J Neurophysiol
88: 627– 638, 2002; 10.1152/jn.00879.2001.
Presynaptic Current Changes at the Mossy Fiber–Granule Cell
Synapse of Cerebellum During LTP
ARIANNA MAFFEI,1 FRANCESCA PRESTORI,1 PAOLA ROSSI,1 VANNI TAGLIETTI,1 AND
EGIDIO D’ANGELO1,2
1
Department of Molecular/Cellular Physiology and Instituto Nazionale Fisica della Materia, University of Pavia, 27100
Pavia, Italy; and 2Department of Evolutionary and Functional Biology, University of Parma, 43100 Parma, Italy
Received 25 October 2001; accepted in final form 28 March 2002.
There is open debate about the expression mechanism of
long-term potentiation (LTP), an enhancement of synaptic
transmission suggested to take part in learning and memory in
the mammalian brain (Bliss and Collingridge 1993; Hawkins et
al. 1993; Malenka and Nicoll 1999). A major question is
whether LTP depends on pre- or postsynaptic changes. The
involvement of presynaptic expression mechanisms during
LTP has been indirectly supported by quantal analysis of
postsynaptic responses (Bekkers and Stevens 1990; Malgaroli
and Tsien 1992) and by the involvement of retrograde messengers backpropagating from the postsynaptic induction site
(Garthwaite et al. 1988; Schuman and Madison 1991). Recently, enhanced presynaptic protein expression and uptake of
fluorescent dyes have been proposed as evidence for increased
vesicle cycling during LTP (Malgaroli et al. 1995; Nayak et al.
1996; Zakharenko et al. 2001). Enhanced neurotransmitter
release could also involve changes in presynaptic terminal
currents; e.g., an increase in Ca2⫹ or a decrease in K⫹ currents
as observed in some forms of plasticity in Aplysia and other
invertebrates (Hawkins et al. 1993; Kandel and Schwartz
1982). Although Ca2⫹ and K⫹ currents control neurotransmitter release at central mammalian synapses also (e.g., Ishikawa
and Takahashi 2001; Lærum and Storm 1994), no changes in
presynaptic terminal excitability have been reported so far
during LTP.
A prerequisite for this investigation is that stable longlasting recordings are established from presynaptic terminals
and that a simultaneous monitoring of the afferent volley and
postsynaptic response is obtained. To this aim we have performed extracellular focal recordings (Del Castillo and Katz
1956), which have previously been used to measure presynaptic currents at the neuromuscular junction (Angaut-Petit et al.
1989; Brigant and Mallart 1982; Del Castillo and Katz 1956;
Katz and Miledi 1965; Mallart 1985) and spine currents in
cultured neurons (Forti et al. 1997). With focal recordings, we
have investigated neurotransmission and LTP at the cerebellar
mossy fiber– granule cell synapse. Cerebellar mossy fibers
form large glutamatergic terminals that contact numerous granule cells (D’Angelo et al. 1995; Eccles et al. 1967; Garthwaite
and Brodbelt 1989; Palay and Chan-Palay 1974), and their
high-frequency stimulation causes N-methyl-D-aspartate (NMDA)
receptor-dependent LTP (Armano et al. 2000; D’Angelo et al.
1999; Hansel et al. 2001). Similar to a few other examples in
the mammalian brain (Borst and Sackmann 1998; Geiger and
Address for reprint requests: E. D’Angelo, Department of Physiology and
Pharmacology, Section of General Physiology, University of Pavia, Via Forlanini 6, 27100 Pavia, Italy (E-mail: [email protected]).
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.
INTRODUCTION
www.jn.org
0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
627
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
Maffei, Arianna, Francesca Prestori, Paola Rossi, Vanni Taglietti,
and Egidio D’Angelo. Presynaptic current changes at the mossy
fiber– granule cell synapse of cerebellum during LTP. J Neurophysiol
88: 627– 638, 2002; 10.1152/jn.00879.2001. The involvement of presynaptic mechanisms in the expression of long-term potentiation
(LTP), an enhancement of synaptic transmission suggested to take
part in learning and memory in the mammalian brain, has not been
fully clarified. Although evidence for enhanced vesicle cycling has
been reported, it is unknown whether presynaptic terminal excitability
could change as has been observed in invertebrate synapses. To
address this question, we performed extracellular focal recordings in
cerebellar slices. The extracellular current consisted of a pre- (P1/N1)
and postsynaptic (N2/SN) component. In ⬃50% of cases, N1 could be
subdivided into N1a and N1b. Whereas N1a was part of the fiber volley
(P1/N1a), N1b corresponded to a Ca2⫹-dependent component accounting for 40 –50% of N1, which could be isolated from individual mossy
fiber terminals visualized with fast tetramethylindocarbocyanine perchlorate (DiI). The postsynaptic response, given its timing and sensitivity to glutamate receptor antagonists [N2 was blocked by 10 ␮M
[1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) and SN by 100 ␮M APV ⫹50 ␮M 7-Clkyn], corresponded to granule cell excitation. N2 and SN could be
reduced by 1) Ca2⫹ channel blockers, 2) decreasing the Ca2⫹ to Mg2⫹
ratio, 3) paired-pulse stimulation, and 4) adenosine receptor activation. However, only the first two manipulations, which modify Ca2⫹
influx, were associated with N1 (or N1b) reduction. LTP was induced
by ␪-burst mossy fiber stimulation (8 trains of 10 impulses at 100 Hz
separated by 150-ms pauses). Interestingly, during LTP, both N1 (or
N1b) and N2/SN persistently increased, whereas P1 (or P1/N1a) did not
change. Average changes were N1 ⫽ 38.1 ⫾ 31.9, N2 ⫽ 49.6 ⫾ 48.8,
and SN ⫽ 59.1 ⫾ 35.5%. The presynaptic changes were not observed
when LTP was prevented by synaptic inhibition, by N-methyl-Daspartate and metabotropic glutamate receptor blockage, or by protein
kinase C blockage. Moreover, the presynaptic changes were sensitive
to Ca2⫹ channel blockers (1 mM Ni2⫹ and 5 ␮M ␻-CTx-MVIIC) and
occluded by K⫹ channel blockers (1 mM tetraethylammmonium).
Thus regulation of presynaptic terminal excitability may take part in
LTP expression at a central mammalian synapse.
628
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
amplitude was taken as a measure of N1 and N2. Except for exemplar
recordings shown in Figs. 1–3, amplitude calibration was omitted
because signal amplitude depended on factors unrelated to membrane
currents. Data in the text are reported as means ⫾ SD.
Mossy fibers and their terminals were identified under fluorescence
optics (CCD camera; PCO Sensicam, Martinsried, Germany and
monochromator; TILL Photonics, Kelheim, Germany) 30 – 60 min
after placing a crystal of fast tetramethylindocarbocyanine perchlorate
(DiI; emission wavelength 564 nm; Molecular Probes, Leiden, The
Netherlands) on the mossy fiber bundle. After locating a presynaptic
terminal and its axon, the microscope was switched to Nomarsky
optics, and a recording (2- to 4-␮m diameter) and a stimulating (1-␮m
diameter) pipette were positioned with digitally controlled piezoelectric manipulators (Physik Instrumente, Waldbronn, Germany), allowing recordings to be made from individual mossy fiber terminals.
LTP was induced by ␪-burst stimulation (TBS; 8 bursts of 10
impulses at 100 Hz repeated every 250 ms) after 20 min of control
stimulation at 0.1 Hz (Armano et al. 2000; D’Angelo et al. 1999).
RESULTS
METHODS
Focal current recordings from the cerebellum granular layer
Focal current recordings (Brigant and Mallart 1982; Del Castillo
and Katz 1956; Forti et al. 1997; Katz and Miledi 1965; Mallart 1985)
were performed in acute 250-␮m-thick cerebellar slices obtained from
19-to 22-day-old Wistar rats, as reported previously (Armano et al.
2000; D’Angelo et al. 1995, 1999). Slices were maintained in standard
Krebs solution at 30°C. Unless otherwise stated, the solutions contained the GABAA receptor blocker bicuculline (10 ␮M; Sigma, St.
Louis, MO). Drugs were either perfused in the bath [6-cyano-7nitroquinoxalene-2,3-dione (CNQX), 1,2,3,4-tetrahydro-6-nitro-2,3dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX), 2amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid,
(RS)-1-amino-indan-1,5-dicarboxylic acid (AIDA; Tocris Cookson),
ClS-adenosine, TTX (Sigma)] or through a local pipette [␻-CTxMVIIC (Bachem, Heidelberg, Germany) and GÖ 6983 (Calbiochem)]
with a 50- to 100-␮M tip diameter. The mossy fiber bundle was
stimulated with a bipolar tungsten electrode via a stimulus isolation
unit (0.2-ms voltage pulses at 0.1 Hz), and a patch-pipette (5–10 ␮m)
was gently positioned on the slice until an “active spot” was detected
(Del Castillo and Katz 1956; Katz and Miledi 1965). Once the
recording pipette contacted the slice, a “gap resistance” nearly doubled the circuit load. This gap resistance was monitored by measuring
current deflections generated by small voltage steps throughout the
recordings (recordings with ⬎ ⫾5% changes in gap resistance were
rejected). Inward membrane currents collected from below the electrode appeared as upward deflections and were termed N (negative)
conforming to the nomenclature of cerebellar field recordings (Eccles
et al. 1967; Garthwaite and Brodbelt 1989). Patch-clamp technology
was adopted to ensure a wide signal-to-noise ratio and recording
bandwidth. Focal extracellular currents were recorded with an Axopatch 200-B amplifier in the voltage-clamp mode (0 mV command
potential) at a 10-kHz cutoff frequency (⫺3 dB) and were sampled
with a Digidata 1200B interface at 50 ␮s/point (Axon Instruments,
Union City, CA). Patch-pipettes ensured low tip resistance (1–2 M⍀
with Krebs filling solution), and fire-polishing was used to improve
the gap resistance, increasing the current measured by the amplifier.
Capacitive coupling and stimulus artifact were reduced by using
thick-walled borosilicate (hard-glass) capillaries (Hingelberg, Malsfeld, Germany) and by Sylgard-coating the pipettes close to their tips.
The extracellular signals were monitored on-line with LTP101M
software (kindly provided by Dr. William Anderson; University of
Bristol, UK), converted to an appropriate format and analyzed off-line
with pClamp software. Signal quality was improved by averaging 10
consecutive tracings, and the stimulus artifact (which was isolated by
applying 1 ␮M TTX at the end of the recordings) was subtracted. Peak
Focal currents (Brigant and Mallart 1982; Del Castillo and
Katz 1956; Forti et al. 1997; Katz and Miledi 1965; Mallart
1985) were obtained by positioning the recording electrode in
the granular layer while stimulating the mossy fiber bundle
within the folium [intrafolial stimulation (IFS); Figs. 1A and
2A)]. The focal current typically comprised four components
termed P1, N1, N2, and SN, resembling those recorded with
grease-gap recordings in vitro (Garthwaite and Brodbelt 1989)
and field recordings in vivo (Eccles et al. 1967) (Figs. 1A and
2A). In 46% of cases, N1 showed a single peak at 1.2 ⫾ 0.4 ms
(n ⫽ 34; for example, see Fig. 1C), whereas in the remaining
cases, N1 displayed a main peak at 0.7 ⫾ 0.3 ms (N1a) and a
secondary peak at 1.5 ⫾ 0.2 ms (N1b; n ⫽ 40; for examples, see
Figs. 1A, 2B, and 3D). N2 arose at 2.1 ⫾ 0.3 ms (n ⫽ 74) and
peaked at 3.3 ⫾ 1.1 ms (n ⫽ 74). N2/SN apparently changed
from an excitatory postsynaptic potential (EPSP) to an EPSPspike complex as the stimulus intensity was increased (Fig.
1A). A comparison with voltage tracings recorded in patch
clamp, whole cell recordings showed that N2/SN was in phase
with EPSP-spike complexes measured postsynaptically from
granule cells (Armano et al. 2000; D’Angelo et al. 1995, 1999)
(Fig. 1B), whereas P1/N1 fell within the synaptic delay.
To determine whether the focal current (especially N1) received a contribution from Purkinje cells (Eccles et al. 1967),
in some experiments, either the Purkinje cell layer was removed (IFScut), or mossy fibers were stimulated through their
collateral branches in neighboring folia [transfolial stimulation
(TFS)], thus preventing Purkinje cell axon activation (Fig. 1C).
In these experiments, the focal current was similar to that
obtained by IFS, as reflected by the similarity of the corresponding N1/N2 ratios [IFScut 1.28 ⫾ 1.2 (n ⫽ 5), TFS 1.45 ⫾
0.6 (n ⫽ 4), IFS 1.43 ⫾ 0.8 (n ⫽ 13)]. Another way to unveil
the contribution of Purkinje cells is to cause their inhibition
through molecular layer interneurons (Eccles et al. 1967). This
was done in experiments wherein bicuculline was omitted from
the extracellular solution and parallel fibers were activated by
a second stimulating electrode molecular layer stimulation
(MLS). Although molecular layer conditioning caused a strong
granule cell inhibition through Golgi cells (see following text
and Fig. 1D), no changes were observed in N1.
J Neurophysiol • VOL
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
Jonas 2000), the large size of cerebellar mossy fibers makes
them suitable candidates for measuring extracellular currents.
In the present study, we show that during LTP, the current
generated by cerebellar mossy fiber terminals was persistently
increased. The presynaptic change depended on the same
mechanisms that determined LTP induction in granule cells,
including activation of the NMDA receptor, metabotropic glutamate receptor (GluR)-1, and protein kinase C (PKC)
(D’Angelo et al. 1999; Masgrau et al. 2001; Rossi et al. 1996).
The presynaptic change was correlated with the intensity of
LTP, was sensitive to Ca2⫹ channel blockers, and was occluded by K⫹ channel blockers. Moreover, although it occurred after manipulations of presynaptic Ca2⫹ influx, no presynaptic change followed direct manipulation of neurotransmitter
release through paired-pulse stimulation (PPS) or adenosine receptor activation. These results provide direct evidence that presynaptic excitability changes, which may enhance neurotransmission during LTP, take place at cerebellar glomerular synapses.
PRESYNAPTIC CURRENT AT MOSSY FIBER–GRANULE CELL SYNAPSE
629
Golgi cells may contribute to shape the focal current, because both their axons and dendrites enter the glomeruli, making connections with granule cells and mossy fibers, respectively (Eccles et al. 1967). In fact, in the absence of bicuculline,
mossy fiber stimulation elicited a large inhibitory response 3–5
ms after the stimulus (Fig. 1D), which decayed with a time
course reflecting granule cell inhibition. However, N1 was
unaffected. These results indicate that the focal current elicited
by IFS mostly reflected mossy fiber and granule cell excitation.
J Neurophysiol • VOL
Unless differently stated, IFS will be adopted in the following
experiments.
Pharmacological and functional properties of the focal
current
The nature of pre- and postsynaptic waveforms was clarified
by pharmacological experiments. Because granular layer excitation is glutamatergic (Garthwaite and Brodbelt 1988), the
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 1. Focal recordings from the cerebellum granular layer. A: extracellular focal currents were recorded from the granular
layer after stimulation of mossy fiber bundle within the folium [intrafolial stimulation (IFS)]. Five single tracings are superimposed
at each of 2 different stimulation intensities (5 and 10 V); corresponding average currents, after artifact subtraction, are at bottom
(see METHODS for details). Focal current consisted of a sequence of waves termed P1, N1, N2, and SN. N1 could be divided into 2
components, N1a and N1b. Unless stated otherwise IFS was at 10 V. B: copy of the focal current in A superimposed on current-clamp
recording obtained in whole cell patch-clamp configuration from a granule cell (holding potential ⫺68 mV) demonstrates
relationship between extra- and intracellular waveforms. Note that P1/N1, which corresponds to a presynaptic signal, falls within
the excitatory postsynaptic potential (EPSP) synaptic delay. Arrows, stimulation. C: schematic representation of cerebellar circuit
and recording configurations used in this study. Mossy fibers (mf) contact numerous granule cell (grC) and Golgi cell (GoC)
dendrites. Axons of grC, parallel fibers (pf), activate Golgi cells, Purkinje cells (PC), and inhibitory interneurons (ii) in the
molecular layer. In addition to mf, the granular layer is traversed by climbing fibers (cf) and PC axons. ⫹, excitation; ⫺, inhibition.
Circular shaded area, enlargement of area covered by the focal electrode. Focal current obtained by IFS of mossy fiber bundle is
compared with those obtained with transfolial stimulation (TFS), which selectively activates mossy fibers through their axonal
collaterals in neighboring folia, or with IFS after surgical removal of Purkinje cells (IFScut). Responses generated by IFS, TFS, and
IFScut were similar. Average tracings are scaled to N1 peak (P1 has been partially blanked). D: molecular layer stimulation
(MLS)-IFS and time course of extracellular current inhibition after MLS. N1 remained stable for every tested MLS-IFS interval,
whereas N2 showed a strong inhibition at 10 ms MLS-IFS interval, and the inhibition recovered at longer conditioning intervals.
Arrow, inhibitory wave. N1 appeared as a single wave in C and D.
630
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
FIG. 3. Focal recordings from visualized mossy fiber terminals. A: mossy fiber and its terminal visualized with fast tetramethylindocarbocyanine perchlorate (DiI). Inset, terminal at higher magnification; calibration bars, 4 ␮m. B: stimulating and recording
pipettes were positioned with Nomarsky optics on the points where the fluorescent image revealed a mossy fiber and its terminal
(profile is redrawn from A and overlaid). Single tracings from terminal show a prominent inward current. Terminal current was not
measurable after small displacement of the recording pipette from the terminal. Plot at right shows sharp dependence of the positive
peak of the terminal current to changes in stimulation intensity. C: terminal current was reduced by perfusion with 1 mM Ni2⫹.
Subtracting Ni2⫹ from the control tracing yielded the current, depending on Ca2⫹ channel activation (control-Ni2⫹).
J Neurophysiol • VOL
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 2. Pharmacological and functional properties
of focal currents. A: application of N-methyl-D-aspartate (NMDA) receptor antagonists APV (100 ␮M) and
7-Cl-kyn (50 ␮M) reduced SN more markedly than
N2. Addition of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX; 10 ␮M) fully
blocked the postsynaptic response. B: signal remaining after glutamate receptor blockage corresponded to
presynaptic current. N1a was almost unaffected by
application of 1 mM Ni2⫹, whereas N1b was reduced.
Tracing at bottom, Ni2⫹-sensitive current (controlNi2⫹). C: both N1 and N2/SN were reduced by reducing the Ca2⫹/Mg2⫹ ratio from 2 mM/1.2 mM to 0.1
mM/3.1 mM (the balance of divalent cations was
maintained). D: N2/SN, but not N1, were reduced by
10 ␮M Cl-adenosine. P1 did not change during pharmacological manipulations.
PRESYNAPTIC CURRENT AT MOSSY FIBER–GRANULE CELL SYNAPSE
J Neurophysiol • VOL
different mechanisms, only those involving a regulation of
Ca2⫹ influx were associated with a presynaptic current change.
Focal recordings from visualized mossy fiber terminals
The emergence of the terminal current into N1 was directly
demonstrated by recordings from an extracellular patch-pipette
positioned on cerebellar mossy fiber terminals visualized with
fast DiI (Fig. 3, A and B). Thirty to sixty minutes after positioning a dye crystal on the mossy fiber bundle, individual
mossy fibers and their terminals were clearly distinguishable
with fluorescence optics. The mossy fiber terminals showed a
typical digitated shape, with smallest and largest diameters of
4.0 ⫾ 1.2 and 5.2 ⫾ 1.4 ␮m (cf. Eccles et al. 1967; Palay and
Chan-Palay 1974).
After stimulation of the afferent axon, recordings from visualized terminals showed a triphasic extracellular current with
a major negative peak. The current had three notable properties. First, it arose in an all-or-none manner by increasing the
stimulus intensity (Fig. 3B, right). The presence of a nonzero
signal at low stimulus intensities may indicate either electrotonic spread of subthreshold depolarization along the afferent
axon or activation of neighboring axons with a lower activation
threshold. Second, the current vanished after a few micrometers of pipette displacement outside the terminal. Finally, the
current inward peak occurred at 1.2 ⫾ 0.4 ms (n ⫽ 6; Fig. 3C)
and was reduced by 45.9 ⫾ 21.4% (n ⫽ 5) by 1 mM Ni2⫹.
These observations are consistent with recordings from a single
mossy fiber terminal with marginal contribution, if any, of
other excitable elements. The terminal current showed timing
and Ca2⫹ sensitivity identical to N1 or N1b, confirming that
composite waveforms included a sizeable component generated by mossy fiber terminals.
LTP is associated with a presynaptic current increase
LTP was induced by TBS of the mossy fiber bundle (Armano et al. 2000; D’Angelo et al. 1999; Hansel et al. 2001).
After TBS, both N2 and SN increased signaling potentiation in
non-NMDA and NMDA receptor-dependent responses, respectively. Strikingly, the presynaptic current also increased
after TBS, whereas no changes were observed in control recordings (Fig. 4A).
N1 and N2/SN potentiation developed along a similar time
course (Fig. 4B). Thirty minutes after TBS, N1, N2, and SN
were enhanced by 38.1 ⫾ 31.9, 49.6 ⫾ 48.8, and 59.1 ⫾
35.5%, respectively (n ⫽ 7). The relationship between N1 and
N2 amplitude changes 30 min after TBS is shown in Fig. 4C.
Regression over the data shows a linear correlation of N2 over
N1 (P ⬍ 0.05 by F-test), indicating that the presynaptic change
reflects the intensity of LTP.
During LTP, P1 did not change (⫺5.5 ⫾ 31.8%; n ⫽ 7).
Moreover, when N1a could be separated from N1b (Fig. 4D),
N1b increased significantly more than N1a (35.4 ⫾ 9.2 vs.
3.0 ⫾ 13.0%; P ⬍ 0.01 by paired t-test; n ⫽ 3). The increase
in N1b, compared with the stability of P1 and N1a, points to a
specific enhancement in the terminal current.
Presynaptic current increases in mossy fiber terminals
A direct demonstration of the origin of presynaptic current
changes was obtained in recordings from visualized mossy
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
postsynaptic response was investigated with specific glutamate
receptor antagonists. To block NMDA receptors, we co-applied the glutamate site antagonist APV and the glycine site
antagonist 7-Cl-kyn, the combination of which has been shown
to improve NMDA EPSP inhibition in whole cell recordings
(D’Angelo et al. 1995). Figure 2A shows that application of
100 ␮M APV and 50 ␮M 7-Cl-kyn left N2 almost unaffected
while reducing SN by 80.1 ⫾ 16.8% (n ⫽ 8). The subsequent
application of 10 ␮M NBQX (n ⫽ 8) reduced N2 by 88.4 ⫾
19.3% (n ⫽ 8). N2 and SN thus reflected postsynaptic
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
and NMDA receptor activation in granule cells (Armano 2000;
D’Angelo et al. 1995, 1999; Garthwaite and Brodbelt 1989).
The presynaptic current, which was isolated by ionotropic
glutamate receptor blockage, was reduced by 41.7 ⫾ 9.3% by
perfusing a nonspecific Ca2⫹ channel blocker, 1 mM Ni2⫹
(Fig. 2B). In those cases in which N1b could be separated from
N1a, N1b was significantly more reduced than N1a (⫺43.7 ⫾
7.5% versus ⫺9.5 ⫾ 7.3%; n ⫽ 6; P ⬍ 0.01 by paired t-test).
The sensitivity of N1, and specifically of N1b to Ca2⫹ channel
blockers, suggests that the presynaptic response includes a
component originating from presynaptic terminals (see below
and Figs. 3C and 8A). P1/N1a was blocked by TTX, thus
corresponding to the axon volley (data not shown).
The functional relationship of N1 with neurotransmitter release was clarified by using three complementary manipulations. First, reducing the Ca2⫹/Mg2⫹ ratio is known to reduce
presynaptic Ca2⫹ influx and neurotransmitter release (Dodge
and Rahamimoff 1967). When the Krebs solution (1.2 mM
Ca2⫹/1.2 mM Mg2⫹) was substituted with one containing a
50-fold lower Ca2⫹/Mg2⫹ ratio (0.1 mM Ca2⫹/3.1 mM Mg2⫹),
N1, N2, and SN were all considerably reduced (⫺53.3 ⫾
10.4%, ⫺49.4 ⫾ 8.2%, and ⫺70.4 ⫾ 18.4%, respectively; n ⫽
3). Similar results were obtained with the use of two Ca2⫹
channel blockers, Ni2⫹ and ␻-CgTC-MVIIC (see Fig. 8B).
Consistently, N1, N2, and SN increased when K⫹ channels
were blocked by tetraethylammonium (TEA; see Fig. 9), a
manipulation known to cause a secondary Ca2⫹ current enhancement (Angaut-Petit et al. 1989; Brigant and Mallart 1982;
Mallart 1985).
Second, activation of adenosine receptors is known to reduce neurotransmitter release at several peripheral and central
synapses (Fredholm 1995). In these experiments we used Cladenosine (Fig. 2D), a rather selective antagonist of the A1
receptor subtype, which is usually located presynaptically and
is abundantly expressed in the cerebellum. As 10 ␮M Cladenosine was perfused into the bath, N1 remained unchanged,
whereas N2 and SN decreased (⫺0.1 ⫾ 17.2, ⫺27.7 ⫾ 5.0, and
⫺72.1 ⫾ 45.7%, respectively; n ⫽ 3). Although adenosine
receptors might enhance K⫹ and inhibit Ca2⫹ channels (reviewed in Fredholm 1995), the changes observed here are in
keeping with the early proposal that adenosine receptors affect
release through a metabolic pathway independent of transmembrane Ca2⫹ influx (Silinsky 1984).
Third, during PPS (20-ms interpulse interval), which is
known to regulate neurotransmitter release by influencing vesicle turnover and presynaptic Ca2⫹ accumulation rather than
Ca2⫹ influx (Regher and Tank 1991; Wu and Saggau 1994), N2
and SN increased, whereas N1 remained stable (see Fig. 7).
Thus although neurotransmitter release was influenced through
631
632
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
fiber terminals (Fig. 5A). After TBS, the terminal current
showed an increase similar to that measured in N1 (35.2 ⫾
15.8%; n ⫽ 3).
It should also be considered that Purkinje cell discharge may
be altered after repeated parallel fiber and climbing fiber activation causing N1 to increase after TBS (Eccles et al. 1967;
Hansel et al. 2001). However, this possibility was ruled out by
inducing LTP in experiments in which Purkinje cell activation
was prevented by either removing the Purkinje cell layer
(IFScut) or using TFS (Fig. 1). With IFScut and TFS, we
obtained robust LTP both in N1 and N2/SN (Fig. 5B), and a
specific N1b increase could be observed in four of nine experJ Neurophysiol • VOL
iments (e.g., Fig. 5B, inset). A slight reduction in the intensity
of changes compared with those obtained with IFS probably
reflected lower stimulation efficiency, because N1 and N2/SN
were all similarly reduced.
Manipulations that prevent LTP also prevent the presynaptic
current increase
If the presynaptic current increase depends on LTP induction,
then it should be prevented by the same manipulations previously
reported to block cerebellar mossy fiber– granule cell LTP in
patch-clamp recordings (Armano et al. 2000; D’Angelo et al.
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 4. Simultaneous pre- and postsynaptic current increase during long-term potentiation (LTP). A: LTP was induced by
␪-burst stimulation (TBS) in IFS experiments. Recording stability was monitored in experiments of the same duration but without
TBS. Tracings show focal currents 20 min after stimulation began (thin line, just before TBS in LTP experiments) and 30 min
thereafter (thick lines, 30 min after TBS in LTP experiments). Note that both pre- and postsynaptic currents increased after TBS
but not in recordings without TBS. Plots show time course of N1, N2, and SN relative amplitude changes in experiments with TBS
(at the arrow; filled symbols) or without TBS (open symbols). Data points are means ⫾ SE from 7 experiments. B: examples of
N1 and N1b potentiation following TBS in 2 different recordings. Note stability of P1 in both cases. C: relationship between N1 and
N2 amplitude changes. Data were obtained from experiments in A (Œ, Fig. 5B (E), and Fig. 8A (‚). Line shows linear regression
over the data (n ⫽ 19; P ⬍ 0.05 by F-test).
PRESYNAPTIC CURRENT AT MOSSY FIBER–GRANULE CELL SYNAPSE
633
Presynaptic currents are unchanged during PPS
Postsynaptic responses to PPS, by revealing the short-term
depressing and/or facilitating properties of a synapse, have
been largely used to shed light on the mechanism and locus of
neurotransmission changes during LTP (Schultz et al. 1994).
Here, in addition, we have measured the effect of PPS on the
presynaptic terminal current (Fig. 7A). During PPS at 50 Hz
(Fig. 7B), the N2 ratio [(N2(2nd) ⫺ N2(1st))/N2(1st)] was ⫺60.1 ⫾
10.0% (n ⫽ 4), reflecting a prevalence of synaptic depression.
No comparable changes were observed in the N1 ratio
[(N1(2nd) ⫺ N1(1st))/N1(1st) ⫽ ⫺9.9 ⫾ 5.1%; n ⫽ 4)]. The
slightly negative value in the N1 ratio may reflect a contribution of terminal current reduction to short-term depression
(Forsythe et al. 1998). During LTP (Fig. 7B), the N2 ratio
increased, whereas the N1 ratio remained constant. Thus during
LTP, the second pulse in a pair was facilitated without corresponding changes in terminal currents.
Pharmacological properties of presynaptic current changes
during LTP
FIG. 5. Specificity of the presynaptic current increase for the mossy fiber
terminal. A: presynaptic current was recorded from a visualized mossy fiber
terminal by stimulating the afferent axon (cf. Fig. 3). After TBS, terminal current
increased. Plot shows average time course of TBS-induced changes (arrow, TBS;
mean ⫾ SE; n ⫽ 3). B: LTP was induced by TBS in TFS or IFScut experiments.
N1 and N2 showed changes similar to those observed during LTP induced with
IFS. Tracings were taken in control and 30 min following TBS. Plots show the
time course of N1, N2, and SN changes with TFS (filled symbols; mean ⫾ SE; n ⫽
4) or IFScut (open symbols; mean ⫾ SE; n ⫽ 5). Arrows, TBS. Continuous line
was replotted from Fig. 4A to compare changes induced by IFS. Inset, specific N1b
potentiation in TFS experiment.
1999). Indeed, both N1 and N2/SN increases were prevented when
bicuculline was omitted from the extracellular solution to cause
granule cell inhibition through the Golgi cell circuit, thereby
preventing removal of Mg2⫹ block from NMDA receptors (Fig.
J Neurophysiol • VOL
If the presynaptic current increase occurs in the mossy fiber
terminal, then the N1 component sensitive to Ca2⫹ channel
blockers would be specifically enhanced. Indeed, in experiments in which a 5-min, 1 mM Ni2⫹ perfusion was performed
before and after LTP induction (Fig. 8A), Ni2⫹ block of N1
during LTP was enhanced (50.1 ⫾ 22.6 vs. 31.4 ⫾ 20.2%; P ⬍
0.006 by paired t-test; n ⫽ 4). A similar result was obtained
with a peptidic Ca2⫹ channel blocker, 5 ␮M ␻-CTx-MVIIC
(Meir et al. 1999). Because ␻-CTx-MVIIC block is almost
irreversible, different recordings were used for control and
LTP experiments. Application of 5 ␮M ␻-CTx-MVIIC caused
a significantly larger N1 reduction 30 min after LTP induction
than occurred in control recordings (42.2 ⫾ 7 vs. 27.9 ⫾ 4.6%,
P ⬍ 0.01 by unpaired t-test; n ⫽ 4). It should be noted that N2
was also reduced by ␻-CTx-MVIIC and Ni2⫹ and that this
reduction was enhanced during LTP (with both blockers, P ⬍
0.02; n ⫽ 4; Fig. 8B).
Finally, we considered whether LTP could be occluded by
manipulating presynaptic terminal excitability. To this end, we
applied 1 mM TEA, which has been shown to enhance depo-
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
6A). Moreover, both N1 and N2/SN increases (Fig. 6B) were
prevented by blocking NMDA receptors with 100 ␮M APV and
50 ␮M 7-Cl-kyn, type 1 metabotropic glutamate receptors
(mGluR-1) with 15 ␮M AIDA (Shoepp et al. 1999), and PKC
with 10 ␮M GÖ 6983 (Bortolotto and Collingridge 2000). Neither
could changes be observed in N1b when it could be isolated (e.g.,
Fig. 6A, inset). It should be noted that neither AIDA nor GÖ 6983
caused any remarkable changes in basal neurotransmission; the
nature of inhibitory waves disclosed by omitting bicuculline and
the changes caused by APV and 7-Cl-kyn are shown in Figs. 1
and 2.
These observations, in addition to confirming that voltagedependent NMDA receptor activation and the mGluR-1-inositol trisphosphate (IP3)-PKC pathway (Masgrau et al. 2001) are
involved in cerebellar mossy fiber– granule cell LTP, demonstrate that N1 (or N1b) changes are not generated by highfrequency stimulation itself but depend on efficient LTP induction.
634
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
larization and neurotransmitter release at central synapses
(Lærum and Storm 1994; Mallart 1985; Meir et al. 1999). TEA
application reversibly enhanced both N1 and N2 (69.7 ⫾ 25.3
and 74.6 ⫾ 15.0%; n ⫽ 6), thus mimicking the changes
observed during LTP. In the presence of TEA, neither presynaptic nor postsynaptic currents were enhanced by TBS (N1 and
N2 changes were ⫺4.1 ⫾ 12.5 and ⫺1.1 ⫾ 6.9%; n ⫽ 5) (Fig.
9). It should be noted that potentiation induced by TEA differed from K⫹ channel-dependent LTP (Anizkstejn and BenAri 1991; Zakharenko et al. 2001) because TEA had a low
concentration, its effect was reversible (data not shown), and it
did not determine neuron burst discharge.
DISCUSSION
In this study, we show that extracellular focal recordings
provide a means to perform noninvasive, long-lasting pre- and
postsynaptic measurements of mossy fiber– granule cell LTP.
Our main observation is that during LTP, the presynaptic
terminal current persistently increased. The relevance of this
finding is related to the ability to identify the terminal current
and measure it simultaneously with the postsynaptic response,
allowing a direct relationship between pre- and postsynaptic
processes to be established. Before addressing the potential
mechanism of presynaptic current potentiation and its implications for LTP expression, we will consider the nature of
recorded signals.
J Neurophysiol • VOL
The focal current reflects excitation at the mossy
fiber– granule cell relay
The focal current recorded from the granular layer of cerebellar slices after mossy fiber stimulation could be interpreted
based on current knowledge of anatomical, functional, and
pharmacological properties of the cerebellar circuitry (Eccles
et al. 1967; Garthwaite and Brodbelt 1988; Palay and ChanPalay 1974). The presynaptic response (P1/N1) fell within the
synaptic delay (⬍2 ms), and the postsynaptic response (N2/SN)
fell in correspondence with intracellular EPSP-spike complexes of granule cells (D’Angelo et al. 1995). In particular, N2
was a fast non-NMDA receptor-mediated component priming
an action potential, and SN was a slow NMDA receptormediated component. Golgi cells controlled the amplitude of
N2/SN through a large GABAA receptor-mediated hyperpolarizing wave, the effectiveness of which decreased with the
temporal separation between excitation and inhibition. These
observations indicate that the mossy fiber– granule cell relay,
which has a large numerical and volumetric prevalence over
other circuit elements of the granular layer, was the main
source of the focal current.
Both the pre- and postsynaptic components of the focal
current were usually graded with stimulation intensity, indicating recruitment of mossy fibers and transition of granule cell
responses from subthreshold EPSPs to spike firing (see Fig.
1A). Peculiar to focal recordings is that the mossy fiber termi-
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 6. Pharmacological blockage of LTP
and presynaptic current changes: application of
TBS in IFS experiments in different pharmacological conditions. Tracings were taken just before TBS (thin lines) and 30 min thereafter (thick
lines). Plots show time course of N1, N2, and SN
relative amplitude changes (mean ⫾ SE). A: absence of bicuculline (F; n ⫽ 6). Inset, separation
of N1a and N1b. B: perfusion of 100 ␮M APV and
50 ␮M 7-Cl-kyn (E; n ⫽ 5),15 ␮M AIDA (‚;
n ⫽ 5), or 10 ␮M GÖ 6983 (〫; n ⫽ 4). Note the
absence of potentiation (or even a tendency to
depression for F, E, and ‚) in all experimental
conditions.
PRESYNAPTIC CURRENT AT MOSSY FIBER–GRANULE CELL SYNAPSE
635
nal current emerged from N1 as a specific Ca2⫹-sensitive
component, which could be separated as a distinct peak (N1b)
in ⬃50% of cases and could be measured from single mossy
fiber terminals made fluorescent with DiI. The small size and
close temporal contiguity of N1b relative to N1a, combined with
small phase differences in axonal and terminal excitation, can
easily explain the blurring of N1b into N1 in some recordings.
The influence of circuit elements other than the mossy
fiber– granule cell relay, in particular Purkinje cells, was ruled
out by using TFS, by surgically removing the Purkinje cell
layer, and by activating inhibitory interneurons to prevent
Purkinje cell activation. The lack of Purkinje cell signals in
focal recordings obtained from the granular layer is explained
by two considerations. First, focal electrodes collected currents
generated from a restricted area underneath their tips (Brigant
and Mallart 1982; Del Castillo and Katz 1956; Forti et al. 1997;
Katz and Miledi 1965; Mallart 1985; see also Fig. 3), being
therefore unable to detect signals generated by Purkinje cells.
J Neurophysiol • VOL
Second, the granular layer of cerebellar slices receives Purkinje
cell axons from a thin strip rather than from the overlying
cerebellar surface. Thus the contribution of Purkinje cells to
granular layer signals is strongly limited in slices compared
with in vivo cerebellar recordings (Eccles et al. 1967). Golgi
cells discharged too late to influence N1, and their spontaneous
activity was usually not observed, ruling out their potential
contribution to focal currents in present experiments.
LTP at the mossy fiber– granule cell relay
LTP was induced by TBS of the mossy fiber bundle and,
characteristically, included a robust potentiation in the presynaptic terminal current. The presynaptic change was Ca2⫹dependent, as expected from terminal but not axonal currents,
and no changes occurred in the presynaptic volley. Moreover,
the presynaptic change could be directly revealed in visualized
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 7. Paired-pulse stimulation alters post- but not presynaptic current during LTP. A: responses were obtained with
paired–pulse stimulation (a) with interpulse intervals of 20 ms (arrows). Then, responses to the 1st pulse alone (b) were digitally
subtracted to isolate responses to the 2nd pulse (a-b). This procedure facilitates amplitude measurements in the 2nd response. It
should be noted that N2/SN were depressed in the 2nd response in a pair, whereas N1 was almost unchanged. B: paired-pulse
stimulation was performed both before and after LTP was induced with TBS. Both 1st and 2nd responses in the pair were increased
during LTP. However, during LTP, N2 depression in the 2nd response was markedly reduced. C: average time course of the N2
ratio [(N2(2nd) ⫺ N2(1st))/N2(1st)] and N1 ratio [(N1(2nd) ⫺ N1(1st))/N1(1st)] in 4 experiments. Reduction in N2 depression contrasts with
N1 constancy after LTP. SN showed changes similar to those in N2.
636
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
8. Enhanced action of Ca2⫹ channel
blockers on the presynaptic current during
LTP. A: action of 1 mM Ni2⫹ application. (a)
After a 20-min control recording, (b) a 5-min
Ni2⫹ application reduced N1 and N2. (c) Block
was recovered during washing with control solution and (d) TBS-induced LTP. (e) A second
5-min Ni2⫹ application was performed, demonstrating a greater block than during control
stimulation. Inset: comparison of Ni2⫹-sensitive current in control experiments (a-b) and
during LTP (d-e). Plots, time course of N1 and
N2 changes (mean ⫾ SE; n ⫽ 4); bars, Ni2⫹
perfusion. B: application of 5 ␮M ␻-CTxMVIIC or 1 mM Ni2⫹ caused a reduction in
both N1 and N2. Histogram shows N1 and N2
block before and after LTP induction (mean ⫾
SE; n ⫽ 4). SN showed changes similar to
those in N2.
FIG.
J Neurophysiol • VOL
2000), although incomplete PKC inhibition by GÖ 6983 or the
contribution of other kinases cannot be ruled out.
LTP expression showed potentiation in both non-NMDA
and NMDA receptor-mediated responses. LTP was more intense than in voltage-clamp but comparable to current-clamp
recordings, probably reflecting a simultaneous increase in neurotransmitter release and intrinsic granule cell excitability (Armano et al. 2000; D’Angelo et al. 1999). LTP persisted for the
entire duration of the recordings (usually 1 h) but could be
measured for 6 h in five experiments (data not shown). This
confirms the long-lasting nature of potentiation, the observation of which was usually limited to ⬍1 h by using patchclamp recordings from granule cells.
Mossy fiber terminal excitation
To reconstruct the process of presynaptic terminal excitation, one should recall that a depolarizing current runs upward
if it is generated below the electrode or downward if it comes
from neighboring regions. Thus invasion of the terminal by a
depolarizing current coming from the axon causes the first
downward deflection, P1 (Brigant and Mallart 1982). This
activates an inward current in the terminal, which largely
depends on Ca2⫹ channels and accounts for a considerable
fraction of N1 or, more specifically, N1b. Actually, this current
is reduced by Ni2⫹, ␻-CgTx-MVIIC, and by lowering the
extracellular Ca2⫹/Mg2⫹ ratio. The blocking action of
␻-CgTx-MVIIC supports the involvement of N- and P/Q-type
Ca2⫹ channels, which cause neurotransmitter release at numer-
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
mossy fiber terminals and was not influenced by Purkinje cell
discharge. The specificity of presynaptic changes for LTP was
demonstrated by their absence when pharmacological manipulations caused LTP induction failure. Moreover, presynaptic
changes were absent during short-term plasticity elicited by
PPS or when neurotransmitter release was modulated through
adenosine receptors (see Terminal current regulation and the
mechanism of LTP expression).
LTP showed properties consistent with those measured with
patch-clamp recordings from granule cells (Armano et al.
2000; D’Angelo et al. 1999). Although the present experiments
did not allow us to control the locus of drug application, the
preventative action exerted by synaptic inhibition and NMDA
receptor blockers is consistent with a voltage-dependent control of granule cell NMDA receptor-dependent LTP induction
through the Golgi cell circuit. Moreover, neither the mGluR-1
blocker AIDA (Schoepp et al. 1999) nor the PKC inhibitor GÖ
6983 (Bortolotto and Collingridge 2000) significantly affected
basal neurotransmission, tending to exclude their presynaptic
action. Consistently, the mGluR-1-IP3-PKC pathway has been
functionally and biochemically characterized in cerebellar
granule cells (Aronica et al. 1993; Masgrau et al. 2001), where
it has been proposed to influence LTP induction (D’Angelo et
al. 1999; Rossi et al. 1996). It should also be noted that
synaptic inhibition, APV, and AIDA, but not GÖ 6983, turned
the effect of TBS toward long-term depression (see Bliss and
Collingridge 1993; Fig. 6). This may reflect the presence of a
PKC-dependent switch in the metabolic pathways involved in
long-term synaptic plasticity (Bortolotto and Collingridge
PRESYNAPTIC CURRENT AT MOSSY FIBER–GRANULE CELL SYNAPSE
ous central synapses (Meir et al. 1999). K⫹ currents sensitive
to a low TEA concentration, potentially including a Ca2⫹dependent K⫹ current (Mallart 1985; Meir et al. 1999), limit
presynaptic terminal excitation.
The Ca2⫹ and K⫹ channel activation sequence proposed
here is similar to that revealed with focal recordings at the
neuromuscular junction of various vertebrates (Angaut-Petit et
al. 1989; Brigant and Mallart 1982). Ca2⫹ and K⫹ currents
have also been shown to shape the presynaptic action potentials
measured with whole cell recordings in hippocampal mossy
fiber terminals (Geiger and Jonas 2000) and in the calyx of
Held (Borst and Sakmann 1998). However, no Ca2⫹-dependent
components emerged from extracellular recordings in the CA1
region of hippocampal slices (Lærum and Storm 1994), perhaps reflecting smaller size or different membrane properties of
synaptic terminals.
Terminal current regulation and the mechanism of LTP
expression
Both the terminal current and the postsynaptic response
changed conjointly when Ca2⫹ influx was modified by altering
the Ca2⫹/Mg2⫹ ratio or by blocking Ca2⫹ or K⫹ channels.
J Neurophysiol • VOL
However, the postsynaptic response changed independently
from the terminal current after activation of A1 adenosine
receptors, in keeping with the cAMP-mediated reduction in
Ca2⫹ sensitivity of vesicular release observed at the neuromuscular junction (Silinsky 1984) and in the rat hippocampus
(Lupica et al. 1992). The postsynaptic response changed independently from the terminal current, also during PPS, which
modifies the number of available vesicles and their release
probability (see Lupica et al. 1992; Regher and Tank 1991;
Schultz et al. 1994). Thus terminal currents were functionally
related to neurotransmitter release through their Ca2⫹-dependent component.
During LTP, the Ca2⫹-dependent terminal current increased.
Occlusion by TEA suggests that this was due to a K⫹ current
reduction (Kandel and Schwartz 1982), raising Ca2⫹ relative to
K⫹ currents. Thus a simple hypothesis is that during LTP, an
increased Ca2⫹ influx raises neurotransmitter release (Hawkins
et al. 1993; Ishikawa and Takahashi 2001; Kuba and Kumamoto 1990). A presynaptic Ca2⫹ accumulation seems less
probable (Regher and Tank 1991; Wu and Saggau 1994) because the paired-pulse facilitation ratio of the presynaptic response did not change with LTP. Moreover, the volley was
stable and retrograde plasticity enhancing presynaptic neuron
excitability (Ganguly et al. 2000) could not take place because
the soma of neurons where mossy fibers are generated was not
included in the slice preparation.
During LTP, the correlation of pre- and postsynaptic
changes supports a causal relationship between terminal current and neurotransmitter release, and the change in pairedpulse facilitation of the postsynaptic response suggests a presynaptic locus of expression (Schultz et al. 1994). The
hypothesis that neurotransmitter release is enhanced bears several mechanistic implications, including an increase in the
frequency of minis, a decreased failure rate, and a decreased
excitatory postsynaptic current variability (Bekkers and
Stevens 1990; Bliss and Collingridge 1993; Hawkins et al.
1993; Malenka and Nicoll 1999; Malgaroli and Tsien 1992).
Because presynaptic changes depend on LTP induction in
granule cells, a retrograde messenger has to diffuse backwards
to the mossy fiber terminal to cause the presynaptic change
(Schuman and Madison 1991). Nitric oxide, which is released
by granule cells on NMDA receptor stimulation (Garthwaite et
al. 1988), might play this role.
We point out that, although the properties of presynaptic
current changes consistently support a presynaptic locus, we
cannot exclude that postsynaptic changes such as an increased
intrinsic excitability (Armano et al. 2000; Hansel et al. 2001) or
receptor expression (Malenka and Nicoll 1999) could also
contribute to mossy fiber LTP expression.
Conclusions
Our results indicate that, similar to what has been observed
in invertebrates, changes in presynaptic terminal excitability
occur in certain vertebrate synapses during long-term synaptic
plasticity (Hawkins et al. 1993; Kandel and Schwartz 1982).
This observation, together with evidence of increased vesicle
cycling (Nayak et al. 1996; Zakharenko et al. 2001), reinforces
the hypothesis that presynaptic changes enhance neurotransmitter release during LTP. However, it is unknown whether
these mechanisms take place at all brain synapses or whether
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
⫹
FIG. 9. Occlusion of LTP and presynaptic current changes by K channel
blockers. Involvement of K⫹ channels was investigated by 1 mM tetraethylammonium (TEA) application. After a control recording period (a), application
of 1 mM TEA increased N1 and N2 (b). TBS (arrow) could not induce any
potentiation either in N1 or N2 (c). Plots, time course of N1 and N2 changes;
bar, TEA perfusion (mean ⫾ SE; n ⫽ 3). SN showed changes similar to those
in N2.
637
638
MAFFEI, PRESTORI, ROSSI, TAGLIETTI, AND D’ANGELO
they subserve specific regulatory actions. We surmise that
presynaptic current plasticity can simultaneously regulate
transmission to the numerous granule cells impinging on each
mossy fiber terminal (28 on average in the rat; see Fig. 1C)
(Eccles et al. 1967). This would contribute to spatial processing
of mossy fiber discharge in the cerebellar granular layer and to
motor control (Eccles et al. 1967; Medina and Mauk 2000).
We thank Dr. Lia Forti for her comments on the manuscript. This work was
supported by European Community grants QLRT-2000 – 02256 and
PL-976060, by Instituto Nazionale Fisica della Materia Progetto Rict́rca Avanzata-Cady, and by Ministerio dell’ Università e della Ricerca Scientifica e
Tecnologica.
REFERENCES
J Neurophysiol • VOL
88 • AUGUST 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
ANGAUT-PETIT D, BENOIT E, AND MALLART A. Membrane currents in lizard
motor nerve terminals and nodes of Ranvier. Pflügers Arch 415: 81– 87,
1989.
ANIZKSTEJN L AND BEN-ARI Y. Novel form of long-term potentiation produced
by a K⫹ channel blocker in the hippocampus. Nature 349: 67– 69, 1991.
ARONICA E, CONDORELLI DF, NICOLETTI F, DELL’ALBANI P, AMICO C, AND
BALAZS R. Metabotropic glutamate receptors in cultured cerebellar granule
cells: developmental profile. J Neurochem 60: 559 –565, 1993.
ARMANO S, ROSSI P, TAGLIETTI V, AND D’ANGELO E. Long-term potentiation
of intrinsic excitability at the mossy fiber– granule cell synapse of rat
cerebellum. J Neurosci 20: 5208 –5216, 2000.
BEKKERS JM AND STEVENS CF. Presynaptic mechanism for long-term potentiation in the hippocampus. Nature 346: 724 –728, 1990.
BLISS TVP AND COLLINGRIDGE GL. A synaptic model of memory: long-term
potentiation in the hippocampus. Nature 361: 31–39, 1993.
BORST JGG AND SAKMANN B. Calcium current during a single action potential
in a large presynaptic terminal of the rat brainstem. J Physiol (Lond) 506:
143–157, 1998.
BORTOLOTTO ZA AND COLLINGRIDGE GL. A role for protein kinase C in a form
of metaplasticity that regulates the induction of long-term potentiation at
CA1 synapses of the adult rat hippocampus. Eur J Neurosci 12: 4055– 4062,
2000.
BRIGANT JL AND MALLART A. Presynaptic currents in mouse motor endings.
J Physiol (Lond) 333: 619 – 636, 1982.
D’ANGELO E, DE FILIPPI G, ROSSI P, AND TAGLIETTI V. Synaptic excitation of
individual rat cerebellar granule cells in situ: evidence for the role of NMDA
receptors. J Physiol (Lond) 484: 397– 413, 1995.
D’ANGELO E, ROSSI P, ARMANO S, AND TAGLIETTI V. Evidence for NMDA and
mGlu receptor-dependent long-term potentiation of mossy fibre– granule
cell transmission in rat cerebellum. J Neurophysiol 81: 277–287, 1999.
DEL CASTILLO J AND KATZ B. Localization of active spots within the neuromuscular junction of the frog. J Physiol (Lond) 132: 630 – 649, 1956.
DODGE FA JR AND RAHAMIMOFF R. Co-operative action a calcium ions in
transmitter release at the neuromuscular junction. J Physiol (Lond) 193:
419 – 432, 1967.
ECCLES JC, ITO M, AND SZENTAGOTHAI J. The Cerebellum as a Neuronal
Machine. Berlin: Springer Verlag, 1967.
FORSYTHE ID, TSUJIMOTO T, BARNES-DAVIES M, CUTTLE MF, AND TAKAHASHI
T. Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse. Neuron 20: 797– 807, 1998.
FORTI L, BOSSI M, BERGAMASCHI A, VILLA A, AND MALGAROLI A. Loose-patch
recordings of single quanta at individual hippocampal synapses. Nature 388:
874 – 878, 1997.
FREDHOLM BB. Purinoreceptors in the nervous system. Pharmacol Toxicol 76:
228 –239, 1995.
GANGULY K, KISS L, AND POO M. Enhancement of presynaptic neuronal
excitability by correlated presynaptic and postsynaptic spiking. Nature Neurosci 3: 1018 –1026, 2000.
GARTHWAITE J, CHARLES SL, AND CHESS-WILLIAMS R. Endothelium-derived
relaxing factor release on activation of NMDA receptors suggests role as
intercellular messenger in the brain. Nature 336: 385–388, 1988.
GARTHWAITE J AND BRODBELT AR. Synaptic activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway in
adult and immature rat cerebellar slices. Neurosci 29: 401– 412, 1989.
GEIGER JRP AND JONAS P. Dynamic control of presynaptic Ca2⫹ inflow by
fast-inactivating K⫹ channels in hippocampal mossy fiber boutons. Neuron
28: 927–939, 2000.
HANSEL C, LINDEN DJ, AND D’ANGELO E. Beyond parallel fiber LTD: the
diversity of synaptic and non-synaptic plasticity in the cerebellum. Nature
Neurosci 4: 467– 475, 2001.
HAWKINS RD, KANDEL ER, AND SIEGELBAUM SA. Learning to modulate transmitter release: themes and variations in synaptic plasticity. Annu Rev Neurosci 16: 625– 665, 1993.
ISHIKAWA T AND TAKAHASHI T. Mechanisms underlying presynaptic facilitatory effect of cyclothiazide at the calix of Held of juvenile rats. J Physiol
(Lond) 533: 423– 431, 2001.
KANDEL ER AND SCHWARTZ JH. Molecular biology of learning: modulation of
transmitter release. Science 218: 433– 443, 1982.
KATZ B AND MILEDI R. Propagation of electrical activity in motor nerve
terminals. Proc R Soc Lond B Biol Sci 161: 453– 482, 1965.
KUBA K AND KUMAMOTO E. Long-term potentiation in vertebrate synapses: a
variety of cascades with common subprocesses. Prog Neurobiol 34: 197–
269, 1990.
LÆRUM H AND STORM JF. Hippocampal long-term potentiation is not accompanied by presynaptic spike broadening, unlike synaptic potentiation by K⫹
channel blockers. Brain Res 637: 349 –355, 1994.
LUPICA CR, PROCTOR WR, AND DUNWIDDIE TV. Presynaptic inhibition of
excitatory synaptic transmission by adenosine in rat hippocampus: analysis
of unitary EPSP variance measured by whole-cell recordings. J Neurosci 12:
3753–3764, 1992.
MALENKA RC AND NICOLL RA. Long-term potentiation—a decade of progress?
Science 285: 1870 –1874, 1999.
MALGAROLI A, TING AE, WENDLAND B, BERGAMASCHI A, VILLA A, TSIEN RW,
AND SCHELLER RH. Presynaptic component of long-term potentiation visualized at individual hippocampal synapses. Science 268: 1624 –1628, 1995.
MALGAROLI A AND TSIEN RW. Glutamate-induced long-term potentiation of
the frequency of miniature synaptic currents in cultured hippocampal neurons. Nature 357: 134 –139, 1992.
MALLART A. A calcium-activated potassium current in motor nerve terminals
of the mouse. J Physiol (Lond) 368: 577–591, 1985.
MASGRAU R, SERVITJIA JM, YOUNG KW, PARDO R, SARRI E, NAHORSHI SR,
AND PICATOSTE F. Characterization of the metabotropic glutamate receptors
mediating phospholipase C activation and calcium release in cerebellar
granule cells: calcium-dependence of the phospholipase C response. Eur J
Neurosci 13: 248 –256, 2001.
MEDINA JF AND MAUK MD. Computer simulation of cerebellar information
processing. Nat Neurosci Suppl 3: 1205–1211, 2000.
MEIR A, GINSBURG S, BUTKEVICH A, KACHALSKY SG, KAISERMAN I, AHDUT R,
DEMIRGOREN S, AND RAHAMIMOFF R. Ion channels in presynaptic nerve
terminals and control of transmitter release. Physiol Rev 79: 1019 –1088,
1999.
NAYAK AS, MOORE CI, AND BROWING MD. Ca2⫹/calmodulin-dependent protein kynase II phosphorilation of the presynaptic protein synapsin I is
persistently increased during long-term potentiation. Proc Natl Acad Sci
USA 93: 15451–15456, 1996.
PALAY SL AND CHAN-PALAY V. Cerebellar Cortex. Berlin: Springer Verlag,
1974.
REGHER WG AND TANK DW. The maintenance of LTP at hippocampal mossy
fiber synapses is independent of sustained presynaptic calcium. Neuron 7:
451– 459, 1991.
ROSSI P, D’ANGELO E, AND TAGLIETTI V. Differential long-lasting potentiation
of the NMDA and non-NMDA synaptic currents induced by metabotropic
and NMDA receptor coactivation in cerebellar granule cells. Eur J Neurosci
8: 1182–1189, 1996.
SCHOEPP DD, JANE DE, AND MONN JA. Pharmacological agents acting at
subtypes of metabotropic glutamate receptors. Neuropharmacol 38: 1431–
1476, 1999.
SCHULTZ PE, COOK EP, AND JOHNSTON D. Changes in paired-pulse facilitation
suggest presynaptic involvement in long-term potentiation. J Neurosci 14:
5325–5337, 1994.
SCHUMAN EM AND MADISON DV. A requirement for the intercellular messenger nitric oxide in long-term potentiation. Science 254: 1503–1506, 1991.
SILINSKY EM. On the mechanism by which adenosine receptor activation
inhibits the release of acetylcholine from motor nerve endings. J Physiol
(Lond) 346: 243–256, 1984.
WU LG AND SAGGAU P. Presynaptic calcium is increased during normal
synaptic transmission and paired pulse-facilitation, but not in long-term
potentiation in area CA1 of hippocampus. J Neurosci 14: 645– 654, 1994.
ZAKHARENKO SS, ZABLOW L, AND SIEGELBAUM SA. Visualization of changes in
presynaptic function during long-term synaptic plasticity. Nature Neurosci
4: 711–717, 2001.