A low-affinity antagonist reveals saturation and desensitization in mature synapses in the auditory brain stem.
ABSTRACT Postsynaptic receptor desensitization has been observed to contribute to depression in immature synapses. However, it is not clear whether desensitization persists and causes depression in mature synapses. We investigate this issue at the endbulb of Held, the synapse made by auditory nerve (AN) fibers onto bushy cells (BCs) of the anteroventral cochlear nucleus, where depression could influence the processing of sound information. Experiments using cyclothiazide (CTZ) have implicated desensitization in endbulbs from postnatal day 16 (P16) to P21 mice, but application of γ-D-glutamylglycine (DGG) did not reveal desensitization in endbulbs >P22. To reconcile these findings, we have studied the effects of both CTZ and DGG on endbulbs from P5 to P40 CBA/CaJ mice. In paired-pulse protocols, both CTZ and DGG reduced depression in all ages at intervals <10 ms, consistent with their effects preventing desensitization. However, DGG increased depression at intervals >20 ms, consistent with DGG's use to prevent saturation. DGG application revealed receptor saturation even under conditions of very low release probability. Preventing desensitization by CTZ occluded the effects of DGG on desensitization and revealed the effects of saturation at short intervals. We developed an approach to separate DGG's effect on saturation from its effect on desensitization, which showed that desensitization has an impact during bursts of auditory nerve activity. Dynamic-clamp experiments indicated that desensitization can reduce BC spike probability and increase latency and jitter. Thus desensitization may affect sound processing in the mature auditory system.
- SourceAvailable from: Miloslav Sedlacek[Show abstract] [Hide abstract]
ABSTRACT: Feed-forward inhibition (FFI) represents a powerful mechanism by which control of the timing and fidelity of action potentials in local synaptic circuits of various brain regions is achieved. In the cochlear nucleus, the auditory nerve provides excitation to both principal neurons and inhibitory interneurons. Here, we investigated the synaptic circuit associated with fusiform cells (FCs), principal neurons of the dorsal cochlear nucleus (DCN) that receive excitation from auditory nerve fibers and inhibition from tuberculoventral cells (TVCs) on their basal dendrites in the deep layer of DCN. Despite the importance of these inputs in regulating fusiform cell firing behavior, the mechanisms determining the balance of excitation and FFI in this circuit are not well understood. Therefore, we examined the timing and plasticity of auditory nerve driven FFI onto FCs. We find that in some FCs, excitatory and inhibitory components of FFI had the same stimulation thresholds indicating they could be triggered by activation of the same fibers. In other FCs, excitation and inhibition exhibit different stimulus thresholds, suggesting FCs and TVCs might be activated by different sets of fibers. In addition, we find that during repetitive activation, synapses formed by the auditory nerve onto TVCs and FCs exhibit distinct modes of short-term plasticity. Feed-forward inhibitory post-synaptic currents (IPSCs) in FCs exhibit short-term depression because of prominent synaptic depression at the auditory nerve-TVC synapse. Depression of this feedforward inhibitory input causes a shift in the balance of fusiform cell synaptic input towards greater excitation and suggests that fusiform cell spike output will be enhanced by physiological patterns of auditory nerve activity.Frontiers in Neural Circuits 07/2014; 8:78. · 2.95 Impact Factor
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ABSTRACT: Spherical bushy cells (SBCs) in the anteroventral cochlear nucleus respond to acoustic stimulation with discharges that precisely encode the phase of low-frequency sound. The accuracy of spiking is crucial for sound localization and speech perception. Compared to the auditory nerve input, temporal precision of SBC spiking is improved through the engagement of acoustically evoked inhibition. Recently, the inhibition was shown to be less precise than previously understood. It shifts from predominantly glycinergic to synergistic GABA/glycine transmission in an activity-dependent manner. Concurrently, the inhibition attains a tonic character through temporal summation. The present study provides a comprehensive understanding of the mechanisms underlying this slow inhibitory input. We performed whole-cell voltage clamp recordings on SBCs from juvenile Mongolian gerbils and recorded evoked inhibitory postsynaptic currents (IPSCs) at physiological rates. The data reveal activity-dependent IPSC kinetics, i.e., the decay is slowed with increased input rates or recruitment. Lowering the release probability yielded faster decay kinetics of the single- and short train-IPSCs at 100 Hz, suggesting that transmitter quantity plays an important role in controlling the decay. Slow transmitter clearance from the synaptic cleft caused prolonged receptor binding and, in the case of glycine, spillover to nearby synapses. The GABAergic component prolonged the decay by contributing to the asynchronous vesicle release depending on the input rate. Hence, the different factors controlling the amount of transmitters in the synapse jointly slow the inhibition during physiologically relevant activity. Taken together, the slow time course is predominantly determined by the receptor kinetics and transmitter clearance during short stimuli, whereas long duration or high frequency stimulation additionally engage asynchronous release to prolong IPSCs.Frontiers in Neural Circuits 01/2014; 8:145. · 2.95 Impact Factor
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ABSTRACT: Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.PLoS Genetics 12/2014; 10(12):e1004823. · 8.17 Impact Factor
98:807-820, 2007. First published 16 May 2007;
Joachim Hermann, Michael Pecka, Henrique von Gersdorff, Benedikt Grothe and
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Synaptic Transmission at the Calyx of Held Under In Vivo–Like
Joachim Hermann,1Michael Pecka,1Henrique von Gersdorff,1,2Benedikt Grothe,1and Achim Klug1
1Ludwig-Maximilians-University, Munich, Germany; and2The Vollum Institute, Oregon Health and Science University, Portland, Oregon
Submitted 29 March 2007; accepted in final form 13 May 2007
Hermann J, Pecka M, von Gersdorff H, Grothe B, Klug A.
Synaptic transmission at the calyx of Held under in vivo–like activity
levels. J Neurophysiol 98: 807–820, 2007. First published May 16,
2007; doi:10.1152/jn.00355.2007. One of the hallmarks of auditory
neurons in vivo is spontaneous activity that occurs even in the absence
of any sensory stimuli. Sound-evoked bursts of discharges are thus
embedded within this background of random firing. The calyx of Held
synapse in the medial nucleus of the trapezoid body (MNTB) has been
characterized in vitro as a fast relay that reliably fires at high stimulus
frequencies (?800 Hz). However, inherently due to the preparation
method, spontaneous activity is absent in studies using brain stem
slices. Here we first determine in vivo spontaneous firing rates of
MNTB principal cells from Mongolian gerbils and then reintroduce
this random firing to in vitro gerbil brain stem synapses at near-
physiological temperature. After conditioning synapses with afferent
fiber stimulation for 2 min at Poisson averaged rates of 20, 40, and 60
Hz, we observed a number of differences in the properties of synaptic
transmission between conditioned and unconditioned synapses. Fore-
most, we observed reduced steady-state EPSC amplitudes that de-
pressed even further during an embedded short-stimulation train of
100, 300, or 600 Hz (a protocol that thus simulates in vitro what
probably occurs at the in vivo MNTB after a short sound stimulus in
a silent background). Accordingly, current-clamp, dynamic-clamp,
and loose-patch recordings revealed a number of action potential
failures at the postsynaptic cell during high-frequency–stimulation
trains, although the initial onset of evoked activity was still transmit-
ted with higher fidelity. We thus propose that some in vivo auditory
synapses are in a tonic state of reduced EPSC amplitudes as a
consequence of high spontaneous spiking and this in vivo–like con-
ditioning has important consequences for the encoding of signals
throughout the auditory pathway.
I N T R O D U C T I O N
The calyx of Held is a large synaptic terminal innervating
principal neurons of the medial nucleus of the trapezoid body
(MNTB) (Forsythe 1994; Held 1893; Kuwabara et al. 1991;
Smith et al. 1991). MNTB neurons sign-invert calyceal exci-
tation into glycinergic inhibition to various nuclei in the audi-
tory brain stem (Banks and Smith 1992; Bledsoe et al. 1990;
Moore and Caspary 1983; Spangler et al. 1985; Thompson and
Schofield 2000). In vitro, the signal derived from the calyx
generates large excitatory postsynaptic currents (EPSCs) with
a short synaptic delay (Barnes-Davies and Forsythe 1995;
Borst and Sakmann 1996; Sakaba and Neher 2001; Taschen-
berger et al. 2002). Speed and fidelity of synaptic transmis-
sion are considered very reliable up to several hundred
Hertz in mature animals (Futai et al. 2001; Joshi et al. 2004;
Taschenberger and von Gersdorff 2000; Wu and Kelly
1993), leading to a view of the calyx of Held as a very
reliable relay synapse.
All the in vitro work mentioned earlier was performed in
brain slices. Inherently, auditory brain slice preparations
differ from intact brains in various parameters, including
spontaneous activity. In vivo, neurons of the auditory brain
stem fire spontaneously at frequencies that vary from ?1 to
?100 Hz, a property that results mainly from the dynamics
of the transduction channels in the cochlear hair cells
(Geisler et al. 1985; Hudspeth 1997; Kiang 1965; Liberman
1978; Roberts et al. 1988), resulting in spontaneous firing of
the auditory nerve (Geisler et al. 1985; Liberman 1978).
Spontaneous firing can also be observed in many brain stem
nuclei including the cochlear nucleus (Brownell 1975; Gold-
berg and Brownell 1973; Joris et al. 1994; Schwarz and Puil
1997; Spirou et al. 1990, 2005) and MNTB (Kadner et al.
2006; Kopp-Scheinpflug et al. 2003; Smith et al. 1998;
Sommer et al. 1993).
In an intact brain, MNTB neurons fire spontaneously at
levels, which might be suitable to chronically induce some
forms of short-term plasticity, such as synaptic depression or
facilitation (Schneggenburger et al. 2002; von Gersdorff and
Borst 2002). Sound stimuli, i.e., streams of high-frequency
activity embedded in this spontaneous firing (Klyachko and
Stevens 2006), would then be processed by the synapse on the
background of chronic depression and/or facilitation (Fig. 1A).
Because of the nature of the brain slice preparation, spontane-
ous activity and its potential effects on short-term plasticity
might be lost in standard in vitro recordings (Fig. 1B). If that
were the case, properties of synaptic transmission in the calyx
of Held under in vivo conditions may be different from those
commonly observed in vitro.
This study investigates synaptic transmission in the calyx of
Held under in vivo–like spontaneous activity levels. We first
measured the rates and statistical properties of spontaneous
firing in the MNTB of Mongolian gerbils (Meriones unguicu-
latus) in vivo. Subsequently, we stimulated the afferent fibers
that give rise to calyceal inputs in gerbil MNTB brain slices at
physiological temperature for prolonged periods of time with
stimuli that mimicked the random spontaneous activity as
closely as possible (Fig. 1C). We assessed changes in synaptic
transmission resulting from this long-term stimulation, such as
synaptic currents, the degree of depression, recovery from
depression, and finally the spiking properties of “spontane-
ously active” neurons.
Address for reprint requests and other correspondence: A. Klug, Neurobi-
ology Group, Dept. Biology II, Grosshaderner Strasse 2, 82152 Martinsried,
Germany (E-mail: firstname.lastname@example.org).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
J Neurophysiol 98: 807–820, 2007.
First published May 16, 2007; doi:10.1152/jn.00355.2007.
807 0022-3077/07 $8.00 Copyright © 2007 The American Physiological Societywww.jn.org
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M E T H O D S
In vivo recordings
Auditory responses from 36 single neurons were recorded in 16
Mongolian gerbils (Meriones unguiculatus) of both sexes, aged be-
tween 21 and 60 days. We found no systematic differences in aurality,
firing frequency, threshold, characteristic frequency, or other response
parameters of neurons, which depended on the age of the animal (data
not shown), and thus the data from all 36 neurons were pooled. In our
sample there was also no covariation between spontaneous activity
and auditory threshold or between spontaneous activity and a neuron’s
characteristic frequency (regression analyses; data not shown). For
experimental reasons, the reported in vivo data were recorded from
MNTB postsynaptic neurons, not calyces of Held or globular bushy
cells in the anteroventral cochlear nucleus. The underlying assumption
is that the spontaneous activity in the MNTB is not high enough to
induce synaptic failures at the calyx of Held synapse, such that the
presynaptic spike frequency is identical to the postsynaptic activity.
Data were collected simultaneously for this study and a different
project involving MNTB response properties (data not shown). All
experiments complied with institutional guidelines and were approved
by the appropriate government authorities (Reg. Oberbayern AZ
by an initial intraperitoneal injection (0.5 ml/100 g body weight) of
ketamine (20%) and xylazine (2%, both in physiological NaCl).
During surgery and recordings, a dose of 0.05 ml of the same mixture
was applied subcutaneously in scheduled intervals that were based on
the animal’s body weight. Constant body temperature was maintained
using a thermostatically controlled heating blanket.
Skin and tissue covering the upper part of the skull were removed
and a small metal rod was mounted to the frontal part of the skull
using UV-sensitive dental-restorative material (Charisma, Heraeus
Kulzer, Hanau, Germany). Custom-made earphone holders were at-
tached to the gerbil head, allowing for the safe insertion of earphones
or probe tube microphones into the ear canal. The animal was then
transferred to a sound-attenuated chamber and mounted in a custom-
made stereotaxic instrument (Schuller et al. 1986). The animal’s
position in the recording chamber was standardized with reference to
stereotaxic landmarks on the skull (Loskota et al. 1974). For electrode
Before surgery, animals were anesthetized
penetrations to the MNTB, a small hole of approximately 1 mm2was
cut into the skull lateral to the lambdoid suture. Micromanipulators
were used to position the recording electrode according to landmarks
on the brain surface and a reference point, which was used for all
penetrations. The meninges overlying the cortex were removed and
saline was applied to the opening to prevent dehydration of the brain.
Typical recording sessions lasted 10–14 h. After successful record-
ings, the animal was killed by injection of an overdose of chloral
hydrate (Sigma–Aldrich Chemie, Munich, Germany). The last elec-
trode position was then marked with a current-induced lesion (20 ?A
for 80–120 s). The head was fixated in 4% paraformaldehyde and
prepared for anatomical processing. Transverse sections were cut and
Nissl-stained to verify the recording sites. An example of an anatom-
ical verification is shown in Supplemental Fig. 1C.1The lesion site
can be clearly seen in the center of the left MNTB.
RECORDINGS OF NEURAL ACTIVITY.
corded extracellularly using 10-M? glass electrodes filled with 1 M
NaCl. The recording electrode was advanced under remote control,
using a piezodrive (Inchworm controller 8200, EXFO Burleigh Prod-
ucts Group, Victor, NY). Extracellular action potentials were recorded
by an electrometer (npi electronics, Tamm, Germany or Electro 705,
WPI, Berlin, Germany), a 50/60-Hz noise eliminator (Humbug, Quest
Scientific Instruments, North Vancouver, BC, Canada), a band-pass
filter (VBF/3, Bortolin Kemo, Porcia, Italy), and an amplifier (model
7607, Toellner Electronic Instrumente, Herdecke, Germany) and sub-
sequently fed into the computer by an A/D-converter [RP2-1, Tucker-
Davis Technologies (TDT), Alachua, FL]. Clear isolation of action
potentials from a single neuron (signal-to-noise ratio ?5) was
achieved by visual inspection on a spike-triggered oscilloscope and by
off-line spike-cluster analysis (Brainware, TDT). Two examples of
recorded single-cell spike waveforms are shown in Supplemental Fig.
1, A and B. The unit in supplemental Fig. 1A is an example of a neuron
with a low spontaneous rate (10 Hz), whereas supplemental Fig. 1B
shows an example of a neuron with a very high spontaneous rate (107
Hz). Both units were recorded from the same animal; histological
verification of the recording site is shown in Supplemental Fig. 1C.
Single-unit responses were re-
STIMULUS PRESENTATION AND RECORDING PROTOCOLS.
were generated at a 50-kHz sampling rate using TDT System III.
1The online version of this article contains supplemental data.
spontaneously active. Responses to sound stimuli, indicated by high-frequency bursts, are embedded in the background activity. B: in a typical slice preparation,
the background activity is not present, such that trains of high-frequency stimuli used to imitate responses to sound are embedded in periods of complete silence.
C: our experimental approach attempted to bring the spontaneous activity back into slice preparations. Responses to simulated sound stimuli were tested to obtain
a baseline of synaptic properties, then spontaneous activity was simulated for several minutes, then the same “sound stimuli” were tested again while they were
embedded in background activity. At the end of data collection, the cell was allowed to recover and the same set of simulated sound stimuli was tested again
to assess the level of recovery.
A: illustration of in vivo activity in the medial nucleus of the trapezoid body (MNTB). In the intact brain, MNTB neurons are chronically
808HERMANN, PECKA, VON GERSDORFF, GROTHE, AND KLUG
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Digitally generated stimuli were converted to analog signals (DA3-2/
RP2-1, TDT), attenuated (PA5, TDT), and delivered to earphones
(MDR-EX70LP, Sony, Berlin, Germany).
The standard stimulus was a 200-ms toneburst with a rise/fall time
of 5 ms, presented at a repetition rate of 2 Hz. Stimulus presentation
was randomized. To search for acoustically evoked responses, noise
stimuli were delivered binaurally. When an auditory neuron was
encountered, we first determined its best frequency (BF) and absolute
threshold audiovisually to set stimulus parameters subsequently con-
trolled by the computer. The frequency that elicited responses at the
lowest sound intensity was defined as BF, the lowest sound intensity
evoking a noticeable response at BF as threshold. These properties
were confirmed by off-line analysis of the frequency versus level
response areas. Monaural pure tones to each ear and binaural pure
tones without interaural intensity or time differences were presented
to define the aurality of the neuron. MNTB neurons responded to
stimulation of the contralateral ear only, with a tonic/primary-like
firing pattern, and were not affected by stimulation of the ipsilateral
Spontaneous activity of a neuron was determined by recording
action potentials in several 5-s-long intervals without sound stimula-
tion and averaging the measured firing rate. All quantifications in this
study are based on off-line analysis with the software packages
Brainware (TDT), Matlab (The MathWorks, Natick, MA), and IGOR
(WaveMetrics, Lake Oswego, OR).
In vitro recordings
Slices of brain stem were prepared from Mongolian gerbils (Meri-
ones uniguiculatus) aged 14 to 19 days (posthearing animals). Data
from these different ages were pooled because no age-dependent
variations in synaptic amplitudes, degree of depression, response to
conditioning, or firing probability were observed (data not shown).
rane inhalation (Isofluran Curamed, Curamed Pharma, Karlsruhe,
Germany) and decapitated. The brain stem was dissected out under
ice-cold dissection ringer (125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2,
0.1 mM CaCl2, 25 mM glucose, 1.25 mM NaH2PO4, 25 mM
NaHCO3, 0.4 mM ascorbic acid, 3 mM myoinositol, 2 mM pyruvic
acid; all chemicals from Sigma–Aldrich). Sections (200–250 ?m)
were cut with a vibratome (VT1000S, Leica, Wetzlar, Germany).
Slices were transferred to an incubation chamber containing extracel-
lular solution (ECS) (125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 2
mM CaCl2, 25 mM glucose, 1.25 mM NaH2PO4, 25 mM NaHCO3,
0.4 mM ascorbic acid, 3 mM myoinositol, 2 mM pyruvic acid; all
chemicals from Sigma–Aldrich) and bubbled with 5% CO2-95% O2.
Slices were incubated for 1 h at 37°C, after which the chamber was
brought to room temperature. Recordings were obtained within 4–5 h
Animals were briefly anesthesized by isoflu-
WHOLE CELL RECORDINGS.
37°C. After incubation, slices were transferred to a recording chamber
and continuously superfused with ECS at 3–4 ml/min through a
gravity-fed perfusion system. MNTB neurons were viewed through a
Zeiss Axioskop 2 FS microscope equipped with DIC optics and a ?40
water-immersion objective (Zeiss, Oberkochen, Germany). Whole
cell recordings were made with an EPC 10 double amplifier (HEKA
Instruments, Lambrecht/Pfalz, Germany). Signals were filtered at
5–10 kHz and subsequently digitized at 20–100 kHz using Patchmas-
ter Version 2.02 software (HEKA). Uncompensated series resistance,
between 5.5 and 15 M?, was compensated to values between 2.1 and
5.8 M? with a lag time of 10 ?s. Potential changes in series resistance
were monitored throughout the recordings and data collection was
discontinued whenever series resistance changed by ?2 M?. All
voltages are corrected for a ?12-mV junction potential.
Patch pipettes were pulled from 1.2-mm borosilicate glass (WPI) or
1.5-mm borosilicate glass (Harvard Instruments, Kent, UK) using a
All recordings were performed at 36–
Sutter P-97 electrode puller (Sutter Instrument, Novato, CA) or a
DMZ Universal Puller (Zeitz Instruments, Munich, Germany). Pi-
pettes were filled with potassium gluconate–based internal solution
for current-clamp recordings (120 mM K-gluconate, 4 mM MgCl2, 10
mM HEPES, 5 mM EGTA, 10 mM tris-phosphocreatine, 4 mM
Na2-ATP, 0.3 mM tris-GTP, 0.5 mM CaCl2; all chemicals from
Sigma–Aldrich) or cesium methanesulfonate–based solution for volt-
age-clamp recordings (125 mM CsMeSO3, 4.5 mM MgCl2, 9 mM
HEPES, 5 mM EGTA, 14 mM tris-phosphocreatine, 4 mM Na2-ATP,
0.3 mM tris-GTP, 1.5 mM CaCl2, all chemicals from Sigma–Aldrich).
During all recordings, 500 nM strychnine hydrochloride (Sigma–
Aldrich) and 20 ?M SR95531 were added to the bath to block
glycinergic and GABAA-ergic inhibition, respectively. During volt-
age-clamp recordings, 5 mM QX-314 (Alomone Labs, Jerusalem,
Israel) was added to the pipette fill to eliminate sodium currents.
STIMULATION OF SYNAPTIC INPUTS.
by midline stimulation of the calyceal input fiber bundle with a 5-M?
bipolar stimulation electrode (matrix electrodes with 270-?m dis-
tance; FHC, Bowdoinham, ME). Stimuli were 100-?s-long square
pulses of 10 to 40 V delivered with an STG 2004 computer-controlled
four-channel stimulator (Multi Channel Systems, Reutlingen, Ger-
many) and a stimulation isolation unit (Iso-flex, AMPI, Jerusalem,
Israel). The stimulator permitted completely independent uploading
and operation of the four channels, allowing the seamless integration
and thus true embedding of simulated auditory signals (i.e., high-
frequency bursts) in the simulated spontaneous activity. Spontaneous
activity was simulated by using 20-, 40-, and 60-Hz Poisson-distrib-
uted stimulus trains (see Figs. 1 and 2, C and E). Sound-evoked
activity was simulated by short trains consisting of 20 stimuli at 100,
300, or 600 Hz.
Synaptic currents were elicited
were simulated with an SM-1 amplifier (Cambridge Conductance,
Cambridge, UK). The 10–90% rise of the current output in response
to a voltage change for this amplifier was 290 ns. Reversal potentials
were set to 0 mV for the excitatory postsynaptic conductances
(EPSGs). The conductance waveforms used were previously recorded
as EPSCs in voltage-clamp mode. After extrapolating the artifacts,
EPSGs were normalized. All conductance values correspond to peak
conductances. In experiments in which background leak was added, a
constant step command was fed from the computer into the conduc-
tance-clamp amplifier by a separate channel and the reversal potential
for this channel was set to ?60 mV. The separately calculated output
of both channels was added together and fed to the HEKA amplifier.
Metrics), MS Excel 2004 (Microsoft, Redmond, WA), and Matlab 7
(The MathWorks). Unless otherwise noted, all errors are reported as
standard error. Statistical significance was tested with a Student’s
t-test, unless otherwise noted. Significant differences are marked with
a single asterisk for values of P ? 0.05 and with a double asterisk for
P ? 0.01.
Data were analyzed in IGOR 5 (Wave-
R E S U L T S
In vivo spontaneous firing rates of MNTB cells
The first goal was to determine the spontaneous firing rates
of MNTB neurons in the intact brain of Mongolian gerbils
(Meriones unguiculatus). Neural activity was recorded in vivo
from single cells in the MNTB with standard extracellular
recording techniques. When a neuron was encountered and
isolated, its basic response features such as aurality, auditory
threshold, and frequency tuning were assessed. Among the 36
neurons from which activity was recorded, thresholds for
sound stimulation ranged from 0 to 60 dB SPL (mean ? 32 ?
809 SYNAPTIC TRANSMISSION AT THE CALYX OF HELD
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by guest on June 1, 2013
2.8 dB SPL) and characteristic frequencies were between 486
Hz and 16.8 kHz. Consistent with known input patterns to the
MNTB, all neurons could be excited when sound was pre-
sented to the ear contralateral to the recording site. None of the
neurons showed any effects of ipsilateral stimulation.
After a neuron’s basic response properties to auditory stim-
ulation were assessed, its spontaneous firing rate in the absence
of sound stimulation was measured over ?50 s and average
discharge rates were calculated and defined as the neuron’s
spontaneous firing rate. Among the 36 neurons, spontaneous
firing rates ranged from 0.15 to 110 Hz (Fig. 2A). The mean
spontaneous rate was 24.9 ? 5.5 Hz. Short clips of spike trains
are shown in Fig. 2B. The spontaneous rates of these neurons
were 16, 40, and 69 Hz, respectively. An analysis of the
interspike intervals (ISIs) revealed that the spikes are near-
Poisson distributed with the exception that very short ISIs (?1
ms) are underrepresented (three ISI histograms in Fig. 2C).
Introducing spontaneous rates into slice preparations of
Based on these in vivo data, three representative frequencies
of spontaneous rates were chosen for stimulation of the in vitro
brain slice preparations: 20, 40, and 60 Hz (Fig. 2D). Distri-
bution of the spike events in each one of these trains was
chosen to be near-Poisson, to imitate the in vivo spontaneous
activity as closely as possible (Fig. 2E). MNTB calyceal input
fibers were stimulated with these spike trains for prolonged
periods of time (?2 min) and voltage-clamp recordings were
performed from MNTB principal neurons. During the 2-min
conditioning, 7,200 Poisson-distributed stimuli were presented
in case of the 60-Hz train, 4,800 stimuli in the case of the
40-Hz train, and 2,400 stimuli in the case of the 20-Hz
Effects of “spontaneous” firing on excitatory synaptic
currents in the calyx of Held
At the beginning of each experiment, a synapse was “rested”
or completely recovered, i.e., no stimuli had been given to the
input fibers for ?5 min. During the 2-min conditioning period
with Poisson-distributed activity, EPSCs depressed substan-
tially with at least two exponential components. The three
graphs in Fig. 3, A–C show EPSC amplitudes of three different
neurons in response to 2-min conditioning stimuli at 20, 40,
and 60 Hz (Fig. 3, A, B, and C, respectively). Each dot in the
graphs represents the amplitude of one EPSC and the solid
lines represent double-exponential fits.
The initial EPSC amplitudes in the three examples were
between about 5 and 9 nA, fairly typical values of rested calyx
of Held/MNTB recordings in animals of this age group (e.g.,
Taschenberger and von Gersdorff 2000; von Gersdorff and
Borst 2002). We term this value the “initial amplitude” or
“A0.” The synaptic current then depressed substantially during
the first few events of the stimulus train (insets in Fig. 3, A–C,
initial steep declines of amplitudes), then declined much
slower (later shallow decline of amplitudes), and then stabi-
lized during the second half of the 2-min train to values
between about 2 and 3 nA.
We were interested in these steady-state values measured
during the second half of the conditioning period because
neuron. Mean spontaneous firing rate among the 36 neurons was 24.9 ? 5.5 Hz. B and C: in vivo recordings of spontaneous activity from 3 MNTB neurons;
clips of original trace (B); interspike interval (ISI) distribution (C). Although the spontaneous firing rates differed between the neurons and were 16, 40, and 69
Hz, the ISI distribution could be described by a single-exponential curve, and thus was near-Poisson-distributed in each case. D and E: based on the results from
B and C, 3 stimulation protocols of simulating spontaneous activity in brain slices at 20, 40, and 60 Hz were created. ISI distribution was designed to be
A: distribution of spontaneous firing rates measured among our sample of 36 neurons. Each dot represents the average spontaneous firing rate of one
810HERMANN, PECKA, VON GERSDORFF, GROTHE, AND KLUG
J Neurophysiol • VOL 98 • AUGUST 2007 • www.jn.org
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