel and Schnegge nburger 2003), which also blocks
BK channels. However, further characterisation is re-
quired to understand the contribution of Ca
2+
-activated K
+
channels to the regulation of transmitter release. In situ
hybridisation and immunohistochemical d ata sug gest that
the related Na
+
-dependent K
+
channels, Slick and Slack
(Bhattacharjee and Ka czmarek 2005), are expressed in t he
MNTB; their function is currently being assessed.
Calcium channels at the calyx of Held
Presynaptic Ca
2+
channels have received considerable atten-
tion, since the calyx of Held preparation offers the means to
study the presynaptic Ca
2+
channels involved in triggering
exocytosis at a glutamatergic synapse directly. Whole-
terminal recordings under conditions suitable for blocking
voltage-gated Na
+
and K
+
channels show peak inward Ca
2+
currents of between 1–2 nA with 2 mM [Ca
2+
]
o
(Borst et al.
1995; Borst and Sakmann 1996, 1998b; Forsythe et al. 1998;
see Fig. 4a). Patch-clamp and immunohistochemical studies
in young rats prior to the opening of the auditory canal (P10)
have reve aled that N-, R- and P-type Ca
2+
channels
contribute to the voltage-dependent Ca
2+
influx (Wu et al.
1999). However, a shift in the balance of the presynaptic
subunits occurs so that, from around P10, the Ca
2+
currents
triggering exocytosis are sensitive only to ω- agatoxin-IVA
(Forsythe et al. 1998), indicating the dominance of the
P-type Ca
2+
channel formed by CaV2.1 subunits. The
sensitivity of the presynaptic Ca
2+
current to ω-agatoxin
IVA is shown in Fig. 4c. Developmental studies have clearly
demonstrated that the switch from mixed N- and P- to P-type
channels takes place at ~P10/11 (Iwasaki and Takahashi
1998; Iwasaki et al. 2000). Although most studies have used
voltage steps to evoke Ca
2+
currents, Borst and Sakmann
(1998b)havestudiedtheactivationofCa
2+
current during a
presynaptic AP with two electrode voltage clamps and have
shown that the peak inward Ca
2+
current occurs shortly after
the AP peak (Fig. 4d). A recent study of transmission
efficacy during synapse development has revealed that
acceleration of the presynaptic AP time-course decreases
Ca
2+
influx, whereas EPSC amplitude increases during
maturation in mice, implying considerable enhancement in
coupling efficacy during calyx of Held development (Yang
and Wang 2006).
The presynaptic P-type Ca
2+
channel shows the classical
bell-shaped current/voltage curve (Fig. 4b) with little
current at voltages negative to −40 mV and peak inward
currents between −20 mV and 0 mV. The terminal does not
possess any transient (“T-type”)Ca
2+
currents as can be
seen by contrasting the Ca
2+
currents evoked in a bushy cell
body (which shows a clear T-type Ca
2+
current) with the
same voltage protocols delivered to a calyceal terminal
(Fig. 4e, lower traces). The presyn aptic P-type current is
subject to several activity-dependent modulations. A form
of Ca
2+
-dependent inactivation (Forsythe et al. 1998) can
be seen from the initial decay of the current in the largest
(−15 mV step) current trace in Fig. 4a. The inactivation
depends on the presence of extracellular Ca
2+
and, by
analogy with the modulation of recombinant P/Q Ca
2+
channels (DeMaria et al. 2001), this could be mediated by
Ca
2+
/calmodulin binding. Ca
2+
-current inactivation contrib-
utes to synaptic depression following prolonged high-
frequency activity (Forsythe et al. 1998) and during the
onset of repetitive stimulation (Xu and Wu 2005). At
short interva ls, a short-term Ca
2+
-dependent facilitation
lasting up to 100 ms occurs, which is distinct from the
voltage-dependent relief of G-protein inhibition (Borst
and Sakmann 1998a; Cuttle et al. 1998) and is mediated
318 Cell Tissue Res (2006) 326:311–337

by frequenin/NCS-1 (neuronal Ca
2+
sensor 1; Tsujimoto
et al. 2002). CaV2.1 knock-out mice, which lack func-
tional P-type channels, maintain transmission at the calyx
of Held through comp ensation by N-type channels
(Inchauspe et al. 2004;Ishikawaetal.20 05). Differences
between transmission in the knock-out and wildtype
animal have given important clues to the physiological
function of P-type channels. Although peak Ca
2+
current
is lower i n the CaV2.1 k nock-out, the major difference is
the absence of Ca
2+
-dependent facilitation of the N-type
Fig. 4 Presynaptic calcium currents. a Ca
2+
currents evoked from a
prepulse to of −120 mV show no activation until step depolarisati ons
are positive to −40 mV. The current activates rapidly and exhibits
marked inactivation at the more positive steps (−15 mV). b The
current-voltage relationship (for the same terminal as in a) shows
steep voltage-dependent activation, with peak inward currents b eing
elicited at around −15 mV; an apparent reversal potential is observed
at around +45 mV. c In animals older than P10, most of the
presynaptic Ca
2+
current is blocked by ω-agat oxin-IVA. Large
depolarisations ( double ar row) relieve the block. A high dose
(200 nM) blocks around 97% o f the current and the remainder is
blocked by cadmiu m (50 μM). a–c Reprinted with permission from
Elsevier, from Forsythe et al. (1998). d The Ca
2+
current elicited by
a presynaptic AP at 36°C. Top Two-electrode voltage-clamp was
made on a single calyx of Held nerve terminal by using a measu red
AP as a voltage-clamp command waveform. Middle Total current.
Bottom Ca
2+
current as the difference current. Taken, with
permission, from Borst and Sakmann (1 998b). e Ca
2+
currents of
bushy cell body and calyx of Held terminal with identical voltage
protocols, stepping from −100 mV to either −50 mV or −10 mV.
Note that the transient C a
2+
current is only apparent in the bushy cell
body and no current is evoked at −50 mV in the calyx (HP holding
potential). Modified, with permissio n, from Doughty et al. (1998).
f The metabotropic glutamate receptor agonist L-AP4 reduces the
amplitude of the presynaptic Ca
2+
current. Single traces are super-
imposed and the complete I/V is shown below. Reprinted, with
permission, from Takahashi et al. (1996), copyright 1996 AAAS
Cell Tissue Res (2006) 326:311–337 319

presynaptic current, suggesting that this facilitation is
dependent on the expression of P-type Ca
2+
channels
(Inchauspe et al. 2004;Ishikawaetal.2005).
Postsynaptic glutamate receptors
and their developmental regulation
The calyx of Held is a glutamatergic synapse, and early
postsynaptic voltage-clamp recordings have shown a fast
component of the EPSC that is sensitive to the AMPA/
kainate-receptor antagonist CNQX, and a slow component
blocked by the NMDA-receptor antagonist AP-5 (Forsythe
and Barnes-Davies 1993). The fast EPSC component is also
blocked by GYKI 52466 showing that it is mediated
exclusively by AMPA receptors (Futai et al. 2001). In
young animals (P8–P10), NMDA-receptor-mediated syn-
aptic conductance is comparable to, or larger, than the
AMPA component (von Gersdorff et al. 1997; Joshi and
Wang 2002) but, with postnatal maturation, the NMDA-
receptor-mediated EPSC is down regulated (Taschenb erger
and von Gersdorff 2000; Futai et al. 2001; Joshi and Wang
2002), with only a small NMDA EPSC remaining after
P20. At the same time, the decay time constant of AMPA-
receptor-mediated EPSCs and miniature EPSCs (mEPSCs)
is speeded up during development (Taschenberger and von
Gersdorff 2000; Joshi and Wang 2002; Joshi et al. 2004),
reaching values of ~0.3 ms after P20 in rats and mice (Futai
et al. 2001; Yamashita et al. 2003; Fernández-Chacón et al.
2004). The fast AMPA EPSC decay is caused by the fast
rates of AMPA-receptor deactivation and desens itisation,
which are probably determined by the high expression
levels of the AMPA-receptor “flop” spli ce variants in these
neurons, as revealed by single-cell polymerase chain
reaction (Geiger et al. 1995; Koike-Tani et al. 2005). The
fast decay of AMPA EPSCs is also observed in
glutamatergic synapses made by auditory fibres in the
chick nucleus magnocellularis (Zhang and Trussell 1994)
and on bushy and stellate cells in the mammalian cochlear
nucleus (Isaacson and Walmsley 1995; Gardner et al.
1999). Fast AMPA-receptor signalling is seen as an
adaptation for the preservation of timing information in
these auditory circuits (Trussell 1999).
Quantal properties of transmission (N, p, q)
Since the discovery by Katz and colleagues that, at the
neuromuscular junction, chemical synaptic transmission is
quantal (Katz 1969), a major goal has been to understand
the regulation of quantal parameters underlying transmis-
sion at a given synapse. The quantal hypothesis states that
the amplitude of a postsynaptic current (PSC; or postsyn-
aptic potential) is determined by the product of the quantal
amplitude q, the number of release sites N and the
probability p that release occurs at each site:
PSC ¼ q  N
 p ð1Þ
The quantal amplitude q is a measure of membrane
current induced by neurotransmitter release from a single
presynaptic vesicle. The presynaptic factors N and p are
dimensionless. The mathematical derivation of this bino-
mial theory for quantal release (Quastel 1997; Scheuss and
Neher 2001 ) postulates N independent release sites, from
each of which exactly one or no release event may occur
per AP. The “release site” in this definition is equal to the
physical docking site of an individual vesicle. It is
important to note, however, that a “ release site” in the
binomial model is not identical to an active zone. A
morphologically defined active zone usually has more than
one docked vesic les (range: 3–8; see above), most of which
are thought to be fusion-competent (Schikorski and Stevens
2001). Thus, a given stimulus could release zero, one, or
several vesicles at an individual active zone: hence,
“multivesicular release” (Wadiche and Jahr 2001). During
multivesicular release, postsynaptic receptors become in-
creasingly saturated, so that the postsynaptic conductance
change will not grow linearly with the second, third, ... nth
vesicle released simultaneously at the same acti ve zone
(Auger et al. 1998; Meyer et al. 2001). Thus, several
released vesicles from a given active zone interact
postsynaptically because of the limited number of postsyn-
aptic receptors (Matveev and Wang 2000). Work on the
calyx of Held has shown that N, depending on the means
taken to minimise postsynaptic receptor saturation and on
the type of stimulus used to evoke release (AP-stimulation
versus direct presynaptic depolarisation or Ca
2+
uncaging),
often lies between two biologically relevant numbers: the
number of active zones (N
az
) and the number of readily
releasable vesicles (N
ves
).
The quantal size q at the calyx of Held has been
determined from spontaneous EPSCs (either in the absence
or presence of TTX) and amplitude histograms generally
show means between 30–35 pA at room temperature and at
a holding potential of approximately −70 mV, with coefficients
of variation of 0.3–0.5 (see Fig. 5a; Sahara and Takahashi
2001; see also Chuhma and Ohmori 1998; Schneggenburger
et al. 1999; Meyer et al. 2001; Taschenberger et al. 2005).
Spontaneous EPSCs might be multiquantal if presynaptic
APs trigger release; however, the application of 1 μM TTX
does not influence the frequency or amplitude of sponta-
neous EPSCs at the calyx of Held (Ishikawa et al. 2002)
and, hence, spontaneous EPSC recorded in the absence of
TTX are probably also true mEPSCs. In rats older than
P8–P10, mEPSCs are ~50 pA (Taschenberger et al. 2005)
and an increase in temperature to ~37°C leads to an
320 Cell Tissue Res (2006) 326:311–337

increase of the quantal amplitude by ~50% (Kushmerick
et al. 2006; M. Postle thwaite, M. H ennig, B. P. Graham
and I.D. Forsythe, submitted for publication). Release
from non-calyceal te r m i n a l s m a y make a sm a l l c o n t r ibu -
tion to the mEPSCs recorded in the MNTB principal
cells. However, sub-threshold depolarisations of the
calyx (Sahara and Takahashi 2001)ordialysiswith
strongly Ca
2+
-buffered solutions to increase presynaptic
[Ca
2+
]
i
beyond base line (Sun et al. 2002; Lou et al . 2005)
increases mEPSC frequency in simultaneous pre- and
postsynaptic recor dings, indicating that the mEPSCs
occurring at rest are to a large part generated by the calyx.
The calyceal EPSC evoked by a single afferent fibre
stimulation is in the range of 4–8 nA, although currents in
excess of 15 nA are not uncommon. The values are more
than two orders of magni tude larger than the amplitude of a
single mEPSC. To a first approximation, then, the quantal
content mm¼ N  pðÞof an evoked EPSC is large (150–
250; Borst and Sakmann 1996; Schneggenburger et al.
1999). However, in the event of pooling of transmitter
between neighbouring active zones, as might occur by spill-
over of glutamate (DiGregorio et al. 2002), quanta may add
up non-linearly and the quantal content might be different.
To investigate quantal size during evoked transmission,
methods of non-stationary EPSC variance analysis (Scheuss
and Neher 2001) have been applied at the calyx of Held.
Meyer et al. (2001) have used a method in which the
decrease of EPSC amplitudes during short-term depression
Fig. 5 Quantal parameters of
synaptic transmission at the
calyx of Held. a Amplitude
distribution of spontaneous
“miniature” EPSCs (mEPSCs).
Sample traces are shown right.
Taken, with permission, from
Sahara and Takahashi (2001).
b Variance-mean analysis of
evoked EPSCs under conditions
of high release probability
(15 mM [Ca
2+
]
o
). The variance-
mean plot shows a maximum
(right). A parabola was fitted to
the four right-most lying data
points (right). Extrapolation to
maximal EPSC amplitudes gave
an estimate of the binomial
parameter N. Taken, with per-
mission, from Meyer et al.
(2001); copyright 2001 by the
Society for Neuroscience.
c Probing the size of a pool of
readily releasable vesicles by
strong presynaptic depolarisa-
tions. A presynaptic depolarisa-
tion to 0 mV for 50 ms,
preceded by a brief pre-pulse to
+80 mV, evoked a presynaptic
Ca
2+
current (I
Ca
) and a large
postsynaptic EPSC (~16 nA).
Deconvolution of the EPSC
with the waveform of the
underlying “quantal” mEPSC
gave the cumulative release rate
(bottom), which was fitted with
a double-exponential function
(bottom, dotted line). Reprinted,
with permission from Elsevier,
from Sakaba and Neher (2001b)
Cell Tissue Res (2006) 326:311–337 321

is used to lead the synapse repetitively through various
states of release probability (Fig. 5 b). Under normal
recording conditions with 2 mM [Ca
2+
]
e
, the variance-mean
relationship of peak EPSC amplitu des during depression
induced by a 10-Hz train is linear, indicating that the release
probability is quite low at 2 mM [Ca
2+
]
e
. With the limiting
condition of low p, the variance-m ean data should only
cover the linearly rising phase of a parabola, and the slope
should be equal to the underlying quantal size. The slopes
averaged ~25–30 pA, which is only slightly smaller than
the mean of amplitude distributions of spontaneous
mEPSCs, which is ~31 pA (Meyer et al. 2001). With a
similar EPSC variance-mean approach, Taschenberger et al.
(2005) have determined the quantal size during evoked
transmission to be somewhat higher at ~50 pA. There is,
therefore, no reason to assume that sub-linear quantal
summation occurs during evoked EPSCs under conditions
of normal release probability (2 mM [Ca
2+
]
e
). Thus, we can
conclude that a single AP evoking an EPSC of 4–8nAis
caused by the release of between 150–250 quanta from the
presynaptic terminal.
This still leaves open the question regarding the number
of independent units N mediating release at the calyx of
Held. The binomial parameter N can be estimated from
EPSC variance-mean plots by extrapolating to the maximal
EPSC amplitude (Silver 2003). Since the EPSC varia nce-
mean relationship is linear at normal release probability
(see above), Meyer et al. (2001) enhanced the release
probability by increasing [Ca
2+
]
e
from 2 mM to 15 mM;
this leads to a five-fold to seven-fold potentiation of the
EPSC amplitude. At 15 mM [Ca
2+
]
e
, a maximum in the
EPSC variance-mean plot has been observed in many cases
(Meyer et al. 2001) and the parabolic fit has been
extrapolated to the maxi mal EPSC amplitude (Fig. 5b,
right panel). Dividing this value by the quantal size should
give an estimate of N, which was found to be ~600 on
average between individual cells. Considering indications
of multivesicular release and AMPA-receptor saturation
(see above), Meyer et al. (2001) interpreted this value
of N as an upper-limit of the number of functional active
zones that contribute to transmission at the calyx of Held,
rather than representing a true “single vesicle release
constraint” at each active zone , as postulated at other
synapses based on EPSC variance-mean analysis (Korn
et al. 1981; Silver et al. 2003). Although the cell-to-cell
variability of the estimated parameter N is large (~200 to
more than 1000; Meyer et al. 2001), there is remarkable
agreement with the number of active zones estimated in the
EM studies (~500; Sätzler e t al. 2002; Taschenberger
et al. 2002; see above).
How m any readily re leasable vesicles are available at
the calyx of Held? An early attempt to estimate the
readily releasable pool employed a method based on
cumulative EPSC amplitudes during 100 Hz stimulation
(Schneggenburger et a l. 1999). High-frequency stimula-
tion leads t o strong depression of EPSC amplitudes at the
calyx of Held (Borst et al. 1995; Wang and Kaczmarek 1998;
Schneggenburger et al. 1999; see also below). If depression
is primarily caused by a presynaptic mechanism related
to the depletion of a readily releasable pool, then back-
extrapolation of the cumulative EPSC amplitude to the
onset time of the stimulus train should give an estimate
of the readily releasable pool. This method gave values
of ~600 vesicles (Schneggenburger et al. 1999) or ~800
vesicles (Bollmann et al. 2000). However, it later became
apparent that the depression induced by 100-Hz trains was
not purely presynaptic (Scheuss et al. 2002;Wongetal.
2003; see below). Correcting for the decrease in quantal size
caused by postsynaptic desensitisation suggests that ~900
vesicles are released during the first five pulses of a 100-Hz
train (Scheuss et al. 2002).
Direct stimulation of the presynaptic nerve terminal with
prolonged presynaptic depolarisations has shown that the
number of readily releasable vesicles at the calyx of Held is
even larger. Sakaba and Neher (2001a) have made
simultaneous pre- and postsynaptic voltage-clamp record-
ings under conditions aimed at isolating volt age-gated Ca
2+
currents. Using long (50 ms) presynaptic depolarisations
that evoke EPSCs of 10 nA or larger (see also Wu and
Borst 1999), they analysed the time-course of quantal
release rates by EPSC deconvolution (Neher and Sakaba
2001) and found that ~3,000 vesicles are released in two
kinetically distinct release phases, with time constants of
~2 ms and ~30 ms, respectively (Fig. 5c; Sakaba and Neher
2001b). The double-exponential time-course was inter-
preted as representing release from two classes of readily
releasable vesicles, which are some times called FRP (“fast-
releasing pool”) and SRP (“slow-releasing pool”). The
reason for the different release kinet ics of FRP and SRP
vesicles are not known at present; this may be caused either
by a differential vesicle-to-Ca
2+
-channel localisation on the
nanometer scale (Meinrenken et al. 2002) or by differences
in the Ca
2+
sensitivity between FRP and SRP vesicles, as
observed in chromaffin cells (Voets 2000; for a review, see
Sorensen 2004).
Presynaptic capacitance measurements after inducing
presynaptic Ca
2+
currents also suggest the release of a large
number of vesicles (~3,300–5,000; Sun and Wu 2001)with
a time constant of about 3 ms. Similarly, release evoked by
Ca
2+
uncaging, which raises [Ca
2+
]
i
to ~10–15 μM, has a
fast- and slow-release component with a total release of
~3,000 v esi cle s as estim at ed by EPSC deconvolut ion
(M. Wölfel, X. Lou and R. Schneggenburger, submitted) or
~4,000 vesicles when estimated by presynaptic capacitance
measurements (Wölfel and Schneggenburger 2003). Thus,
there is broad agreement across several studies that strong
322 Cell Tissue Res (2006) 326:311–337

direct Ca
2+
stimuli of the presynaptic nerve terminal
stimulates the release of ~3,000–4,000 vesicles at the calyx
of Held, probably in more than one kinetic release
component.
The large number of readily releasable vesicles from
functional studies reflects the overall “giant” structure of
the calyx of Held, with several hundred presynaptic active
zones in EM reconstructions (~300–700 active zones;
Sätzler et al. 2002; Taschenberger et al. 2002). The number
of readily releasable vesicles as defined in functional
studies (~3,000–4,000; see above) is somewhat larger than
the EM estimates of morphologically docked vesicles,
which were ~1,100–2,800, depending on the exact distance
of vesicles from the membrane and on postnatal age
(Sätzler et al. 2002; Taschenberger et al. 2002). Neverthe-
less, considering that the functional pool size is variable
between cells (Wölfel and Schneggenburger 2003) and that
ultrastructural analysis can only reconstruct one or a few
calyces, the agreement between the functional and the
morphological data is reasonable. If a single AP releases
~150–200 vesicles (see above), then the average release
probability of a given readily releasable vesicle (p
ves
) must
be low (~200/3,000 or 5%–7%). Such a small release
probability p
ves
has consequences for our understanding of
the mechanisms of short-term plasticity at the calyx of Held
(see below).
Presynaptic Ca
2+
signalling and the intracellular Ca
2+
sensitivity of synaptic vesicle fusion
The good accessibility of the calyx of Held to whole-cell
recording has also been used for combined electrophysio-
logical and Ca
2+
-imaging studies investigating presynaptic
Ca
2+
dynamics in a single nerve terminal. Ca
2+
imaging has
shown that the spatially averaged, free Ca
2+
concentration
([Ca
2+
]
i
) signal in the calyx has an amplitude of ~400 nM
and decays with a time constant of 80 – 100 ms (Helmchen
et al. 1997). The fast decay of [Ca
2+
]
i
, which is also
apparent after brief trains of presynaptic stimuli (~40 ms;
Billups and Forsythe 2002) is caused by effective Ca
2+
-
extrusion mechanisms, such as Na
+
-Ca
2+
exchangers, Ca
2+
-
ATPas es in the plasma membrane (Kim et al. 2005 ) and
uptake of Ca
2+
into mitochondria (Billups and Forsythe
2002). In addition, slow Ca
2+
binding to the Ca
2+
-binding
protein parvalbumin, which is present in calyces of Held
(Felmy and Schneggenburger 2004), further accelerates the
decay of spatially averaged [Ca
2+
]
i
(M. Müller, B.
Schwaller and R. Schneggenburger, submitted).
Since transmitter release occurs at the membrane in close
proximity to voltage-gated Ca
2+
channels, the “local”
intracellular Ca
2+
signal relevant for vesicle fusion and
transmitter release must be substantially higher than the
spatially averaged [Ca
2+
]
i
. Indeed, theoretical work in the
1980s has shown that the fast time-course (~1 ms) of
transmitter release during an AP in the nerve terminal can
only be explained by a similarly rapid rise and decay of the
local Ca
2+
signal (Chad and Eckert 1984; Simon and Llinás
1985; Yamada and Zucker 1992; Roberts 1994). Neverthe-
less, the relationship between the presynaptic intracellular
Ca
2+
-concentration ([Ca
2+
]
i
) and transmitter release was
unknown for CNS synapses until recently. The appl ication
of simultaneous pre- and postsynaptic patch-clamp mea-
surements, combined with presynaptic Ca
2+
uncaging,
has been used to study the intracellular Ca
2+
requirements
for vesicle fusion at the calyx of Held (Bollmann et al.
2000; Schneggenburger and Neher 2000; Felmy et al.
2003b; Wölfel and Schneggenburger 2003; Bollmann and
Sakmann 2005; Lou et al. 2005).
Figure 6ashowsaCa
2+
-uncaging experiment at the calyx
of Held (Schneggenburger and Neher 2000). The good
accessibility of the calyx to whole-cell patch-clamp recordings
was used to load the nerve terminal with a mixture of a Ca
2+
-
loaded light-sensitive Ca
2+
chelator (DM-nitrophen) and a
suitable low-affinity Ca
2+
indicator (fura-2FF in the case of
Fig. 6a). A brief flash of light (~1 ms; Schneggenburger and
Neher 2000)oraUV-laserpulse(Bollmannetal.2000)then
photolyzed part of the DM-nitrophen, leading to a rapid
increase in [Ca
2+
]
i
that returned slowly (t
1/2
:~150ms)to
baseline (Fig. 6a, left). Such step-like [Ca
2+
]
i
elevations
triggered transmitter release that was measured as an EPSC
in simultaneous postsynaptic whole-cell recording (Fig. 6a,
right). The amount and the kinetics of release depended on the
[Ca
2+
]
i
reached after the flash.
During Ca
2+
uncaging, spatial gradients of [Ca
2+
]
i
as
occur during the opening of presynaptic Ca
2+
channels are
avoided. Since the Ca
2+
-loaded DM-nitrophen is most
probably homogeneously distributed in the cytosol, Ca
2+
uncaging shoul d generate homog eneous [Ca
2+
]
i
elevations
(Naraghi et al. 1998). Thus, the [Ca
2+
]
i
measured after
uncaging is equal to the [Ca
2+
]
i
signal that drives
transmitter release. An estimate of the local Ca
2+
transient
at the site of vesic le fusion can then be obtained by back-
calculation from the meas ured Ca
2+
sensitivity in a “reverse
approach” (for a review, see Schneggenburger and Neher
2005). First, the relationship between transmitter release
rate and presynaptic [Ca
2+
]
i
is measured and fitted with a
kinetic model of Ca
2+
binding and vesicle fusion, taking
into account the kinetic parameters of transmitter release,
such as Ca
2+
-dependent synaptic delay. The models
incorporate five Ca
2+
-binding steps, since the relationship
between transmitter release and [Ca
2+
]
i
is highly non-linear,
with a slope of ~4–5 in a double-logarithmic data plot
across a range of ~2–8 μM [Ca
2+
]
i
(see also Fig. 6 b, right).
The parameters of the model can then be used to predict the
time-course and amplitude of the local Ca
2+
signal as
Cell Tissue Res (2006) 326:311–337 323

324 Cell Tissue Res (2006) 326:311–337

“seen” by an average readily releasable vesicle. A brief
local Ca
2+
signal of 10–25 μM amplitude, with a half-width
of ~0.5 ms is compatible with transmitter release following
a presynaptic AP (Bollmann et al. 2000; Schneggenburger
and Neher 2000).
Comparison of this transient local Ca
2+
signal at the
release site with the spatially averaged [Ca
2+
]
i
signal of the
whole terminal (~400 nM; see above) shows that the local
Ca
2+
signal is about 20–40 times higher than the spatially
averaged signal and that it decays ~50–100 times faster.
The brief duration of the back-calculated local Ca
2+
signal
(~0.5 ms) was recently confirmed more directly (Bollmann
and Sakmann 2005). In this study, Ca
2+
uncaging induced
by laser-pulses was used to produce rapidly decaying
[Ca
2+
]
i
transients by including millimolar concentrations
of the slow Ca
2+
buffer EGTA in the presynaptic pipette
solution. The fluorescence change of a low-affinity Ca
2+
indicator was measured after the laser-pulses, and the
[Ca
2+
]
i
transient, which was slightly faster than the
measured fluorescence change, was back-calculated accord-
ing to kinetic modelling. EPSC amplitu de and rise-time
(reflecting the amount and kinetics of transmitter release)
depended on the width of the presynaptic [Ca
2+
]
i
transient.
Brief [Ca
2+
]
i
transients with a half-width of less than
0.5 ms were needed to produce EPSCs with a similarly
rapid rising phase as those produced during a presynaptic
AP (Bollmann and Sakman n 2005).
Recently, the intracellular Ca
2+
requirements for low rates
of asynchronous transmitter release have been investigated at
the calyx of Held (Lou et al. 2005;seeFig.6b). The
presynaptic termin al was dialysed with strongly Ca
2+
-
buffered pipette solutions aimed at clamping the resting
[Ca
2+
]
i
to values between 50 nM and 800 nM and the
effective [Ca
2+
]
i
was measured with an indicator dye. This
showed that increasing [Ca
2+
]
i
above the resting value of
~30 nM in the calyx led to a clear increase in the frequency
of spontaneous mEPSCs (Fig. 6b, left) and, thus, that
“spontaneous” release was not completely independent of
[Ca
2+
]
i
. Plotting mEPSC frequency as a function of [Ca
2+
]
i
gave a slope of less than 1 in the range of low [Ca
2+
]
i
(Fig. 6b, right). Interestingly, the intracellular Ca
2+
depen-
dency of mEPSC frequency was shown to be contiguous
with the [Ca
2+
]
i
dependency of release evoked by weak
flashes (Fig. 6b, right, open circles) and with the peak release
rates observed after flashes that elevated [Ca
2+
]
i
to >2 μM
(Fig. 6b, right, clos ed circles; Lo u et al. 2005). The
authors concluded that the same Ca
2+
-sensing mecha-
nism mediated both asynchronous release close to resting
[Ca
2+
]
i
and transient release with Ca
2+
uncaging steps to
higher [Ca
2+
]
i
. In order to explain t he strongly reduced
Ca
2+
cooperativity at low [Ca
2+
]
i
,an“allosteric” model
was prop osed in w hich vesicle fusion could occ ur a t low
rates in the absence of Ca
2+
binding, althoug h binding of
an increasing number of Ca
2+
ions progressively in-
creased the ve sicle fus ion rate co nstants (Lo u et al.
2005). This model is a nalogous to allosteric models for
ligand-gated ion channel activation (e.g. for cyclic-
nucleotide gated channels; L i et al. 1997), where
evidence for ion channel opening in the absence of
ligand binding ha s been obtaine d.
The finding that the Ca
2+
cooperativity in triggering
vesicle fusion is low around resting [Ca
2+
]
i
(~1) and that it
increases to a value of ~4 with [Ca
2+
]
i
stimuli of higher
amplitudes (Fig. 6b, right) is also likely to be of particular
functional relevance. If a high cooperativity mechanism
operated close to baseline [Ca
2+
]
i
, then small sub-micro-
molar elevations of residual [Ca
2+
]
i
would produce strong
increases in transmitter release. This would be highly
undesirable as it would generate excess ive “tonic” turn-
over of transmitter quanta at a synapse designed to transmit
information phasically, locked to each presynaptic AP. The
data by Lou et al. (2005) also reveal an amazing dynamic
range covered by the Ca
2+
regulation of transmitter release:
from a “spontaneous” release rate of ~1 Hz at resting
[Ca
2+
]
i
to a peak transmitter release of ~300 vesicles/ms
during the AP (Schneggenburger and Neher 2000;
Taschenberger et a l. 2005). Thus, the presynaptic AP
transiently increases the rat e of transmitter release by
Fig. 6 Intracellular Ca
2+
sensitivity of synaptic vesicle fusion.
a Presynaptic Ca
2+
uncaging at the calyx of Held. Left, top A calyx
filled with fura-2FF and Ca
2+
-loaded DM-nitrophen imaged at low
and high resolution during and after the experiment, respectively. Left,
bottom Three flashes with different intensities elevated the presynaptic
intracellular Ca
2+
concentration ([Ca
2+
]
i
) to ~8, 12 and 25 μM. Right,
top The EPSCs evoked by these [Ca
2+
]
i
elevations. Right, bottom
From the EPSCs, the transmitter release rates were determined by
EPSC deconvolution. Adapted, with permission from MacMillan,
from Schneggenburger and Neher (2000). b Ca
2+
dependency of
transmitter release over an extended range of presynaptic [Ca
2+
]
i
. Left
Simultaneous pre- and postsynaptic recordings were made at resting
[Ca
2+
]
i
in the presynaptic terminal (top) or with strongly Ca
2+
-
buffered solutions aimed at elevating the presynaptic [Ca
2+
]
i
above
resting values (middle, bottom). Note that elevating [Ca
2+
]
i
above
baseline leads to an increased transmitter release rate, as is apparent by
the increased mEPSC frequency. Right The Ca
2+
sensitivity of
asynchronous release measured by infusing terminals with strongly
Ca
2+
-buffered solutions (open symbols; see b, left) is contiguous with
the Ca
2+
sensitivity as measured by Ca
2+
uncaging (filled symbols).
The data were fitted with an “allosteric” model of Ca
2+
activation of
vesicle fusion (black line). Adapted, with permission from MacMillan,
from Lou et al. (2005). c Botulinus toxin A (BotTx A), which
specifically cleaves the SNARE-protein SNAP-25, induces a right-
ward-shift of the intracellular Ca
2+
sensitivity of vesicle fusion. Left
Ca
2+
-uncaging stimulus (arrow), followed by a strong presynaptic
depolarisation in a control cell. Middle Same protocol applied in a
calyx recorded with BotTx A added to the presynaptic patch pipette.
Right Relationship between release rates (normalised to the number of
readily releasable vesicles) and presynaptic [Ca
2+
]
i
after the flash is
shifted to the right in the presence of BotTx A. Adapted, with
permission, from Sakaba et al. (2005), copyright 2005 AAAS
R
Cell Tissue Res (2006) 326:311–337 325

~300,000-fold over resting values or by more than five orders
of magnitude for a [Ca
2+
]
i
elevation of approximately two to
three orders of magnitude (Fig. 6b, right). This is only
possible with a highly non-linear mechanism coupling the
rise of [Ca
2+
]
i
to transmitter release. Synaptotagmin-1 has
been identified as the Ca
2+
sensor for a fast component of
transmitter release in hippocampal neurons (Geppert et al.
1994; Fernández-Chacón et al. 2001). At the calyx of Held,
where synaptotagmin-1 is not expressed, the close homo-
logue synaptotagmin-2 might play this role (Pang et al.
2006). However, the molecular mechanism responsible for
the high cooperativity of Ca
2+
in vesicle fusion is still
unknown and needs to be addressed in future work.
A recent study has analysed the molecular determinants
of Ca
2+
-induced vesicle fusion by perfusing calyces with
various botulinum and tetatanus neurotoxins (Sakaba et al.
2005). These toxins proteolytica lly cleave SNAREs at
specific sites and thereby inhibit transmitter release (for a
review, see Humeau et al. 2000). When Sakaba et al. (2005)
included BotTx C1 or tetanus toxin (TeT), which specifi-
cally cleaves syntaxin or synaptobrevin, respectively, in the
presynaptic recording pipette, release was reduced but the
remaining release has similar kinetics (an “all-or-none”
effect of the toxins). On the other hand, in the presence of
BotTx A, which cleaves off the last nine amino acids of
SNAP-25, release in response to a Ca
2+
-uncaging step to
about 10 μM[Ca
2+
]
i
was nearly abolished, although higher
levels of Ca
2+
uncaging could almost fully rescue release in
the presence of BotTx A (Fig. 6c, middle; Sakaba et al.
2005). Analysis of the release rate versus [Ca
2+
]
i
relation-
ship over a range of ~3–60 μM [Ca
2+
]
i
showed that BotTx
A induced an approximately four-fold rightward shift of the
intracellular Ca
2+
sensitivity of vesicle fusion, without a
change in the apparent Ca
2+
cooperativity as revealed by
the similar slopes in the double-logarithmic plots (Fig. 6c,
right). Thus, interfering with the integrity of the presynaptic
SNARE-complex can lead to a decrease in the Ca
2+
sensitivity of release.
Mechanisms of short-term plasticity
Repetitive stimulation of afferent fibres leads to a pro-
nounced frequency-dependent depression of EPSCs at the
calyx of Held synapse (Borst et al. 1995; von Gersdorff et
al. 1997; Wang and Kaczmarek 1998). Thus, the calyx is a
depressing synapse but facilitation of the second EPSC
amplitude is sometimes observed in response to high-
frequency trains (e.g. Schneggenburger et al. 1999). The
facilitation can be uncovered by lowering the initial release
probability with low extracellular [Ca
2+
] (Barnes-Davies
and Forsythe 1995; Borst et al. 1995) or by lowering the
quantal output during the first stimulation in paired pre- and
postsynaptic whole-cell recording (Sakaba and Neher
2001a; Felmy et al. 2003b) and under conditions where
postsynaptic desensitisation has been minimised (Wong et
al. 2003). Felmy et al. (2003a,b) have studied the
mechanism of short-term facilitation and found, by using
Ca
2+
uncaging, that the intracellular Ca
2+
sensitivity of
vesicle fusion is unchanged during facilitation. Following
prolonged high-frequency stimulation, the calyx of Held
shows a pronounced post-tetanic potentiation of transmitter
release, which is mediated by a mechanism dependent on
residual Ca
2+
, but different from that implicated in short-term
facilitation (Habets and Borst 2005; Korogod et al. 2005).
Thus, short-term plasticity at the calyx of Held appears to be
similar to that at the neuromuscular junction, with depression
prevailing during high-frequency trains at normal release
probability and a transient overshoot of transmission follow-
ing such trains (Liley and North 1953; Elmquist and Quastel
1965). Longer-lasting forms of plasticity have so far not been
apparent at the calyx of Held.
Direct whole-cell recordings from the nerve terminal
allow assessment of whether changes in the AP wave form
or changes in A P-media ted Ca
2+
influx contribute to
synaptic depression. During the strong depression induced
by 100-Hz trains, AP amplitude decreases slightly and
becomes broader; however, presynaptic voltage-clamp
experiments show similar Ca
2+
-current integrals activated
by early and late AP waveforms (Borst and Sakmann
1999), suggesting that changes in AP waveform may not
contribute to the depression of release during brief high-
frequency trains. At lower frequencies (2–30 Hz), the Ca
2+
current decreas ed with repetitive stimulation because of
Ca
2+
-current inactivation (Xu and Wu 2005). Although the
relative reduction of Ca
2+
current is small (Fig. 7a, right),
with the high-power relationship between Ca
2+
current and
release (3.6 as measured by Xu and Wu 2005), even a small
decrease is expected to be highly efficient in modulating
transmitter release and the decrease expected by a simple
3.6
th
power relationship predicted the observed depression
of EPSC amplitudes (Fig. 7a, right; Xu and Wu 2005).
Thus, Ca
2+
-current inactivation, which was first shown to
mediate the “deep” depression observed after prolonged
high-frequency stimulation at the calyx of Held (Forsythe et
al. 1998), also contributes to the depression observed
during the onset of low-to-intermediate frequency trains
(2–30 Hz).
Depletion of a readily releasable vesicle pool was also
postulated to contribute to depression at the calyx of Held
(von Gersdorff et al. 1997; Schneggenburger et al. 1999;
Weis et al. 1999). In the following years it became clear,
however, that the AMPA-R desensitisation that had been
observed earlier at glutamatergic chick endbulb synapses
(Trussell et al. 1993; Otis et al. 1996) might play a larger
role in depression at the calyx of Held than initially
326 Cell Tissue Res (2006) 326:311–337

suspected. Neher and Sakaba (2001) have f ound that
cyclothiazide, which very effectively slows the rate of
desensitisation of AM PA-R in MNTB principal cells
(Koike-Tani et al. 2005), reduces the depression of EPSCs
observed in simultaneous pre- and postsynaptic recordings.
Using EPSC fluctuation a nalysis o f the peak EPSC
amplitudes during 100-Hz trains at elevated release prob-
ability, Scheuss et al. (2002) estimated that, during the third
to fifth EPSC in a 100-Hz train, the quantal amplitude q
was reduced to ~35% of its initial value (see Fig. 7c). The
reduction of q was less strong in the presence of CTZ; with
CTZ and kynurenic acid (a low-affinity AMPA-R antago-
nist), q was stable throughout the first five pulses of a 100-
Hz train. Thus, the reduction of q during the second to the
fifth stimulus of a 100-Hz train is probably caused by
desensitisation of postsynaptic AMPA-Rs. Taschenberger et
al. (2002) and Wong et al. (2003) have also shown, by
using the low-affinity fast-off-rate antagonist, kynurenic
acid (and CTZ in some cases), that postsynaptic mecha-
nisms contribute to depression (Fig. 7b). The postsynaptic
contribution to depression is reduced with developmental
maturation (Taschenberger et a l. 2002, 2005), perhaps
because changes in calyx morphology allow faster diffusion
of glutamate from the synaptic cleft (Renden et al. 2005).
The extent to which vesicle pool depletion contributes to
depression remains unclear. Several different mechanisms of
depression are present at the calyx of Held (see above) and
with a large pool of readily releasable vesicles (see above;
Schneggenburger and Neher 2000; Sakaba and Neher 2001a;
Sun and Wu 2001; Wölfel and Schneggenburger 2003), the
role of vesicle pool depletion early during high-frequency
trains seems less significant. However, a key finding is that
Fig. 7 Mechanisms of short-term depression at the calyx of Held.
a Inactivation of presynaptic Ca
2+
current contributes to synaptic
depression. Left A presynaptic AP (top) was used as a voltage-clamp
command waveform and was applied twice with an interval of 500 ms.
The second pulse (red traces) evoked a smaller Ca
2+
current (middle)
and a smaller EPSC (bottom). Middle I
Ca
(top)andEPSCs(bottom)
evoked by 10 short presynaptic depolarisations at 2 Hz. Note the
decrease in I
Ca
and EPSCs from the second depolarisation onwards.
Right The relative I
Ca
and EPSC amplitudes plotted as a function of
stimulus number (black symbols). Because of the high</