 |
 Previous Article | Next Article 
The Journal of Neuroscience, April 1, 2001, 21(7):2434-2441
In Vitro Eye-Blink Classical Conditioning Is NMDA
Receptor Dependent and Involves Redistribution of AMPA Receptor
Subunit GluR4
Joyce
Keifer
Neuroscience Group, Division of Basic Biomedical Sciences,
University of South Dakota School of Medicine, Vermillion, South Dakota
57010
 |
ABSTRACT |
The classically conditioned vertebrate eye-blink response is a
model in which to study neuronal mechanisms of learning and memory. A
neural correlate of this response recorded in the abducens nerve can be
conditioned entirely in vitro using an isolated
brainstem-cerebellum preparation from the turtle by pairing trigeminal
and auditory nerve stimulation. Here it is reported that conditioning
requires that the paired stimuli occur within a narrow temporal window of <100 msec and that it is blocked by the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid. Moreover, there
is a significant positive correlation between the levels of
conditioning and greater immunoreactivity with the glutamate receptor 4 (GluR4) AMPA receptor subunit in the abducens motor nuclei, but not
with NMDAR1 or GluR1. It is concluded that in vitro
classical conditioning of an abducens nerve eye-blink response is
generated by NMDA receptor-mediated mechanisms that may act to modify
the AMPA receptor by increasing GluR4 subunits in auditory nerve synapses.
Key words:
eye blink; conditioning; in vitro; turtle; NMDA; GluR4
| |
INTRODUCTION |
The classically conditioned
eye-blink reflex is an established model for studies of the cellular
mechanisms that underlie learning and memory (Bloedel and Bracha, 1995 ;
Kim and Thompson, 1997). In this paradigm, an eye-blink reflex in
response to a tone can be evoked when the tone is repeatedly paired
with an air puff to the cornea that normally elicits the blink
response. A neural correlate of the eye-blink reflex can be classically conditioned entirely in vitro using a brainstem preparation,
with or without the cerebellum, from the turtle (Keifer et al., 1995; Anderson and Keifer, 1997, 1999). Activity recorded in the abducens nerve, which contains motor neuronal projections to the extraocular muscles controlling movement of the nictitating membrane and eyelid during a blink (Keifer, 1993), can be conditioned by pairing a trigeminal nerve unconditioned stimulus (US) with an auditory nerve
conditioned stimulus (CS). These electrical stimuli represent analogs
of the corneal air puff and tone used in behavioral studies, but in
this case, they are applied directly to the cranial nerves. This
classically conditioned abducens nerve response shows gradual acquisition of conditioned responses (CRs) to paired stimuli and extinction when the stimuli are unpaired. Acquisition of CRs is rapid,
taking place in ~2-3 hr and thereby allowing in vitro
studies of this poorly understood learning process.
Long-term potentiation (LTP), a form of activity-dependent synaptic
plasticity that is likely to have a role in associative learning, has
been shown to mediate a cellular analog of classical conditioning of
the siphon-withdrawal reflex of Aplysia (Murphy and
Glanzman, 1997). The Hebbian-like form of LTP recorded from Aplysia, which has been extensively studied in neurons from
hippocampal slices, requires a narrow temporal relation between the
paired stimuli, activation of postsynaptic NMDA receptors, and
intracellular Ca2+ entry (Malenka, 1994;
Murphy and Glanzman, 1996, 1997; Bi and Poo, 1998; Soderling and
Derkach, 2000). In the hippocampus, LTP is also associated with the
modulation of AMPA receptors (Liao et al., 1995; Barria et al., 1997;
Hayashi et al., 2000; Soderling and Derkach, 2000). One proposed
mechanism for enhanced synaptic strength during learning-related forms
of plasticity is the induction of functional AMPA receptors at
"silent synapses" that contained previously only NMDA receptors
(Malenka and Nicoll, 1997). Synapses that contain NMDA receptors alone
are silent because at normal resting potentials the receptor or
channel is blocked by extracellular Mg2+.
During LTP, it is thought that AMPA receptors are delivered to synapses
from nonsynaptic sites, rendering them to be functional. In support of
this hypothesis, activity-dependent redistribution of AMPA receptors
has been observed directly in cultured hippocampal neurons using
fluorescence microscopy (Liao et al., 1999; Lissin et al., 1999; Shi et
al., 1999). In this study, it is shown that in vitro
classical conditioning of a vertebrate eye-blink reflex requires that
the CS and US occur within a narrow temporal window of <100 msec and
is NMDA receptor dependent. Moreover, conditioning is associated with
upregulation of the AMPA receptor subunit glutamate receptor 4 (GluR4),
but not of NMDAR1 or GluR1, in the abducens motor nuclei. These
observations suggest that there is an NMDA-dependent redistribution of
AMPA receptor subunits during conditioning, findings that are
consistent with the silent-synapse hypothesis.
Parts of this paper have been published previously (Keifer,
2000).
| |
MATERIALS AND METHODS |
Conditioning procedures. Freshwater pond turtles
Chrysemys picta were anesthetized by hypothermia and
decapitated. The brainstem-cerebellum was bathed in physiological
saline (2-4 ml/min) containing (in mM): 100 NaCl, 6 KCl, 40 NaHCO3, 2.6 CaCl2, 1.6 MgCl2, and 20 glucose, which was oxygenated with 95% O2/5%
CO2 and maintained at room temperature
(22-24°C) (Keifer et al., 1995; Anderson and Keifer, 1997, 1999).
Suction electrodes were used for stimulation and recording of cranial
nerves. The US was a 2× threshold single-shock stimulus applied to the
trigeminal nerve; the CS was a 100 Hz, 1 sec train stimulus applied to
the ipsilateral posterior root of the eighth nerve that was ~75% of
the threshold amplitude required to produce activity in the nerve. This
latter nerve will be referred to as the auditory nerve. Neural activity
was recorded from the ipsilateral abducens nerve. Timing parameters
required to produce conditioning were investigated by using a CS-US
interval of 10 msec (n = 5), 20 msec (n = 6), 60 msec (n = 5), 80 msec (n = 5), 100 msec (n = 5), 300 msec (n = 4), 500 msec (n = 1), 1 sec (n = 3), and 2 sec
(n = 3). The CS-US interval is defined as the time
between the offset of the CS and the onset of the US. The intertrial
interval between the CS and the US was 30 sec. A pairing session
consisted of 50 CS-US presentations followed by a 30 min rest period
in which there was no stimulation. Seven additional preparations were
tested for extinction of CRs in response to CS-US intervals of 300 msec to 2 sec after they were initially conditioned at the 0 msec
interval. CRs were defined as abducens nerve activity that occurred
during the CS and had an amplitude of at least 25% of the
unconditioned response (UR). The stimulus protocol was designed
with some modification to be similar to those used in behavioral
studies of the rabbit (see Keifer et al., 1995). Those experiments have
typically used a CS ranging between 250 and 500 msec, although a 1 sec
CS produces conditioning. A 1 sec CS was chosen for the in
vitro turtle preparation because of its cooler temperature and
relatively slower conduction time. Auditory nerve fibers in the turtle
are most sensitive to stimuli between 100 and 500 Hz, and therefore a
CS of 100 Hz was used. Intertrial intervals were 30 sec, the average
interval used in behavioral studies. The range of optimal conditioning
parameters for this preparation has not yet been exhaustively examined.
In addition to the pairing-specific acquisition of abducens nerve CRs,
this in vitro preparation exhibits other features observed during conditioning experiments in rabbits. These include suppression of the UR during application of the paired stimuli and a shift in the
latency of the CR toward the occurrence of the US with training
(Anderson and Keifer, 1997).
Pharmacology. The NMDA receptor antagonist
D,L-2-amino-5-phosphonovaleric acid (AP-5; 100 µM; Tocris) was dissolved in physiological saline and
perfused through the bath. In some experiments, application was
performed throughout the conditioning procedure to test for effects on
the induction of CRs. In other experiments, AP-5 was applied after the
end of the third pairing session to test for effects on CR expression
after conditioning had been obtained. In these experiments, AP-5 was
perfused through the bath for two additional pairing sessions and was
washed out for 40 min, and conditioning was resumed in normal saline to
test for recovery. A high-threshold auditory nerve-evoked abducens
nerve response was also tested for habituation in AP-5. A
high-intensity (~4-5× threshold) train stimulus applied to the
auditory nerve evokes a burst discharge in the ipsilateral abducens
nerve at monosynaptic latencies (Keifer et al., 1995). This response is
thought to represent the blink component of a neural analog of the
behavioral "startle" response, and it normally habituates to
repeated stimuli. Here, AP-5 (100 µM) was applied to the
bath, and as during the conditioning procedure, a 100 Hz, 1 sec train
stimulus (the same as the CS but of greater amplitude) was applied to
the auditory nerve in blocks of 50 stimuli per session, at an interval
of 30 sec, and followed by a 30 min rest period.
Immunocytochemistry. After the physiological experiments,
brainstem-cerebellum preparations were immersion fixed in cold 3% paraformaldehyde (Keifer and Carr, 2000). Tissue sections were cut at
30 µm and were preincubated in 10% normal goat serum for 1 hr
followed by incubation in primary antibody overnight at 4°C with
gentle shaking. The primary antibodies used were a monoclonal antibody
raised in mouse that recognizes NMDAR1 and polyclonal antibodies raised
in rabbit that recognize GluR1 or GluR4 (PharMingen, San Diego, CA).
The GluR1 and GluR4 antibodies recognize both flip and flop splice
variants. An antibody that recognizes GluR2/3 in turtles has been
obtained recently, and these results will be described elsewhere.
Concentrations of primary antibodies were 1:100, except for NMDAR1 that
was 1:1000. Triton X-100 (0.1%) was used during incubation with
primary antibodies to GluR4. After the primary antibody, sections were
incubated in a secondary antibody for 1-2 hr using a concentration of
1:100. The secondary antibodies were indocarbocyanine-3 (Cy3) or
Cy2-conjugated goat anti-mouse or goat anti-rabbit IgGs (Jackson
ImmunoResearch, West Grove, PA) that were used to visualize the primary
antibodies. The primary antibodies developed in mammals were likely to
have maintained their specificity in turtles because glutamate receptor
subunits are highly conserved and immunoblot analysis shows that they
recognize similar molecular weight structures in birds compared with
mammals. Moreover, the pattern of staining in the turtle brainstem is
similar to that described for rats (Keifer and Carr, 2000). The
secondary antibodies were tested for their specificity by several
procedures described in Keifer and Carr (2000).
Digital images of immunofluorescence from tissue sections containing
the principal and accessory abducens motor nuclei were captured using a
video camera-equipped Zeiss Axioskop Mot-2 microscope. Quantitative
image analysis was performed by using Adobe Photoshop software. The
histogram feature calculates the average luminosity level and SD of the
pixels in the selected field. Values were obtained for label in the
principal and accessory abducens motor nuclei, and background
values taken from unlabeled areas were subtracted from these for
every section analyzed to control for variations in staining.
Generally, 6-10 sections were analyzed and averaged for each case. The
analysis was performed blind to the results of the conditioning
experiment. StatView software was used for statistical analysis.
Western blot analysis. Turtle brainstems or mouse hippocampi
were lysed in Laemmli buffer and centrifuged, and the supernatant was
loaded on a 10% polyacrylamide minigel (Bio-Rad, Hercules, CA) and
separated by gel electrophoresis. Proteins were transferred from the
gel by electroblotting onto polyvinylidene difluoride membranes in
blocking buffer containing 5% nonfat milk for 30 min. The membranes
were incubated with the primary antibody to GluR4 (1:100 dilution),
washed, and incubated in HRP-conjugated goat anti-rabbit IgG secondary
antibody (Vector Laboratories, Burlingame, CA). Immunoreactivity was
visualized by chemiluminescence.
| |
RESULTS |
Classical conditioning of the in vitro abducens nerve
response was obtained previously by using a delay conditioning paradigm (Keifer et al., 1995; Anderson and Keifer, 1997, 1999). During this
procedure, a 1-sec-duration CS is applied to the auditory nerve and is
followed immediately by a single-shock stimulus applied to the
trigeminal nerve (i.e., a CS-US interval of 0 msec; the CS-US
interval is defined as the time between the offset of the CS and the
onset of the US). In the present study, the temporal parameters of the
CS and US required to produce conditioning of the abducens nerve
response were investigated by changing the CS-US interval to values
ranging from 10 msec to 2 sec (Fig.
1A). When the
percentage of preparations that conditioned using these CS-US
intervals was examined, the results showed that there is a narrow
temporal window between the occurrence of the CS and US in which
conditioning can be produced. These results are summarized in Figure
1B. Preparations presented with CS-US intervals
ranging from 300 msec to 2 sec failed to produce CR acquisition
(n = 11; Fig. 1B). No CRs were
recorded from any of these preparations during five sessions of
pairing. Extinction of CRs also occurred in response to these CS-US
delays (Fig. 2). After conditioning had
been obtained initially using the 0-msec-delay stimulus paradigm (n = 7; Fig. 2, pairing sessions 1-3), the
CS-US delay was changed from 300 msec to 2 sec, and the percentage of
recorded CRs decreased (Fig. 2, pairing sessions
4-6). Reacquisition was obtained when the stimulus
paradigm was returned to the 0 msec delay (Fig. 2, pairing
sessions 7-10). These data suggest that intervals from 300 msec
to 2 sec do not support abducens nerve CRs. However, when the CS and US
were separated by an interval of not >100 msec (n = 26), the percentage of preparations that conditioned gradually increased as the interval became smaller
(R2 = 0.87; p = 0.0001) until conditioning peaked using an interval of 20 msec (Fig.
1B). There was no significant correlation in the
average percentage of CRs that were recorded among experiments within
the 100 msec interval (R2 = 0.05; p = 0.28). Thus, precise timing constraints
between the CS and US are required to produce abducens nerve
conditioning.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 1.
A, Schematic diagram illustrating
the classical conditioning paradigm. The 1 sec duration CS applied to
the auditory nerve precedes the single-shock US applied to the
ipsilateral trigeminal nerve. The delay between the offset of the CS
and the onset of the US ranged from 10 msec to 2 sec. B,
The percentage of preparations that exhibited conditioning plotted as a
function of the CS-US interval. CS-US intervals of 300 msec to 2 sec
failed to produce conditioning in all preparations tested. However,
when the CS and US were separated by not >100 msec, the percentage of
preparations that conditioned gradually increased as the interval
became smaller and peaked at a CS-US interval of 20 msec. Each
circle represents the mean of at least five
experiments.
|
|
View larger version (13K):
[in this window]
[in a new window]
|
Figure 2.
Summary data from seven preparations that had been
conditioned initially to a mean of 34% CRs using a CS-US interval of
0 msec (filled circles; pairing sessions
1-3). Intervals of 300 msec to 2 sec resulted in extinction of
CRs to a mean of 4% (open circles; pairing
sessions 4-6). Resumption of paired stimuli at the 0 msec interval resulted in reacquisition of abducens nerve CRs to a mean
of 24% (filled circles; pairing sessions
7-10).
|
|
To begin to analyze cellular mechanisms of conditioning in this
preparation, we tested the hypothesis that classical conditioning of
the abducens nerve response was mediated by NMDA receptors. Previous
studies have shown that the turtle blink reflex pathways use both NMDA
and AMPA receptors (Keifer, 1993; Keifer and Carr, 2000). In normal
physiological saline, the abducens eye-blink response has a
short-latency, short-duration burst component and a later,
long-duration component (Fig.
3A, upper trace).
Dual-component blink responses have been observed in all vertebrate
species that have been studied (Pellegrini et al., 1995). Bath
application of the NMDA receptor antagonist AP-5 blocked the
long-duration component of the reflex while leaving the short-duration
component unaffected, as is shown in Figure 3A (compare the
UR in the lower trace with that in the upper
trace) (Keifer, 1993). The long-duration component of the reflex
originates from the medullary reticular formation and is not necessary
for conditioning, nor is it affected by the occurrence of the CR
(Keifer, 1993; Anderson and Keifer, 1999). In the present experiments,
AP-5 was used to determine whether induction of CRs was blocked when
training was performed in the presence of the drug or whether the
expression of CRs was disrupted when the drug was applied after
conditioning had been obtained. When preparations were initially
conditioned in the presence of AP-5 (100 µM),
none demonstrated abducens nerve CRs in four sessions of CS-US pairing
at 0 msec delay (n = 9; Fig. 3B). In
experiments in which conditioning was conducted in normal physiological
saline, CRs were recorded in the abducens nerve (Fig. 3A,
upper trace, arrow). When AP-5 was applied to the bath after
CRs had been acquired in normal saline (Fig. 3C,
pairing sessions 1-3), they were significantly attenuated
(Fig. 3A, lower trace, C,
pairing sessions 4, 5; p < 0.001). The CRs
were again recorded when AP-5 was washed out of the bath (Fig.
3C, pairing sessions 6, 7). It
is notable that the percentage of CRs after AP-5 washout was
approximately the same as that before the drug application. This
suggests that CRs were not reacquired during this phase of conditioning
but rather that their expression had recovered. Application of 100 µM AP-5 does not affect habituation, a
nonassociative form of learning. When a high-intensity stimulus is
applied to the auditory nerve, it evokes a monosynaptic abducens nerve
discharge that is believed to represent the blink component of a neural
analog of the behavioral startle response. This response habituates in
normal saline to repeated stimuli (Keifer et al., 1995). The effect of
AP-5 appears to be specific to associative learning because this
compound failed to block habituation of this high-threshold auditory
nerve-evoked abducens nerve response. In AP-5, the mean percentage of
responses in each stimulus session gradually habituated (94, 66, 36, 14, and 4%; n = 6), similar to responses recorded in
normal saline (92, 66, 36, 18, and 4%; n = 3). These
data suggest that abducens nerve classical conditioning requires NMDA
receptor activation for both the induction and expression of CRs.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 3.
A, Abducens nerve recordings are
shown during CS-US pairing in normal saline (upper
trace) and in 100 µM AP-5 (lower
trace). In normal saline, the CR (arrow) and UR
are recorded as burst discharges, and the UR demonstrates short- and
long-duration components. In AP-5, CRs are generally not recorded, and
the long-duration portion of the UR is blocked, leaving only the
short-duration component. Each arrowhead indicates the
start of the 1 sec CS; the dot indicates the US. The
CS-US delay in these recordings was 10 msec. B,
Preparations that were presented with the conditioning protocol in the
presence of AP-5 (hatched horizontal bar) failed
to exhibit CRs in four sessions of CS-US pairing using a 0 msec delay
(n = 9). C, Bath application of AP-5
blocks the expression of abducens nerve CRs. Data from nine experiments
are summarized. During the initial pairing sessions, a mean of
~60-70% CRs was recorded (filled vertical
bars; pairing sessions 1-3).
Application of AP-5 (hatched horizontal bar) resulted in
attenuation in the percentage of CRs to a mean of 6% (hatched
vertical bars; pairing sessions 4, 5).
Approximately 60% of CRs on average were again recorded after washout
of the drug (filled vertical bars; pairing
sessions 6, 7). Double asterisks indicate
p < 0.001 (ANOVA).
|
|
The recent findings that the functional properties of AMPA receptors
are dependent on their subunit composition (Ozawa et al., 1998) and
that this composition can be highly labile under some conditions (Liao
et al., 1999; Lissin et al., 1999; Shi et al., 1999) are particularly
relevant to studies of conditioning. Changes in number, distribution,
or subunit composition of the postsynaptic receptors are viable
mechanisms for synapse modification that may occur during learning. To
investigate whether alterations in receptor subunit composition occur
during conditioning of the in vitro abducens nerve response,
immunocytochemistry and light microscopy were used to localize NMDA and
AMPA receptor subunits in the untrained turtle brainstem and cerebellum
and in preparations that had undergone the conditioning procedure.
Glutamate receptor subunit immunocytochemistry was performed on three
groups of brainstem-cerebellum preparations: untrained preparations
[from Keifer and Carr (2000)] that were placed immediately in
fixative (n = 5), preparations that underwent the
conditioning procedure but failed to acquire CRs (n = 2), and preparations that successfully acquired various levels of CRs
(n = 12). Results from untrained preparations showed intense to moderate label for NMDAR1 and GluR1, and minimal label for
GluR4, in the principal and accessory abducens motor nuclei (Keifer and
Carr, 2000). Immunoreactivity levels of untrained versus trained
preparations that failed to exhibit CRs were not significantly
different (principal abducens, p = 0.68; accessory abducens, p = 0.61; ANOVA). However, in preparations
that exhibited acquisition of CRs, there was a visible and significant
increase in AMPA receptor subunit immunoreactivity for GluR4 in the
abducens motor neurons (Fig. 4).
Photomicrographs show a clear increase in GluR4 immunoreactivity in the
principal abducens motor neurons after conditioning (Fig.
4B) as compared with an unconditioned preparation
(Fig. 4A). Similar results were observed for the
accessory abducens nucleus. Quantitative image analysis of these
immunocytochemical data was performed to examine the relationship
between the degree of glutamate receptor subunit immunoreactivity of
the abducens motor nuclei and the level of conditioning (Fig.
5). Images of the abducens motor nuclei
from all preparations tested were analyzed using software that
calculated the average luminosity of the pixels in the selected field.
Analysis of NMDAR1 immunoreactivity in both the principal and accessory
abducens motor nuclei (Fig. 5A,D) failed to show a
significant relationship between the level of NMDAR1 and the level of
abducens nerve CRs (R2 = 0.001, p = 0.93, and
R2 = 0.009, p = 0.73, respectively). Immunoreactivity for GluR1 also failed to show a
significant correspondence with the level of conditioning; however,
there was a trend toward reduced GluR1 immunoreactivity and a greater
number of CRs in the accessory abducens nucleus (Fig. 5B,E;
principal abducens, R2 = 0.02, p = 0.53; accessory abducens,
R2 = 0.17, p = 0.08). On the other hand, there was a significant positive correlation
between GluR4 immunoreactivity in the principal and accessory abducens
motor nuclei and the expression of CRs (Fig. 5C,F;
R2 = 0.74, p = 0.0001, and R2 = 0.42, p = 0.009, respectively). Thus, it appears that the
GluR4 subunit, but not NMDAR1 or GluR1, is upregulated in the
abducens motor nuclei during classical conditioning of the abducens
nerve eye-blink response.
View larger version (109K):
[in this window]
[in a new window]
|
Figure 4.
A, Photomicrograph of GluR4
immunoreactivity visualized by using a Cy2-conjugated secondary
antibody in the principal abducens motor nucleus from an untrained
preparation. B, A conditioned preparation that exhibited
98% CRs in the last pairing session. The arrows in
B indicate intensely GluR4-immunopositive abducens motor
neurons from the conditioned preparation that are not apparent in the
untrained preparation. Scale bar, 100 µm.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Figure 5.
The intensity of immunoreactivity of the principal
(A-C) and accessory (D-F)
abducens motor nuclei is plotted as a function of the level of
conditioning. Each circle represents an individual case;
closed circles are untrained preparations, and
open circles are preparations that underwent the
conditioning procedure. Values plotted on the y-axis are
in arbitrary numbers that represent average pixel luminosity. The total
number of CRs recorded from individual preparations is plotted on the
x-axis. R2 values are
given in the text.
|
|
To verify the specificity of the GluR4 antibody in turtles, Western
blot analysis was performed on turtle brainstem tissue and, for
comparison, on mouse hippocampi. Immunoblots showed a single band that
migrated to an Mr of ~108,000 kDa
for both the turtle and the mouse tissue (Fig.
6). This is similar in size to that
reported previously for rat (Petralia and Wenthold, 1992) and bird
(Levin et al., 1997) brain. Moreover, these findings are consistent
with our previous immunocytochemical studies of the turtle brainstem
that show a pattern of immunostaining with antibodies to NMDAR1, GluR1,
and GluR4 similar to that described in rats (Keifer and Carr,
2000).
View larger version (59K):
[in this window]
[in a new window]
|
Figure 6.
Western blot analysis of SDS gels of turtle
(T) brainstem and mouse (Ms)
hippocampal tissue using antibodies to GluR4. For both the turtle and
mouse tissue, a single band of Mr = ~108,000 kDa migrated on the gels. Molecular weight markers are on
the left.
|
|
| |
DISCUSSION |
The present results support the hypothesis that vertebrate
eye-blink classical conditioning involves NMDA receptor-mediated mechanisms and modification of the AMPA receptor. Indirect evidence suggests that classical conditioning of the mammalian eye-blink response involves NMDA receptor-dependent synaptic plasticity (Servatius and Shors, 1996; Kishimoto et al., 1997). Moreover, by the
use of autoradiography to examine the hippocampus in rabbits that had
been classically conditioned, the number of NMDA receptors was found to
be unchanged with respect to that in unconditioned animals, whereas
AMPA receptors were significantly increased (Tocco et al., 1991). These
findings are consistent with the present results, although in this
study it is unclear whether there are more AMPA receptors or more
existing receptors that have incorporated GluR4 subunits. These initial
findings on the mechanisms that underlie conditioning in this
preparation are reminiscent of those that produce other types of
synaptic plasticity such as LTP and long-term depression (LTD). First,
there are strict timing constraints for the generation of LTP, LTD, and
classical conditioning. Bi and Poo (1998) showed that a critical window
of <40 msec was required between presynaptic and postsynaptic stimuli
to generate either LTP or LTD in hippocampal cultures. Similar timing
constraints of <60 msec between stimuli have been reported for
synaptic modification in the electrosensory lobe of the electric fish
(Bell et al., 1997). In the in vitro turtle
brainstem-cerebellum, the CS and US must occur within ~100 msec to
produce conditioning, and in hippocampectomized rabbits, an interval of
300 msec, but not 500 msec, can support eye-blink conditioning (Moyer
et al., 1990). It appears that the associative requirements for LTP,
LTD, and classical conditioning all fall within a relatively narrow
temporal range. Second, activation of NMDA receptors is required to
produce some forms of LTP and LTD and classical conditioning. Induction of Hebbian LTP and LTD requires conjunctive presynaptic and
postsynaptic activity to activate NMDA receptor function and allow Ca
2+ entry (Malenka, 1994; Soderling and
Derkach, 2000). Classical conditioning also requires NMDA receptor
function (Servatius and Shors, 1996; Kishimoto et al., 1997; Murphy and
Glanzman, 1997) (the present study). Furthermore, the importance of
Ca2+ influx in associative changes in
synaptic efficacy was demonstrated by showing that postsynaptic
injection of Ca2+ chelators attenuated
conditioning in Aplysia (Murphy and Glanzman, 1996; Bao et
al., 1998). An instrumental role for intracellular Ca2+ in the generation of eye-blink
conditioning has yet to be shown. Third, evidence suggests that there
is a modification of the AMPA receptor during LTP (Liao et al., 1995;
Barria et al., 1997; Hayashi et al., 2000; Soderling and Derkach, 2000)
and perhaps LTD (Lüthi et al., 1999). Receptor modifications may
include phosphorylation that enhances AMPA receptor currents (Barria et
al., 1997) or redistribution of AMPA receptor subunits at synaptic
sites (Liao et al., 1995; Lissin et al., 1999; Shi et al., 1999;
Hayashi et al., 2000). Such AMPA receptor alterations may also occur
during classical conditioning (Tocco et al., 1991) (the present study). Further studies, such as protein analysis, will be required to provide
more detail regarding changes in postsynaptic receptors and their
underlying mechanisms during in vitro eye-blink conditioning.
Previous studies of in vitro classical eye-blink
conditioning from the turtle have suggested that mechanisms underlying
acquisition of CRs can be separated from those that generate the
appropriate timing of CRs (Anderson and Keifer, 1997, 1999). Anderson
and Keifer (1999) demonstrated that abducens nerve CR acquisition could
be obtained from a highly reduced preparation consisting of an
~4-mm-thick section of brainstem tissue extending from the trigeminal
to the glossopharyngeal nerve and containing a portion of the abducens
eye-blink reflex circuitry. In these preparations, the UR has only the
short-duration component, similar to that produced in the presence of
AP-5. The long-duration component of the reflex, which is mediated by
NMDA receptors, is conveyed through the medullary reticular formation
that is eliminated by removal of the medulla. The properties of
acquisition and extinction of CRs in reduced brainstems are similar to
those in brainstem-cerebellum preparations; however, quantitative
analysis reveals that the CRs have abnormally short onset latencies.
These findings are similar to those of Perrett et al. (1993) who
found that short-latency CRs were produced after lesions of the
cerebellar cortex in rabbits. Furthermore, in vitro
preparations lacking a cerebellum show an attenuation in the
training-related shift in CR onset latency to later in the CS (Anderson
and Keifer, 1999). These data suggest that there are mechanisms for CR
acquisition within the brainstem abducens eye-blink circuitry, whereas
the learned timing of CRs is controlled by the cerebellum.
Taken together, the findings from in vitro classical
eye-blink conditioning in the turtle allow the outline of a model of the cellular events that may lead to CR acquisition (Fig.
7). From the data above, the model
assumes that acquisition of CRs occurs locally within the blink reflex
circuitry. This assumption is justified based on the fact that
brainstem preparations demonstrate conditioning (Anderson and Keifer,
1999) and on preliminary data suggesting that brainstem conditioning is
NMDA receptor dependent and also results in increased GluR4 in the
abducens nuclei (J. Keifer, unpublished observations). Both
physiological and immunocytochemical studies confirm the presence of
NMDA and AMPA receptors in the abducens blink reflex pathway (Keifer,
1993; Keifer and Carr, 2000). Although the model shown in Figure 7
emphasizes the abducens motor neurons as a site of conditioning, the
principal sensory trigeminal nucleus cannot be excluded. Also, the
present model does not address the findings related to timing of CRs
that appear to be a function of the cerebellar circuitry (Anderson and
Keifer, 1997, 1999). Tract-tracing studies suggest that inputs from the trigeminal and auditory nerves to the abducens motor nuclei are direct
(Herrick and Keifer, 1998), and recordings of reflex and single-unit
latencies show that they are likely to be monosynaptic (Keifer, 1993;
Keifer et al., 1995; Herrick and Keifer, 1998). The trigeminal
nerve-evoked blink reflex produced by the US is mediated predominantly
by AMPA receptors. The long-duration, NMDA receptor-mediated component
of the reflex is conveyed by a pathway through the medullary reticular
formation and is not necessary for conditioning (Keifer, 1993; Anderson
and Keifer, 1999). Other polysynaptic pathways linking trigeminal nerve
inputs with abducens motor neurons in the brainstem cannot be excluded
and may include the principal sensory trigeminal nucleus. The
high-threshold auditory nerve-evoked startle reflex is AMPA receptor
mediated because it is blocked by CNQX. However, a decrement in its
amplitude in AP-5 in some cases suggests a weak NMDA receptor-mediated
component (Keifer, unpublished observations). One parsimonious
explanation for these results is that NMDA receptors are located at
auditory nerve synaptic junctions and that AMPA receptors are located
extrasynaptically. With this arrangement, NMDA receptors alone would be
activated by the weak CS and thus produce no response, whereas a strong stimulus to the nerve may cause glutamate to spill over to nearby AMPA
receptors and produce the startle response. This scenario for
trigeminal and auditory nerve synapses onto abducens motor neurons is
illustrated in Figure 7.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 7.
A model showing proposed postsynaptic mechanisms
underlying classical conditioning of the in vitro
abducens nerve response in the turtle. Evidence suggests that inputs
from the US and CS directly contact abducens motor neurons. The US is
conveyed primarily by AMPA receptors (triangles);
synapses from the CS contain mainly NMDA receptors
(squares), but AMPA receptors are present
extrasynaptically. During conditioning, the strongly depolarizing US is
proposed to drive AMPA receptors away from trigeminal nerve synapses
into nearby auditory nerve synapses (dashed arrows) that
are normally silent to the weak CS input. Accumulation of AMPA
receptors allows depolarization of auditory nerve synapses in response
to glutamate release, allowing Ca2+ entry through
NMDA receptors. A series of unknown biochemical events, indicated by
the question mark, leads to a modification of the GluR4
AMPA receptor subunit, and these events are targeted to auditory nerve
synapses and underlie the generation of the CR. Small filled
circles represent synaptic vesicles.
|
|
During conditioning, the present results show that NMDA receptors must
be activated, presumably to allow intracellular
Ca2+ entry, and that there is upregulation
of the GluR4 subunit of the AMPA receptor in the abducens motor nuclei.
Assuming the synapse-receptor arrangement in Figure 7, there must be a
mechanism for the auditory nerve CS to activate postsynaptic NMDA
receptors or channels. Although many scenarios may be considered, one
possibility is that existing extrasynaptic or intracellular AMPA
receptors are driven away from synapses that are depolarized by the US
and move into nearby silent auditory nerve synapses (Fig. 7). Lissin et al. (1999) have shown in cultured neurons that GluR1 subunits rapidly
redistribute away from synapses after application of glutamate. In
time, accumulation of AMPA receptors results in sufficient depolarization of the synapse by the CS so that NMDA receptors are
released from their Mg2+ block and allow
intracellular Ca2+ entry as conditioning
proceeds. Intracellular Ca2+ initiates a
series of as yet unknown biochemical events that result in enhanced
incorporation of the AMPA receptor subunit GluR4 into auditory nerve
synapses that underlies the CR. The requirement for paired CS-US input
for conditioning can be accommodated by the model. Stimulation of the
US alone will not generate conditioning because, even if the AMPA
receptors are redistributed, NMDA receptors will not be activated by
the CS to allow Ca2+ entry. Stimulation of
the CS alone or of the CS before the US will have no effect because
these synapses are normally silent to weak input. The model is
necessarily speculative because not enough is known about the cellular
events leading to eye-blink conditioning, and there are many
possibilities. The model assumes that conditioning takes place within
the abducens motor neurons, in part because they receive convergent CS
and US inputs. However, this may not be the case. The principal sensory
trigeminal nucleus may also participate in conditioning of the blink
reflex, but connections with the abducens motor neurons are as yet
unspecified. Another point to consider when revising the model is that
the results show weakly GluR4-immunopositive abducens motor neurons in
unconditioned preparations and intensely immunopositive neurons in
those that conditioned. This finding leaves open the question of
whether GluR4 subunits are newly synthesized during conditioning or
whether there is a post-translational modification of the C terminal that allows them to be better recognized by the antibodies.
The finding that the GluR4 subunit of the AMPA receptor was modified
during conditioning was unexpected, especially in view of the reports
of activity-dependent modification of the GluR1 subunit (Barria et al.,
1997; Liao et al., 1999; Lissin et al., 1999; Shi et al., 1999).
However, the present data do not exclude redistribution of GluR1 or
NMDAR1 during conditioning. Immunostaining has revealed that the GluR4
subunit is especially dense in the auditory brainstem, particularly
among nuclei that are contacted by auditory nerve fibers (Rubio and
Wenthold, 1997). Additionally, AMPA receptors that contain GluR4
generate currents that decay rapidly and thereby mediate fast neuronal
transmission, a necessary feature of the auditory system (Mosbacher et
al., 1994). Tract-tracing studies in turtles have shown that the
abducens motor nuclei are directly contacted by the auditory nerve
(Herrick and Keifer, 1998). Therefore, it is plausible that GluR4 AMPA
receptor subunits are targeted to auditory nerve synapses by an NMDA
receptor-dependent process to strengthen the CS input during
conditioning that results in the generation of CRs.
| |
FOOTNOTES |
Received Sept. 18, 2000; revised Dec. 15, 2000; accepted Jan. 8, 2001.
This work was supported by a Regents Research Award and National
Institutes of Health Grants MH 58709 and P20 RR15567 that is
designated as a Center of Biomedical Research Excellence. I thank Curt
W. Anderson for participation in some of these experiments and James C. Houk, Ronald Lindahl, and N. Traverse Slater for helpful comments on a
previous version of this manuscript. I also thank Tim Clark for
generating the Western blots.
Correspondence should be addressed to Dr. Joyce Keifer, Neuroscience
Group, Division of Basic Biomedical Sciences, University of South
Dakota School of Medicine, 414 East Clark Street, Vermillion, SD 57069. E-mail: jkeifer{at}usd.edu.
| |
REFERENCES |
-
Anderson CW,
Keifer J
(1997)
The cerebellum and red nucleus are not required for in vitro classical conditioning of the turtle abducens nerve response.
J Neurosci
17:9736-9745[Abstract/Free Full Text].
-
Anderson CW,
Keifer J
(1999)
Properties of in vitro turtle conditioned abducens nerve responses in the absence of the sustained component of the UR.
J Neurophysiol
81:1242-1250[Abstract/Free Full Text].
-
Bao J-X,
Kandel ER,
Hawkins RD
(1998)
Involvement of presynaptic and postsynaptic mechanisms in a cellular analog of classical conditioning at Aplysia sensory-motor neuron synapses in isolated cell culture.
J Neurosci
18:458-466[Abstract/Free Full Text].
-
Barria A,
Muller D,
Derkach V,
Griffith LC,
Soderling TR
(1997)
Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation.
Science
276:2042-2045[Abstract/Free Full Text].
-
Bell CC,
Han VZ,
Sugawara Y,
Grant K
(1997)
Synaptic plasticity in a cerebellum-like structure depends on temporal order.
Nature
387:278-281[Medline].
-
Bi G-Q,
Poo M-M
(1998)
Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type.
J Neurosci
18:10464-10472[Abstract/Free Full Text].
-
Bloedel JR,
Bracha V
(1995)
On the cerebellum, cutaneomuscular reflexes, movements control and the elusive engrams of memory.
Behav Brain Res
68:1-44[ISI][Medline].
-
Hayashi Y,
Shi S-H,
Esteban JA,
Piccini A,
Poncer J-C,
Malinow R
(2000)
Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction.
Science
287:2262-2267[Abstract/Free Full Text].
-
Herrick JL,
Keifer J
(1998)
Central trigeminal and posterior eighth nerve projections in the turtle Chrysemys picta studied in vitro.
Brain Behav Evol
51:183-201[Medline].
-
Keifer J
(1993)
In vitro eye-blink reflex model: role of excitatory amino acids and labeling of network activity with sulforhodamine.
Exp Brain Res
97:239-253[ISI][Medline].
-
Keifer J
(2000)
In vitro classical conditioning in turtles is NMDA receptor-dependent and involves redistribution of AMPA receptor subunit GluR4.
Soc Neurosci Abstr
26:718.
-
Keifer J,
Carr MT
(2000)
Immunocytochemical localization of glutamate receptor subunits in the brain stem and cerebellum of the turtle, Chrysemys picta.
J Comp Neurol
427:455-468[ISI][Medline].
-
Keifer J,
Armstrong KE,
Houk JC
(1995)
In vitro classical conditioning of abducens nerve discharge.
J Neurosci
15:5036-5048[Abstract].
-
Kim JJ,
Thompson RF
(1997)
Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning.
Trends Neurosci
20:177-181[ISI][Medline].
-
Kishimoto Y,
Kawahara S,
Kirino Y,
Kadotani H,
Nakamura Y,
Ikeda M,
Yoshioka T
(1997)
Conditioned eyeblink response is impaired in mutant mice lacking NMDA receptor subunit NR2A.
NeuroReport
8:3717-3721[Medline].
-
Levin MD,
Kubke MF,
Schneider M,
Wenthold R,
Carr CE
(1997)
Localization of AMPA-selective glutamate receptors in the auditory brainstem of the barn owl.
J Comp Neurol
378:239-253[ISI][Medline].
-
Liao D,
Hessler NA,
Malinow R
(1995)
Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice.
Nature
375:400-404[Medline].
-
Liao D,
Zhang X,
O'Brien R,
Ehlers MD,
Huganir RL
(1999)
Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons.
Nat Neurosci
2:37-43[ISI][Medline].
-
Lissin DV,
Carroll RC,
Nicoll RA,
Malenka RC,
von Zastrow M
(1999)
Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons.
J Neurosci
19:1263-1272[Abstract/Free Full Text].
-
Lüthi A,
Chittajallu R,
Duprat F,
Palmer MJ,
Benke TA,
Kidd FL,
Henley JM,
Collingridge GL
(1999)
Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction.
Neuron
24:389-399[ISI][Medline].
-
Malenka RC
(1994)
Synaptic plasticity in the hippocampus: LTP and LTD.
Cell
78:535-538[ISI][Medline].
-
Malenka RC,
Nicoll RA
(1997)
Silent synapses speak up.
Neuron
19:473-476[ISI][Medline].
-
Mosbacher J,
Schoepfer R,
Monyer H,
Burnashev N,
Seeburg PH,
Rupersberg JP
(1994)
A molecular determinant for submillisecond desensitization in glutamate receptors.
Science
266:1059-1062[Abstract/Free Full Text].
-
Moyer JR,
Deyo RA,
Disterhoft JF
(1990)
Hippocampectomy disrupts trace eye-blink conditioning in rabbits.
Behav Neurosci
104:243-252[ISI][Medline].
-
Murphy GC,
Glanzman DL
(1996)
Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+.
Proc Natl Acad Sci USA
93:9931-9936[Abstract/Free Full Text].
-
Murphy GG,
Glanzman DL
(1997)
Mediation of classical conditioning in Aplysia californica by long-term potentiation of sensorimotor synapses.
Science
278:467-471[Abstract/Free Full Text].
-
Ozawa S,
Kamiya H,
Tsuzuki K
(1998)
Glutamate receptors in the mammalian central nervous system.
Prog Neurobiol
54:581-618[ISI][Medline].
-
Pellegrini JJ,
Horn AKE,
Evinger C
(1995)
The trigeminally evoked blink reflex. I. Neuronal circuits.
Exp Brain Res
107:166-180[ISI][Medline].
-
Perrett SP,
Ruiz BP,
Mauk MD
(1993)
Cerebellar cortex lesions disrupt learning-dependent timing of conditioned eyelid responses.
J Neurosci
13:1708-1718[Abstract].
-
Petralia RS,
Wenthold RJ
(1992)
Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain.
J Comp Neurol
318:329-354[ISI][Medline].
-
Rubio ME,
Wenthold RJ
(1997)
Glutamate receptors are selectively targeted to postsynaptic sites in neurons.
Neuron
18:939-950[ISI][Medline].
-
Servatius RJ,
Shors TJ
(1996)
Early acquisition, but not retention, of the classically conditioned eyeblink response is N-methyl-D-aspartate (NMDA) receptor dependent.
Behav Neurosci
110:1040-1048[ISI][Medline].
-
Shi S-H,
Hayashi Y,
Petralia RS,
Zaman SH,
Wenthold RJ,
Svoboda K,
Malinow R
(1999)
Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.
Science
284:1811-1816[Abstract/Free Full Text].
-
Soderling TR,
Derkach VA
(2000)
Postsynaptic protein phosphorylation and LTP.
Trends Neurosci
23:75-80[ISI][Medline].
-
Tocco G,
Devgan KK,
Hauge SA,
Weiss C,
Baudry M,
Thompson RF
(1991)
Classical conditioning selectively increases AMPA receptor binding in rabbit hippocampus.
Brain Res
559:331-336[Medline].
Copyright © 2001 Society for Neuroscience 0270-6474/01/2172434-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
M. Mokin, Z. Zheng, and J. Keifer
Conversion of Silent Synapses Into the Active Pool by Selective GluR1-3 and GluR4 AMPAR Trafficking During In Vitro Classical Conditioning
J Neurophysiol,
September 1, 2007;
98(3):
1278 - 1286.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Keifer, Z.-Q. Zheng, and D. Zhu
MAPK Signaling Pathways Mediate AMPA Receptor Trafficking in an In Vitro Model of Classical Conditioning
J Neurophysiol,
March 1, 2007;
97(3):
2067 - 2074.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mokin, J. S. Lindahl, and J. Keifer
Immediate-Early Gene-Encoded Protein Arc Is Associated With Synaptic Delivery of GluR4-containing AMPA Receptors During In Vitro Classical Conditioning
J Neurophysiol,
January 1, 2006;
95(1):
215 - 224.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mokin and J. Keifer
Expression of the immediate-early gene-encoded protein Egr-1 (zif268) during in vitro classical conditioning
Learn. Mem.,
March 1, 2005;
12(2):
144 - 149.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Coleman, C. Cai, D. G. Mottershead, J.-P. Haapalahti, and K. Keinanen
Surface Expression of GluR-D AMPA Receptor Is Dependent on an Interaction between Its C-Terminal Domain and a 4.1 Protein
J. Neurosci.,
February 1, 2003;
23(3):
798 - 806.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
