1Unité de Neurosciences Intégratives et Computationnelles, Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France; and 2Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel
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ABSTRACT |
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Ego-Stengel, Valérie, Daniel E. Shulz, Sebastian Haidarliu, Ronen Sosnik, and Ehud Ahissar. Acetylcholine-Dependent Induction and Expression of Functional Plasticity in the Barrel Cortex of the Adult Rat. J. Neurophysiol. 86: 422-437, 2001. The involvement of acetylcholine (ACh) in the induction of neuronal sensory plasticity is well documented. Recently we demonstrated in the somatosensory cortex of the anesthetized rat that ACh is also involved in the expression of neuronal plasticity. Pairing stimulation of the principal whisker at a fixed temporal frequency with ACh iontophoresis induced potentiations of response that required re-application of ACh to be expressed. Here we fully characterize this phenomenon and extend it to stimulation of adjacent whiskers. We show that these ACh-dependent potentiations are cumulative and reversible. When several sensori-cholinergic pairings were applied consecutively with stimulation of the principal whisker, the response at the paired frequency was further increased, demonstrating a cumulative process that could reach saturation levels. The potentiations were specific to the stimulus frequency: if the successive pairings were done at different frequencies, then the potentiation caused by the first pairing was depotentiated, whereas the response to the newly paired frequency was potentiated. During testing, the potentiation of response did not develop immediately on the presentation of the paired frequency during application of ACh: the analysis of the kinetics of the effect indicates that this process requires the sequential presentation of several trains of stimulation at the paired frequency to be expressed. We present evidence that a plasticity with similar characteristics can be induced for responses to stimulation of an adjacent whisker, suggesting that this potentiation could participate in receptive field spatial reorganizations. The spatial and temporal properties of the ACh-dependent plasticity presented here impose specific constraints on the underlying cellular and molecular mechanisms.
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INTRODUCTION |
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The study of the required
conditions for the induction of neuronal plasticity in the adult
primary sensory cortices has led to the implication of neuromodulators
in this process. Acetylcholine (ACh) released in the cortex from fibers
originating in the nucleus basalis magnocellularis (NBM) is a major
candidate (Dykes 1997; Singer 1990
).
Indeed, ACh micro-iontophoresis (Greuel et al. 1988
; Metherate and Weinberger 1989
; Metherate et al.
1987
, 1988a
,b
) or stimulation of the NBM (Bakin and
Weinberger 1996
; Edeline et al. 1994
;
Kilgard and Merzenich 1998b
; Tremblay et al.
1990a
,b
) during the repetitive presentation of a stimulus is
sufficient to induce long-lasting modifications of neuronal responses.
Furthermore, cortical map reorganization and neuronal receptive field
changes in sensory cortices were shown to be blocked by lesions of the cholinergic system (Baskerville et al. 1997
; Bear
and Singer 1986
; Sachdev et al. 1998
) or by
cholinergic antagonists (Maalouf et al. 1998
). Thus
increased levels of ACh in the cortex provide the adequate
neurochemical environment for the induction of plasticity (Dykes
1997
; Singer 1990
).
By contrast, the requirements for ACh during the expression phase of
plasticity have not been extensively studied. In the olfactory cortex,
ACh exerts a differential effect on thalamocortical versus
intracortical pathways (Hasselmo and Bower 1993). Based on these observations, these authors proposed that increased levels of
ACh promote learning of new information by enhancing afferent inputs
and enabling plasticity, whereas decreased cholinergic levels
facilitate retrieval (Hasselmo and Bower 1993
). However, behavioral studies have shown instances in which retrieval of a newly
acquired memory depends on the similarity between the endogenous
neurochemical state that develops after training and the one that
develops during testing [endogenous state-dependent learning
(discussed in Izquierdo 1984
)]. This suggests that at the cellular level, retrieval of an ACh-induced plasticity could be
improved by the presence of ACh during testing (Zornetzer
1978
). We have recently reported that in the barrel cortex of
anesthetized rats, ACh plays a dual role in neuronal plasticity: it is
essential both during the induction and the expression phases
(Shulz et al. 2000
). Herein, we analyzed the effects of
applying consecutive sensori-cholinergic pairing protocols,
investigated the retrieval kinetics, and tested to see if the
ACh-dependent plasticity occurred when stimulating nonprincipal
whiskers as well. The latter analysis was motivated by the fact that
previous studies on plasticity in the barrel cortex using
whisker-pairing protocols have shown that enhancement in response was
prominent for the intact adjacent whisker as well as for the principal
whisker (Armstrong-James et al. 1994
; Diamond et
al. 1993
).
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METHODS |
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Animal preparation
Twenty-four adult male Wistar albino rats weighing 300 ± 25 g obtained from the Animal Breeding Unit of The Weizmann
Institute of Science were used for these experiments. Maintenance,
manipulations, and surgery were according to institutional animal
welfare guidelines that meet the National Institutes of Health
standards. The animals received an injection of atropine methyl nitrate
(0.3 mg/kg im), a derivative of atropine that does not cross the
blood-brain barrier (Weiner 1980), and were anesthetized
with urethan (1.5 g/kg ip). Supplementary doses of urethan (0.15 g/kg
ip) were administered when necessary throughout the experiment to
maintain an adequate level of anesthesia, indicated by the absence of
eyeblink reflex or response to hindpaw pinch. Body temperature was
maintained at 37°C using a temperature-regulated heating pad.
The animal was mounted in a stereotaxic frame with a modified head
holder without ear bars, which allowed free access to the somatosensory
cortex and to vibrissae (Haidarliu 1996). A local anesthetic (lidocaïne, 2%) was injected subcutaneously in all skin incisions. The right scalp and temporal muscle were resected. A
3 × 3 mm craniotomy was made to expose the right
posteromedial barrel subfield (PMBSF;
P1-P4,
L4-L7 from Bregma)
(Chapin and Lin 1984
). The dura was opened. A dental
cement cup was made surrounding the skull opening and was filled with
saline to prevent drying of the cortex. Vibrissae were clipped on the
left side of the snout to a length of 1 cm.
Electrophysiological recording and iontophoresis
Neural activity was recorded extracellularly with a
multi-electrode array composed of one or two tungsten-in-glass
electrodes (TE, 0.2-0.8 M at 1 kHz) and one or two combined
electrodes (CE) mounted within a metallic guide tube (Haidarliu
et al. 1995
). The CEs were composed of a tungsten-in-glass
electrode surrounded by six glass micropipettes for simultaneous
iontophoresis and recording. The six barrels were filled with
acetylcholine chloride (1 M, pH 4.5) and sodium chloride (3 M) for
current balance. In three experiments, one iontophoresis barrel was
filled with atropine sulfate (0.1 M, pH 4.5). Results reported in this
paper do not involve atropine iontophoresis. Retaining currents of
10
nA were used to prevent drugs from leaking. During periods of ejection, balanced 20- to 80-nA currents were applied. The CEs and TEs were lowered independently using a multi-electrode microdrive system. Signals were amplified and filtered for spike activity (0.5-8 kHz).
For each recording electrode, up to three single units were isolated
using a template-matching spike sorter (MSD-2; Alpha-Omega, Nazareth,
Israel). The shape of action potentials was continuously inspected to
ensure that the same neurons were recorded throughout the protocols.
When action potential waveforms could not be discriminated, multi-unit
data were collected either by defining a template encompassing several
waveforms or by amplitude sorting. Spike times were acquired on a
computer at 1 kHz.
Whisker stimulation and pairing protocol
Once units were isolated, vibrissae were at first manually
deflected while monitoring the extracellular signal. For each unit, the
principal whisker was defined as the whisker eliciting the maximal
neuronal response. This whisker was chosen for computer-controlled stimulation. Since the electrodes in the array could be located in
different barrels, in some cases simultaneously recorded units did not
have the same principal whisker. We selected the principal whisker of
units recorded by a CE for subsequent stimulation. Hence for some of
the other units, the stimulated whisker was an adjacent whisker rather
than their principal whisker. We inserted the selected whisker in a
short Teflon tubing attached to a linear electromagnetic vibrator
(Schneider 1988). Stimulation was automatically controlled by the data-acquisition computer and consisted of pulses of
5-ms rise time followed by 5-ms fall time, producing a 160 µm
rostrocaudal deflection at ~5 mm from the follicle of the deflected whisker.
We determined the response to deflections of the vibrissae at temporal frequencies from 2 to 11 Hz (Fig. 1A). For each frequency, stimuli were always applied in blocks of 12 consecutive trains of 4 s + 1-s inter-train interval; each block of stimulation thus lasted 60 s. The temporal-frequency tuning curve (TFTC) of each unit was determined by deflecting the principal vibrissa at different frequencies in the following order: 2, 5, 8, 11, (in a few cases 14), (45-s interval), (14), 11, 8, 5, 2 Hz, with inter-block intervals of 10 s. The 45-s interval was designed to effectively separate the two blocks of stimulation at the highest frequency. Consequently, responses at each frequency were obtained from two blocks of 12 trains of stimuli each. The total number of deflections ranged from 192 at 2 Hz to 1,056 at 11 Hz. In a few cases (n = 19/208), 14 Hz was also tested.
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Two control TFTCs were determined before pairing: one in the absence of iontophoresis and a second one during ACh iontophoresis. We then applied a pairing protocol consisting of one block of 24 trains of stimulation (each of 4 s + 1-s inter-train interval) of the vibrissa at one fixed temporal frequency (5, 8, or 11 Hz) accompanied with ACh iontophoresis. Following pairing, two test TFTCs were determined again, one without ACh and one with ACh.
The temporal stability of response and the eventual effect of the application of ACh during the second control TFTC were tested on 40 units for which the protocol was identical except that the pairing period between the control and test TFTCs was omitted ("unpaired" group). The sequence of stimulation was in those cases: TFTC without ACh, TFTC with ACh, TFTC without ACh, and TFTC with ACh.
Data analysis
Recordings were monitored on-line by inspecting a rate meter for each unit (firing rate as a function of time) and data analysis was performed off-line (Matlab). Units that had a discharge rate less than 2 spikes/s (including the spontaneous activity) in response to deflections of the principal whisker at 2 Hz were considered as unresponsive units and analyzed separately.
Unit responses to stimulation were plotted as raster diagrams (Fig. 1B). The response of a unit to a deflection of the vibrissa was defined as the spike count in a fixed temporal window chosen to contain the entire response (0-60 ms; restricted to 50 ms for 23 units for which an inhibition phase started at 50 ms; see Fig. 1E). Response during stimulation trains was composed of an initial adapting phase during the first 500 ms followed by a steady-state response. Only deflections between 500 and 4,000 ms of each train were included for quantification of the steady-state regime. Conversely, the first deflection of each train was analyzed separately. Peristimulus time histograms (PSTHs) were constructed for each stimulation frequency by averaging the instantaneous firing rate of the unit relative to the onset of deflection of the vibrissa (Fig. 1E). One-millisecond bins were used, and smoothing was achieved by convolution with a right triangle of area 1 and base 4 ms. Note that due to the periodic nature of the stimulation, especially for the higher frequencies, the activity that precedes the stimulus in each PSTH corresponds to the tonic activation of the unit during the stimulus train and cannot be considered as a spontaneous activity. To estimate the decrease of the response within a train and the kinetics of response from train to train, the spike count was averaged respectively for individual deflections across the 24 trains (Fig. 1C) and for deflections in the steady state of each train (Fig. 1D). TFTCs were obtained by plotting the average spike count as a function of the frequency of stimulation.
We looked for specific changes in the response of the unit at each
frequency compared with other frequencies independently of global
modifications of excitability. To this purpose, the relative strength
of the response to a given frequency was quantified by the weighted
ratio WR = (Rf - AvgR)/(Rf + AvgR), where Rf is the response to
stimulation at a given frequency and AvgR is the averaged response to
stimulation at all other frequencies. This ratio, which takes values
from 1 to +1, was calculated independently for each of the 24 trains
of stimuli and for each frequency. To assess the effect of pairing, the
24 values obtained before and after pairing were statistically compared
[2-tailed Kolmogorov-Smirnov (KS), significance level
P < 0.01]. This comparison was performed independently for each frequency and for the two test conditions, without and with ACh. When several pairings were performed on the same
units and to keep the initial state comparable among units, only the
first paired frequency was considered for quantifying the percentage of
modified units. The effect was assessed systematically on the test
period immediately after the last pairing at that frequency. Average
values are displayed as means ± SE unless indicated otherwise.
Histology
At the end of seven experiments, small electrolytic lesions were made at known depths using 3- to 5-µA current applied twice for 2 s through one of the tungsten-in-glass electrodes. The animal was given a lethal dose of thiopentone (0.5 ml ip per animal) and perfused transcardially with saline followed by a fixative solution (2.5% glutaraldehyde, 0.5% paraformaldehyde, and 5% sucrose in 0.1 M phosphate buffer, pH 7.4). Tangential or coronal sections (50 or 60 µm) were cut through the right PMBSF and stained for cytochrome oxidase to visualize barrels. The laminar positions of the lesions in coronal sections were used to establish a correspondence between the depth of the electrode penetration and the layer recorded from. This relation enabled us to estimate the laminar location of each cell from its recording depth.
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RESULTS |
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Two hundred and eight units were recorded in the somatosensory cortex of adult rats during at least one complete stimulation protocol. Twenty-two units were unresponsive to whisker stimulation (see METHODS) and were analyzed separately. Of the remaining 186 units, 134 (62 single units and 72 multi-units) were recorded by a combined electrode (CE) and 52 (24 single units and 28 multi-units) by a tungsten-in-glass electrode (TE).
Spontaneous and evoked activity in control conditions
The spontaneous activity of units was quantified over periods of 45 to 110 s prior to any pharmacological stimulation. Single and multi-units recorded by the TEs had an average spontaneous firing rate similar to single and multi-units recorded by the CEs (TEs: 12.1 ± 2.1 spikes/s; CEs: 12.9 ± 1.4 spikes/s; 2-tailed Student's t-test, P > 0.7), suggesting that the geometry of the combined electrodes did not introduce a sampling bias.
The stimulated whisker was mechanically deflected at frequencies ranging from 2 to 11 Hz. Units responded with phasic increased activity after each deflection. The raster plots displayed in Fig. 2 show the response of a cortical unit to two blocks of stimulation (12 4-s trains each) at 2, 5, 8, and 11 Hz. The first deflection of each train, which in all cases was preceded by a 1-s stimulation-free period, elicited a comparable discharge rate whatever the frequency of stimulation. The following deflection occurred after a variable time interval depending on the stimulus frequency, from 500 ms at 2 Hz to 91 ms at 11 Hz, and produced a smaller response. This decrease in evoked activity from one deflection to the following was prominent for shorter intervals, i.e., higher frequencies of stimulation. After this transient kinetics, the response reached a steady-state level that decreased with increasing stimulation frequencies. Almost all units exhibited these low-pass filter characteristics: in 180/186 cases the response to 5-Hz stimulation was lower than the response to 2-Hz stimulation (this difference reached significance in 98 cases; 1-tailed Mann-Whitney U test, P < 0.01). In a few cases, however, the steady-state response to 5-Hz stimulation was significantly greater than to 2-Hz stimulation (n = 6/186). This specific tuning property was not correlated to other cell parameters (depth, spontaneous and evoked levels of activity). Figure 2 also demonstrates temporal stability of responses during the recording because the responses to stimulation at the same frequency during different blocks (which for 2 Hz for example were done at 10 min interval) were unchanged.
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The response to each deflection was quantified by the number of action potentials in the temporal window 0-60 ms after the onset of deflection. By averaging this spike count across trains, the kinetics of the discharge rate during the train was compared across frequencies. Figure 3A displays the average kinetics for all single units recorded in the barrel corresponding to the stimulated whisker (left, n = 63) and in adjacent barrels (right, n = 13). For both populations, the response to the first deflection of the train was constant across frequencies, whereas the response to following deflections rapidly decreased and stabilized at different plateau values depending on the stimulation frequency. This adaptation phenomenon usually did not occur at 2 Hz (red lines in Fig. 3A), indicating the lack of lasting effect 500 ms after the onset of whisker deflection, and was strongest at 11 Hz. The low-pass filter characteristic was observed both when the principal whisker or an adjacent whisker was stimulated. In the latter case, however, steady-state responses at higher frequencies (8 and 11 Hz) were on average indistinguishable from spontaneous activity.
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We investigated whether the response evolved from one train to the next as stimulation at one fixed frequency was presented. This was done by plotting the steady-state value of the response (calculated as the average of individual deflection responses in the 500- to 4,000-ms window of each train) as a function of the train number. Figure 3B displays the result of this analysis for the same populations as in Fig. 3A. No systematic trend was observed from one train to another or from the first block of stimulation (trains 1-12) to the second block (trains 13-24). This confirms that the adaptation rate and the evoked activity of barrel cortex neurons was stable over the course of a TFTC protocol.
Pairing-induced plasticity of the response to principal-whisker stimulation
A full pairing protocol was applied on 119 units recorded by the CEs. Units for which the principal whisker (n = 105) and an adjacent whisker (n = 14) were stimulated were analyzed separately for assessing the percentage of modifications.
Frequency-specific modifications of response were observed following
pairing of ACh iontophoresis with stimulation of the principal whisker
at a fixed temporal frequency. Figure 4
shows two examples of significant potentiations of the response to
stimulation at the paired frequency. For the cortical unit in Fig.
4A1, submitted to a pairing at 8 Hz, the response to
8-Hz stimulation was enhanced after pairing when tested with ACh
iontophoresis (KS, P < 1.108). The potentiation
was revealed only for the paired frequency and exclusively when
the unit was tested with ACh (KS, P = 0.3 for the test
without ACh). As seen in this example, the modifications of the
response following each deflection could be accompanied by an increase
in the tonic level of activity within the train. In control conditions,
this tonic level was constant with stimulation frequency or increased
concentrations of ACh. Its modification after pairing was thus
unexpected. We quantified this component of the response as the
integrated spike count in the 20 ms preceding each deflection.
Statistical analysis was conducted for this additional set of values.
Both the phasic (due to each whisker deflection) and tonic (due to the
entire train of deflections) components of the response were increased
in the example of Fig. 4A1 (KS, P < 1.10
4 for each
component). In Fig. 4B1, pairing ACh iontophoresis with whisker stimulation at 11 Hz on a different cortical unit also resulted
in a significant enhancement of response at the paired frequency (KS,
P < 1.10
7; P < 1.10
4 for each
component of the response). The TFTCs summarize the frequency
specificity of the potentiation; TFTCs computed before and after
pairing overlap for all frequencies except the paired ones (Fig. 4,
A2 and B2).
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Frequency-specific changes in response were quantified for each unit by calculating the difference (after minus before pairing) in relative strength of the response to stimulation at each frequency (WR). The ratio WR is not affected by global multiplicative changes in responsiveness; changes in WR indicate changes in response at one frequency relative to the responses at other frequencies. The cortical units of Fig. 4 showed an increased absolute response for the paired frequency after pairing and no change for unpaired frequencies. Consequently, the relative strength in response (WR) to the paired frequency was increased and those to the unpaired frequencies were decreased (Fig. 4, A2 and B2, right).
Each unit was tested for statistically significant modifications of its relative response (expressed by WR) at the paired frequency. Overall, 29% of the units had a significantly modified response after pairing when tested with ACh and the majority of these changes were potentiations (18 of 30). Twenty-one percent of the units had a modified response when tested without ACh, and these changes were mainly decreases in response (13 of 22). Similar results were obtained when the analysis was restricted to single units: 8 cells of 53 showed a modified response when tested without ACh, whereas 16 showed a modified response when tested with ACh of which 75% were potentiations.
We studied whether these changes in response resulted from the
fixed-frequency pairing by comparing response modifications obtained
after ACh pairing and response modifications observed when units were
only repeatedly tested without and with ACh (see METHODS).
Figure 5 displays the result of such
repetitive testing for two cortical units. In Fig. 5A, PSTHs
of response before and after pairing show no frequency-specific
modification of response when tested either without ACh or with ACh
(KS, P > 0.05). Similarly for the cell depicted in
Fig. 5B, although a general decrease in response occurred,
we did not observe a significant potentiation of the response at one
frequency compared with others (blue vs. green PSTHs in Fig.
5B; KS, P > 0.05). However, a
frequency-specific ACh-dependent potentiation to stimulation at 5 Hz
was revealed after pairing ACh iontophoresis with stimulation at that
frequency (red vs. blue PSTHs; KS, P < 1.105), indicating that
the effect was caused by the pairing.
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We compared the modifications of response observed for units submitted
to repetitive testing ("unpaired" group) and units submitted to the
pairings ("paired" group). The cumulative distribution of changes
expressed with ACh were significantly different for these two
populations (1-tailed Mann-Whitney U test, P < 0.001) (Shulz et al. 2000) and revealed that the
potentiations of response could be attributed to the effects of the
pairings whereas the depressions could be explained, at least on a
statistical background, by the ACh-induced variability. Moreover, the
TFTC reorganization appeared to be different in the two groups: whereas
changes in the paired group were highly specific to the paired
frequency, and thus exhibited a sharp peak at that frequency, in the
unpaired group, changes were usually distributed across frequencies. To demonstrate this difference in the profile of changes, we averaged response ratio (WR) changes for all units showing a statistically significant potentiation at any frequency, separately for units in the
paired and unpaired groups. As expected, in the paired group, there was
a significant enhancement in response at the paired frequency compared
with changes at other frequencies (ANOVA, P < 0.01).
By contrast, in the unpaired group, changes in response at all
frequencies were similar (ANOVA, P > 0.3), which
indicates that there was no "natural" tendency for spontaneous
potentiations at one particular frequency within the range used here.
Second, we quantified the peak observed in the profile of changes by
calculating for the two groups of cells the difference between the
changes at the paired frequency (for the unpaired group, the maximally enhanced frequency) and the average change at the two neighboring frequencies (i.e., ±3 Hz). This value, which measures the contrast between the response change at the peak and at neighboring frequencies, was significantly higher in the paired group than in the unpaired group
(2-tailed Student's t-test, P < 0.0004).
This result confirms that when changes occurred due to the pairings,
they were specific to the paired frequency, whereas changes observed
after repetitive testing corresponded to global changes in excitability
that generalized to neighboring frequencies (Fig. 5).
Pairing-induced plasticity of the response to adjacent-whisker stimulation
We conducted a separate analysis for the 14 units recorded by CEs,
submitted to at least one pairing protocol, and for which we stimulated
one adjacent whisker instead of the principal whisker. As for
principal-whisker stimulation, frequency-specific and ACh-dependent modifications of response were observed following pairing. The cortical
cell of Fig. 6A exhibited a
weak response to whisker stimulation at 8 Hz in control conditions.
After pairing ACh iontophoresis with stimulation at that frequency, the
response to 8 Hz was enhanced when testing with ACh iontophoresis (KS,
P < 1.105), whereas it was
unchanged when testing without ACh (KS, P = 0.9).
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Population analysis confirmed that the effects of the pairing protocols
were not restricted to units located in the barrel corresponding to the
stimulated whisker. Six units of 14, which were stimulated via a
nonprincipal whisker, showed a significant potentiation of response to
stimulation at the paired frequency when tested with ACh after pairing,
which was significantly more (2,
P < 0.05) than the percentage for principal-whisker
stimulation (42 vs. 17%). For both populations of cells, the
potentiations observed during testing with ACh were maximal for the
paired frequency compared with unpaired frequencies (1-tailed
Student's t-test, P < 0.05), whereas no
significant difference was observed when tested without ACh (1-tailed
Student's t-test, P > 0.15; Fig. 6B). Additionally, both populations of units showed
cumulative and reversible effects and similar kinetics of the
expression of modifications; therefore they were grouped in one large
dataset for the description of these characteristics.
Laminar location of cells expressing pairing-induced potentiations
Histological localization of the recording sites was performed
after seven experiments in which small electrolytic lesions were made
at the end of the recordings. Using the known depths of those
recordings, we established a layer-depth correspondence and used this
relation to estimate the laminar location of other recording sites for
which we had the direct reading of the electrode microdrive. Based on
this estimation, cells expressing a potentiation of the response to the
paired frequency when tested with ACh were exclusively found in layers
IV and V of the barrel cortex (layer IV, n = 14/75;
layer V, n = 10/31). Layers II, III, and VI, which were
less explored in our study, did not show such plasticity (layers II and
III, n = 0/6; layer VI, n = 0/7).
However, the difference in the proportion of potentiated cells across
layers was not significant (2,
P = 0.10).
Pairing-induced modifications are not transferred through the intracortical network
Within the multi-electrode array, units recorded by the CEs were
presumably directly reached by iontophoresed ACh, whereas units
recorded by the TEs were beyond the range of diffusion of ACh
(Haidarliu, Shulz, and Ahissar, unpublished results). Indirect effects of ACh could be mediated, however, through modulation of
network activity. We investigated whether the response of units recorded by the TEs were modified after the pairing protocol. Of 23 single units, only one showed an increased response to stimulation at
the paired frequency when tested with ACh. This was significantly less
than for units recorded by the CEs (2,
P < 0.05); this was consistent with the limited
diffusion volume of ACh in the cortex and suggested that the change in
the activity of units reached by iontophoresed ACh did not induce
significant changes in the activity of distant units through the
cortical network.
Effects of pairing on unresponsive units
Twenty-two units were initially unresponsive to whisker stimulation; 8 of these units were recorded by a CE and submitted to at least one complete pairing protocol (19 protocols were applied in total for the 8 units). We did not observe the appearance of a response to stimulation at any of the tested frequencies in any of these cases when tested with ACh (KS, P > 0.05), suggesting that the response potentiations revealed for initially active units resulted from increases in the discharge rates of the recorded units and not from the addition of de novo responses of previously silent units.
Cumulative effects of consecutive pairing protocols
On 57 units, we performed one to three additional pairing
protocols at the same frequency after the first pairing. We observed cumulative effects until a maximal enhancement of response to stimulation at the paired frequency was reached. Figure
7 displays the results on two cortical
cells submitted to three consecutive pairings at 5 (Fig. 7A)
and 8 Hz (Fig. 7B). In the first example, two pairing
protocols were necessary to reveal ACh-dependent plasticity at the
paired frequency (1st pairing, KS, P > 0.9; 2nd
pairing, KS, P < 0.005 compared with initial control),
and a third pairing further enhanced the potentiation (KS,
P < 1.106). The time course
of the potentiation through the three pairings is depicted in Fig.
7A2. Note that the TFTC as well as the WR value for the
paired frequency remained unchanged when tested without ACh (KS,
P > 0.4 for all pairings) even though tests without and with ACh alternated during the experiment. In the second example, potentiation of the response to stimulation at the paired frequency during ACh iontophoresis was already present after the first pairing (KS, P < 1.10
4) and reached its
maximum after the second pairing (KS, P < 1.10
4). As confirmed by
the consecutive values of WR (Fig. 7B2), the potentiation
was saturated after the second pairing since a third pairing did not
further increase the relative strength of the response of the cell for
the paired frequency. With this unit, the noncholinergic tests revealed
constant WRs (KS, P > 0.2), except for the last
pairing, after which it was significantly reduced (KS,
P < 1.10
5). Whether this
reduction was related to the saturation of the ACh-dependent expression
is not known.
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Of the 57 units tested, 11 units exhibited a significantly increased
response to stimulation at the paired frequency during ACh
iontophoresis after a series of several pairings, whereas only 5 of
these units showed significant modifications after the first pairing.
On average for these 11 units, the relative response to stimulation at
the paired frequency when tested with ACh was significantly increased
after the first pairing (2-tailed Student's t-test,
P < 0.002; Fig. 8,
right) and was further potentiated by the second and third
pairings at the same paired frequency (2-tailed Student's
t-test compared with initial control, 2nd pairing,
P < 1.104, n = 11; 3rd pairing, P < 0.05, n = 4).
In contrast, tests of response without ACh (- - -) or to stimulation
at unpaired frequencies (Fig. 8, left) did not reveal any
change (2-tailed Student's t-test, P > 0.3 in each case).
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Frequency-specificity and reversibility of the modifications
To confirm the specificity of changes for the paired frequency, we
performed pairings at 5, 8, or 11 Hz. These three frequencies were
equally effective in the induction of ACh-dependent potentiations of
response (respectively, 3/10, 14/73, and 7/36 significant
potentiations; 2, P > 0.7).
Furthermore the enhancement of response after pairing at one frequency
could be reversed by a second pairing at a different frequency,
resulting in a relative decrease in response to stimulation at the
initially potentiated frequency and an increase at the newly paired
frequency. Figure 9 displays this switch
in response enhancement for a cortical unit submitted to two
consecutive pairings, first at 8 Hz (Fig. 9A; KS,
P < 0.0002) and then at 11 Hz (Fig. 9B; KS,
P < 1.10
6).
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Overall, 29 units were tested with two different pairing frequencies. In five cases, the response to stimulation at the first paired frequency was potentiated after pairing when tested with ACh. Figure 9C shows the average response differences for paired and unpaired frequencies when tested without and with ACh for these units. After the first pairing, response at the paired frequency under ACh iontophoresis was enhanced compared with unpaired frequencies (1-tailed Student's t-test, P < 0.01). The second pairing induced an ACh-dependent enhancement of response for the newly paired frequency (1-tailed Student's t-test, P < 0.05 compared with all other frequencies) and a decrease in response to stimulation at the initially paired frequency (1-tailed Student's t-test, P < 0.001). These results suggest that the frequency selectivity of units is affected both by potentiations of response to stimulation at paired frequencies and depotentiations of response for other previously enhanced frequencies.
Kinetics of the potentiated responses
Of the 24 cases of significant potentiations, the tonic component of the response was significantly increased in 9 cases (see Fig. 4A for an example of enhanced tonic component and Fig. 5B for potentiation of the phasic response following each deflection only). In four of these nine cases, the increase in the absolute stimulus-locked spike count could be fully explained by the change in the tonic component. These results indicated that the modification was not temporally restricted to the epochs of afferent input activation; rather they suggest a prolonged change in activity during stimulation trains.
Retrieval of the potentiated response required both stimulation at the
paired frequency and the application of ACh. Since stimulation at the
paired frequency was presented in blocks of 12 trains, the potentiation
could in principle either appear de novo during each stimulation train,
in which case the response to the first deflection of the trains should
not be potentiated, or could develop during the entire block, affecting
the response to all deflections in the trains. A comparison of the
potentiations of the responses to the first deflections with those of
the responses to the subsequent deflections revealed similar
potentiations. Figure 10, A
and B, shows the results for the three different groups of
significantly modified units submitted to pairings at 5-, 8-, and 11-Hz
stimulation. Both the response to the first deflection of the train and
the response during the steady state were significantly increased after
pairing when testing with ACh (2-tailed paired Student's
t-test, 1st deflection, P < 1.104, steady state,
P < 1.10
10). Thus the
potentiation has a slow kinetics compatible with the time scale of a
block of stimulation (tens of seconds) rather than that of a single
train (seconds).
|
Sequential averaging of triplets of trains for units potentiated at 8 Hz (Fig. 10C) depicts this slow retrieval kinetics. During the first three trains, the average potentiation was not significant (2-tailed paired Student's t-test, P > 0.5). However, during the remaining nine trains, potentiation was significant for the entire train, including the first deflection (2-tailed paired Student's t-test, P < 0.02 for each 3-train average).
This slow kinetics developed de novo for each block of stimulation at the paired frequency. Figure 11A shows four examples of the time course of steady-state response potentiation when units were tested with ACh at the paired frequency. The potentiation was expressed after a delay of two to five trains at the beginning of each block of stimulation. Averaging across all units expressing a significant potentiation at 8 Hz (Fig. 11B) revealed that the potentiation was statistically significant after a delay of two trains for the first block and three trains for the second block of stimulation (2-tailed paired Student's t-test, P < 0.05). By contrast, no specific time course was observed on the response to other frequencies during ACh iontophoresis. Note however that the response to the first train of stimulation at 11 Hz, which follows the first block of stimulation at the paired frequency, was potentiated after pairing, indicating a prolonged effect of the potentiation between blocks. When units were tested without ACh, no change in response was observed or a depression without a specific time course which occasionally reached significance.
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DISCUSSION |
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Pairing ACh iontophoresis with mechanical deflections of the principal vibrissa at a given frequency produced specific potentiations of the response of barrel cortex neurons selective of the paired frequency, the expression of which depended on the presence of ACh. Herein, we demonstrated that these modifications were frequency-specific, cumulative, could be saturated and reversed. This ACh-dependent potentiation also occurred and at an even higher proportion when an adjacent vibrissa rather than the principal vibrissa of the recorded neuron was stimulated. The potentiation was not immediately expressed on the presentation of the paired frequency but rather required several trains of stimulation at that frequency to be expressed.
Spontaneous and evoked activity in control conditions
Spontaneous activity and responses to mechanical stimulation in
control conditions were similar to those obtained previously (Ahissar et al. 1997, 2000
). Each train of deflections
produced a maximal response to the first deflection in the train
followed by a rapid decrease until the response stabilized at its
steady-state value. Typically, temporal-frequency tuning curves showed
decreasing steady-state responses to increasing frequencies of
stimulation. The decrease in cortical response to deflections within a
train of stimulation agrees with previous studies demonstrating
suppressive effects for this range of inter-stimulus intervals
(Shimegi et al. 1999
; Simons 1985
). This
phenomenon may be partially explained by the decreased afferent
activity from the thalamus in anesthetized animals (Ahissar et
al. 2000
; Diamond et al. 1992
). Thalamocortical and intracortical mechanisms are also likely to participate. A decremental response of the thalamocortical connection arising from
lemniscal thalamic nuclei was described for frequencies above 5 Hz
(Castro-Alamancos and Connors 1996c
). Also, paired-pulse depression has been demonstrated in vitro on individual intracortical excitatory postsynaptic potentials in neocortex (Markram and
Tsodyks 1996
; Thomson et al. 1993
) and could
theoretically result in low-amplitude response to high frequencies of
afferent inputs (Abbott et al. 1997
). Additionally,
intracellular recordings have revealed inhibitory postsynaptic
potentials evoked by vibrissal stimulation that weaken subsequent
responses occurring at inter-stimulus intervals in the time range
studied here (Carvell and Simons 1988
; Moore and Nelson 1998
). The potentiations of response observed after
pairing might result in part from a change in these thalamocortical and intracortical response properties (see following text).
Characteristics and effectiveness of the sensori-cholinergic pairings
Previous studies of ACh-dependent functional plasticity in the
adult rat barrel cortex have mainly employed global manipulations of
the cholinergic innervation, such as lesion of the NBM by the immunotoxin 192 IgG-saporin (Baskerville et al. 1997;
Sachdev et al. 1998
) or cholinergic blockade using
systemically injected atropine sulfate (Maalouf et al.
1998
). The disruption of the cortical cholinergic activity
produced by these protocols consistently resulted in a reduced
plasticity in the barrel cortex, demonstrating the critical role of ACh
in the induction of plasticity. However, these studies did not
investigate the requirement for ACh during the induction and the
expression phases of plasticity. To determine the implication of ACh in
these two phases, we designed a protocol in which the local
concentration of ACh could be increased by iontophoresis. This
technique enabled us to pair a specific sensory stimulation with ACh
and test the response to a range of different stimuli both without and
with ACh for the same units.
During the pairing protocol, mechanical stimulation at a fixed
frequency associated with ACh iontophoresis lasted 2 min, a duration
only twice longer than the duration of a single block at the same
temporal-frequency during the control and test periods. Nonetheless,
this short pairing protocol was sufficient to rapidly induce
plasticity. The effect was not saturated after one pairing; the
modifications could be further enhanced by applying additional pairing
protocols. The short duration of pairing and the cumulative effect of a
series of pairings suggest that we used a relatively "weak"
protocol [for comparison, consider the extensive pairings performed in
Kilgard and Merzenich (1998a)]. This is also supported by the fact that the modifications could be rapidly reversed by applying a second pairing at a different frequency. These
considerations raise the possibility that modifications analogous to
those revealed after pairing might have also been induced during the
control periods in particular during the control period with ACh before pairing, i.e., the second TFTC determination. During that period, stimulation was applied in blocks of 1 min each, first from the lowest
to the highest frequency and then in reverse order. It is possible that
plastic changes were induced for each frequency and immediately
reversed by stimulation at the next frequency. Furthermore some of
these changes may have lasted beyond the duration of the TFTC. To
estimate the incidence of lasting changes following TFTCs with ACh, we
performed experiments during which pairing was not applied but the
control and test periods were maintained. Indeed, unpaired units also
exhibited significant modifications. However, there was a qualitative
difference between the modifications obtained with and without pairing.
Whereas the modifications obtained without pairing were generalized
across a range of frequencies, those obtained after pairing were
specific to the paired frequency. We conclude that even though the
presence of ACh during the TFTC induces modifications, the presentation
of multiple frequencies does not allow any single frequency to be
strongly associated with the ACh.
Special care was taken to ensure that ejection currents did not affect
neuronal activity. First, currents used for drug applications were in
the range of 20-80 nA. These intensities usually do not by themselves
affect neuronal activities (Purves 1981; Shulz et al. 1997
). Second, balanced ejections were applied to minimize extracellular potential changes near the tip of the electrode. Indeed,
our results revealed that the time course of the effects was generally
too slow to be caused by DC effects. Moreover, the blockade of the
effects by atropine (Shulz et al. 2000
) confirmed the
specific action of ACh on cholinergic receptors. Another strong indication that current effects were not involved comes from the fact
that even though ACh was iontophoresed continuously during the test
after pairing, the modifications were frequency specific. The
potentiation revealed after pairing, including the increased background
activity, was only observed within trains of stimulation at the paired
frequency. If these modifications were due to current effects, they
should presumably affect the evoked activity during the entire ACh
iontophoresis period, a phenomenon that we did not observe.
Characteristics of the modifications of response induced by sensori-cholinergic pairings
Modifications observed after pairing were mainly increases of
response to stimulation at the paired frequency, revealed only when ACh
was re-supplied. The response at temporal frequencies other than the
paired frequency was unchanged or changed to a lesser extent. Previous
investigations in the auditory (Bakin and Weinberger
1996; Bjordahl et al. 1998
; Edeline et
al. 1994
; Kilgard and Merzenich 1998b
;
Metherate and Weinberger 1989
, 1990
) and the
somatosensory (Metherate et al. 1987
, 1988b
;
Rasmusson and Dykes 1988
; Tremblay et al.
1990b
) cortex have shown that pairing sensory stimulation with
either ACh iontophoresis or NBM stimulation produces lasting changes in
response for the paired stimulus. Cortical mapping of the primary
auditory area has confirmed these stimulus-specific alterations and has
shown that the cortical area devoted to the paired tone frequency was
increased after extensive pairing of NBM stimulation and tone
presentation (Kilgard and Merzenich 1998a
). In our
study, the cortical area activated by the paired whisker is not
expected to be significantly increased, first because only responses to
a single stimulation frequency were potentiated, and second because we
used a relatively weak pairing. Even though only one third of the
neurons submitted to a pairing showed significant modifications, this
subset of cells exhibited robust modifications and a consistent pattern
of plasticity, including selectivity for the paired frequency,
summation, saturation and reversibility. This percentage of changes may
be due to the experimental methods used, which might not reveal the
whole population potentially expressing plasticity, or alternatively
may result from the fact that ACh influence is restricted to a subset
of cortical cells.
In all the aforementioned studies, the plastic changes were measured
during testing without ACh iontophoresis or stimulation of the NBM. By
contrast in our protocol, the expression of frequency-specific potentiations depended on the presence of ACh during testing. Part of
this discrepancy can be explained by the fact that protocols used
previously in the literature generally employed more massive pairings
that could eventually render the expression of plasticity less
dependent on the presence of ACh. It would be also interesting to
determine whether, in those cases, retesting with ACh unmasks more
robust and consistent changes. The implication of ACh in the expression
of plasticity in the barrel cortex is supported by a study
(Delacour et al. 1990) made in the awake rat using a
whisker-pairing protocol. The pairing resulted in an enhanced response,
the expression of which was blocked by the exogenous application of
atropine, indicating that the effect depended on the effectiveness of
endogenous ACh. As this study was performed on awake animals, ACh might
have been already present at a sufficient concentration at the time of
sensory pairing to induce plasticity, and during tests of response to
express changes (Sarter and Bruno 1997
). The
"control" and "test" periods would then be equivalent to our
"with ACh" condition, whereas the "atropine" periods would correspond to our "without ACh" condition.
Possible mechanisms of the pairing-induced potentiation of response
Possible mechanisms underlying the induction and expression of the plasticity described here are highly constrained by characteristics such as the frequency specificity of the expressed potentiation, the dependence on ACh, and the kinetics observed during retrieval.
In control conditions, barrel cortex neurons exhibited decreased spike counts in response to increasing frequencies of stimulation. This is due to the decrease in evoked activity from one deflection to the following within a train. The cortical mechanisms involved in this low-pass filtering might have been affected by the pairings. In particular, the removal of the rapid decrease in response within trains at the paired frequency should produce an increase in the steady-state response and could contribute to the transformation to a band-pass filtering described here after pairing. However, we report in addition that after pairing, the response to the first deflection of each train was also significantly enhanced, indicating a prolonged effect from one train to another within blocks of stimulation. This increase in response cannot be explained by modifications of the kinetics within trains and must be attributed to an independent mechanism. Moreover, the analysis of the time course of the potentiation of response revealed a delay in its establishment lasting tens of seconds. These results imply that the expression of plasticity under ACh involves a mechanism triggered by the presentation of the paired temporal frequency, developing with a slow kinetics, and, once established, retained during at least 1 s (the inter-train interval).
The enhanced responses could result from plasticity at the level of the thalamocortical connections, the intracortical circuitry, or changes in the intrinsic properties of the cortical cells. ACh diffusion diameter in cortical tissue during prolonged iontophoresis was estimated in the 300-µm range (Haidarliu and Ahissar, unpublished observations), supporting the idea of a localized effect of ACh in our protocol. We also report a lack of expressed modifications in units distant from the iontophoresis electrode (recorded by a TE). Taken together, these findings argue in favor of a local modification induced and expressed by the neuronal microcircuits in the immediate vicinity of the electrode.
Repetitive stimulation at 7-14 Hz of some thalamocortical pathways
induces a rapid enhancement of cortical responses known as the
augmenting response (Castro-Alamancos and Connors 1996a; Morison and Dempsey 1942
). One interesting hypothesis is
that our pairing would result in the involvement of these augmenting responses. However, the augmenting response develops during the first
two to three stimulations of thalamocortical pathways, whereas we did
not observe rapid increases in response within trains but rather rapid
decreases. Also, the augmenting response is modulated by the behavioral
state of the animal (Castro-Alamancos and Connors 1996b
;
Steriade and Morin 1981
) and is abolished during states of arousal, when the cortical release of ACh is high. In our case, on
the contrary, the increased effects were observed during ACh application.
In anesthetized rats, thalamic neurons respond in a low-pass manner
similar to cortical neurons (Ahissar et al. 2000;
Diamond et al. 1992
). Consequently at the cortical
level, all thalamocortical projections that are active at high
frequencies are also presumably active at low frequencies. This implies
that the sets of afferents active for different temporal frequencies of
stimulation strongly overlap. Because of this lack of input
separability, it is hardly conceivable that the frequency specificity
of the modifications could arise from synaptic changes in a subset of
thalamocortical connections. Indeed, such synaptic changes would also
be expressed at frequencies lower than the paired frequency. We did not
find such an asymmetry in the profile of changes after pairing. Our observations are thus more compatible with a participation of intracortical mechanisms (see also Fox et al. 2000
;
Wallace and Fox 1999
).
The expression of these intracortical changes was shown to be specific
to the paired frequency. Several hypotheses concerning the underlying
cellular mechanisms are considered. First, similar to the mechanisms
generating the augmenting response (Castro-Alamancos 1997; Castro-Alamancos and Connors 1996a
),
specific membrane conductances can be activated or de-inactivated
within a precise time window after each spike (or short burst of
spikes), resulting in an increased response to afferent activity at a
specific frequency. However, this mechanism should reset a few hundred
of milliseconds after the end of each train of stimulation. Thus the
response to the first deflection of the following train in our protocol
should not be potentiated since the inter-train interval exceeds that time window. Our observation that the response to the first deflection of each train of stimulation was also potentiated led us to reject this hypothesis.
Second, recent studies have shown that a single synapse may exhibit
different time constants of rapid depression and facilitation so that,
depending on the value of these parameters, it transmits information
with different filtering characteristics (Markram and Tsodyks
1996; Tsodyks and Markram 1997
). In this
framework, stimulation of the whisker at one frequency would be
preferentially propagated through the cortical network by synapses
transmitting efficiently that frequency. Synaptic plasticity was
interpreted by these authors in terms of modifications of the frequency
dependence of transmission (Markram et al. 1998a
). Thus
the band-pass TFTCs observed after pairing could be explained by
band-pass dependence of synaptic transmissions on the frequency of
stimulation. However, the frequency dependence of the net synaptic
effect is usually of a low-pass or high-pass nature (Gupta et
al. 2000
; Markram et al. 1998b
). Modifications
in synaptic time constants could increase or decrease the cutoff
frequencies of these transfer functions but could not create a
band-pass transfer function for an individual synapse. Thus our results
could be explained in this framework only if changes in high- and
low-pass synapses occurred in a coordinated manner in the cortical
network to produce a net band-pass transmission.
Another possibility is that each frequency selectively activates a
subset of neuronal circuits. As we usually did not observe frequency-specific cells for frequencies higher than 2 Hz before pairing, we have to suppose that these circuits are bound together by
other means than an absolute increase in activity, for example, through
synchronization of their different members. Other potential circuits
are those that contain oscillatory units (Ahissar 1998). Frequency-specific changes can be induced in these circuits by changing
their working parameters such as the input-output transfer functions of
the oscillatory units (Ahissar et al. 1997
) or of the
other members of the circuit. In agreement with this explanation is the
observation that oscillatory neurons of the somatosensory cortex often
exhibit slow locking in kinetics (Ahissar, unpublished observations),
with a time scale that is similar to that of the slow retrieval
kinetics observed herein.
Probably the most challenging constraint imposed by our data is that,
whatever the underlying mechanism, its expression depends on ACh. The
differential effect of ACh on the multiple types of interneurons in the
cortex (Kawaguchi 1997; Xiang et al.
1998
) enables switching between different neuronal circuits.
Thus ACh may switch between circuits that are tuned to different
frequencies (Ahissar et al. 1997
) by changing the
balance between recurrent inhibitory pathways (Reyes et al.
1998
). Further studies are needed to distinguish between these
possible explanations.
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ACKNOWLEDGMENTS |
---|
We thank Y. Frégnac for helpful comments on the manuscript.
This work was supported by Programme International de Cooperation Scientifique Centre National de la Recherche Scientifique, Ministère des Affaires Etrangères Français, Association Franco-Israélienne pour la Recherche Scientifique et Technique, Human Frontier Science Program, the Abramson Family Foundation (USA), and the MINERVA Foundation (Germany).
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FOOTNOTES |
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Address for reprint requests: D. E. Shulz, U.N.I.C., UPR CNRS 2191, Institut de Neurobiologie Alfred Fessard, CNRS, 91198 Gif sur Yvette, France (E-mail: shulz{at}iaf.cnrs-gif.fr).
Received 26 September 2000; accepted in final form 21 February 2001.
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REFERENCES |
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