Download Learning-related postburst afterhyperpolarization reduction in CA1

Learning-related postburst afterhyperpolarization
reduction in CA1 pyramidal neurons is mediated
by protein kinase A
M. Matthew Oha,1, Bridget M. McKaya,b, John M. Powerc, and John F. Disterhofta,b,1
aDepartment
of Physiology and bNeuroscience Program, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611-3008; and cQueensland
Brain Institute, The University of Queensland, Brisbane, OLD 4072, Australia
Edited by Richard F. Thompson, University of Southern California, Los Angeles, CA, and approved December 9, 2008 (received for review August 5, 2008)
Learning-related reductions of the postburst afterhyperpolarization (AHP) in hippocampal pyramidal neurons have been shown ex
vivo, after trace eyeblink conditioning. The AHP is also reduced by
many neuromodulators, such as norepinephrine, via activation of
protein kinases. Trace eyeblink conditioning, like other hippocampus-dependent tasks, relies on protein synthesis for consolidating
the learned memory. Protein kinase A (PKA) has been shown to be
a key contributor for protein synthesis via the cAMP-response
element-binding pathway. Here, we have explored a potential
involvement of PKA and protein kinase C (PKC) in maintaining the
learning-related postburst AHP reduction observed in CA1 pyramidal neurons. Bath application of isoproterenol (1 !M), a "-adrenergic agonist that activates PKA, significantly reduced the AHP
in CA1 neurons from control animals, but not from rats that
learned. This occlusion suggests that PKA activity is involved in
maintaining the AHP reduction measured ex vivo after successful
learning. In contrast, bath application of the PKC activator, (–)
indolactam V (0.2 !M), significantly reduced the AHP in CA1
neurons from both control and trained rats, indicating that PKC
activity is not involved in maintaining the AHP reduction at this
point after learning.
hippocampus ! protein kinase C ! trace eyeblink
T
he postburst afterhyperpolarization (AHP) has been repeatedly demonstrated ex vivo to be reduced in hippocampal
pyramidal neurons after hippocampus-dependent learning, such
as the trace eyeblink conditioning (EBC) task (1). Trace EBC is
a hippocampus-dependent task (2–4) that, like others (5, 6),
requires protein synthesis for learning and consolidation of the
memory (7), yet the molecular cascade that underlies this
learning-related AHP alteration after trace EBC has not been
studied or identified.
The postburst AHP is predominantly a Ca2!-dependent K!
current with 2 distinct components (8–10). The SK2 channels
underlie the apamin-sensitive medium AHP (11–13) and have
been shown to be intimately involved in regulating synaptic
plasticity in dendritic spines along with NMDA receptors (14).
The channel underlying the later, slow AHP has yet to be
discovered; however, it is thought to be localized to the apical
and basal dendrites in close proximity to the soma (15, 16).
Because of its somatic localization, the slow AHP may play a
significant role in the final somatic integration of synaptic inputs.
Unless specified, we will be referring to both the medium and
slow components of the AHP throughout the text.
Activation of protein kinases via various neuromodulators
reduces the AHP (10, 17). Specifically, activation of cholinergic
and metabotropic glutamate receptors have been shown to
reduce the AHP via protein kinase C (PKC) and Ca2!/calmodulin-dependent protein kinase II (CaMKII) (18–22). Activation
of monoamine receptors reduces the AHP via protein kinase A
(PKA) (21, 23). In addition to reducing the AHP, all 3 kinases
have been implicated in long-term potentiation (LTP), with PKC
and CaMKII being involved in the early, induction phase and
1620 –1625 ! PNAS ! February 3, 2009 ! vol. 106 ! no. 5
PKA demonstrated to be crucial for the late, protein synthesisdependent phase (6, 24–26).
One of the signaling pathways that leads to protein synthesis
involves PKA-mediated activation of MAPK, and subsequently,
cAMP-response element-binding proteins (CREB) (6, 25, 27).
Transgenic mice with a constitutively active form of CREB have
recently been shown to have CA1 pyramidal neurons with
significantly reduced AHPs (28). Interestingly, EBC is also
significantly impaired by disruption of the cAMP/PKA pathway
in the cerebellum (29), a structure known to be critically involved
in learning this task (30).
PKC levels in the hippocampus have been shown to be
modulated by various learning tasks. Increase in membraneassociated PKC has been demonstrated in the hippocampus and
cerebellum of rabbits after learning EBC (31–33). However, a
decrease in membrane-associated PKC has also been shown in
the hippocampus after spatial learning in rats and mice (34–36).
PKC activation is also thought to underlie odor-discrimination
learning, as a PKC activator occluded the learning-related AHP
reduction in piriform cortical neurons evaluated ex vivo (37).
Although PKC activation has been shown to be capable of
activating MAPK (38), numerous studies have demonstrated
that the cAMP/PKA pathway activation in the hippocampus is
also essential for long-term memory and consolidation of hippocampus-dependent tasks (5, 6, 25). It remains to be determined if PKC and PKA are involved in maintaining the learningrelated AHP reduction in CA1 hippocampal pyramidal neurons
after trace EBC.
We report here that learning the trace EBC task occludes the
PKA-, but not PKC-mediated AHP reduction in CA1 pyramidal
neurons at a timepoint after rats have achieved a behavioral
criterion indicating that they have learned trace EBC well. This
occlusion strongly suggests that PKA activity and its associated
signaling cascade are involved in maintaining the learningrelated AHP reduction observed in CA1 neurons ex vivo at a
timepoint when the initial consolidation of the memory and
related protein synthesis have occurred.
Results
Postburst AHP Is Reduced in CA1 Neurons from Rats that Learned Trace
EBC. All trained rats used for the biophysical experiments
reached a minimum of 60% conditioned responses (CRs) by the
final training session and exhibited significantly more CRs than
the pseudoconditioned animals throughout the training sessions
Author contributions: M.M.O. and J.F.D. designed research; M.M.O. and B.M.M. performed
research; J.M.P. contributed new reagents/analytic tools; M.M.O. and B.M.M. analyzed
data; and M.M.O., B.M.M., and J.F.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To
whom correspondence may be addressed. E-mail: [email protected] or
[email protected].
© 2009 by The National Academy of Sciences of the USA
www.pnas.org"cgi"doi"10.1073"pnas.0807708106
(Fig. 1B). The day after the last training session, postburst AHP
was measured from visually identified CA1 pyramidal neurons in
whole-cell current clamp mode using a 50-Hz train of 8 antidromic action potentials (APs). Similar to other studies that
examined the AHP after trace EBC (39–41), CA1 pyramidal
neurons from trained animals had significantly reduced AHPs ex
vivo (Fig. 2). The AHP peak amplitude (see Fig. 2B) (F2,105 "
9.919, P " 0.0001), the AHP amplitude measured 1 sec after the
end of the last AP (see Fig 2C) (1 sec AHP: F2,105 " 6.688, P "
0.0018) and the integrated AHP area (see Fig. 2D) (F2,105 "
6.607, P " 0.0020) from trained rats were significantly smaller
than those from pseudoconditioned and naïve animals. Additionally, nearly 30% of CA1 neurons from trained animals had
AHP measures smaller than one standard deviation from the
mean of the naïve neurons (see Fig. 2 B–D). Similar to previous
reports, the AHP measurements from pseudoconditioned and
naïve rats were nearly identical (Fisher’s PLSD p’s all #0.2), and
thus, were combined as controls for the rest of the analyses. No
significant learning-related differences were observed for other
membrane properties (Table 1).
Learning Occluded the PKA-Induced AHP Reduction. Isoproterenol
(Iso) is a !-adrenergic agonist that is commonly used to activate
PKA (21, 42, 43) and has been used to suppress the postburst
AHP (21, 39). Thus, we tested for the potential PKA involvement
in maintaining the learning-related AHP reduction by changing
the perfusate to an aCSF with 1 "M Iso and repeating the
biophysical measurements after the baseline measurements were
recorded. We chose 1 "M Iso because we wanted to reduce, not
abolish, the postburst AHP. Repeated ANOVA, using data from
all of the neurons in both training groups to which Iso was
applied, revealed that Iso significantly reduced the AHP in CA1
neurons from control animals and made these values very similar
to those from trained animals (Fig. 3). Iso had no impact on the
AHP measures in CA1 neurons from trained rats (see Fig. 3). To
verify that Iso was reducing the AHP via a PKA-mediated
mechanism, we measured the AHP before bath application of
the PKA inhibitor H 89 (5–10 "M), 10 min after H 89, and 10
min after H 89 and Iso in a few neurons (n " 5) from control
animals. H 89 reduced the peak AHP (6.57 $ 1.25 to 5.78 $ 1.24
mV; paired t test, P % 0.05) but did not affect the 1 sec AHP nor
Oh et al.
NEUROSCIENCE
Fig. 1. Young adult F344xBN rats readily learn trace EBC. (A) Rats were given
2 training sessions a day over 3 days. Hippocampal slices and biophysical
recordings were made the day after the fifth training session. (B) Trained
(Trace, n " 18) rats exhibited significantly more CRs than pseudoconditioned
(Pseudo, n " 10) rats during all training sessions (repeated ANOVA: F(1,5,130) "
10.729, P % 0.0001; unpaired t test: P % 0.05 for session 1, p’s %0.0005 for
sessions 2–5). Additionally, the trained rats exhibited greater CRs over the
training sessions (F4,68 " 13.738, P % 0.0001), whereas the pseudoconditioned
rats remained unchanged throughout the 5 sessions (F4,36 " 0.743, P " 0.569).
There was no difference between the groups in the spontaneous blinks during
the habituation session (P # 0.4).
Fig. 2. Postburst AHP in CA1 pyramidal neurons is significantly reduced after
learning trace EBC. (A) Examples of antidromically evoked AHP are illustrated.
The APs have been truncated for illustration purposes. (B–D Left) Learning had
a significant impact on the peak AHP amplitude (F2,105 " 9.919, P " 0.0001),
amplitude measured 1 sec after the last AP (F2,105 " 6.688, P " 0.0018), and
integrated AHP area (F2,105 " 6.607, P " 0.0020). All 3 measures were significantly reduced in CA1 neurons from trained rats. (B–D Right) In addition to
the reductions, the distribution of the AHP measures were dramatically
shifted to smaller values than one standard deviation from the mean of the
naïve neurons. Of the CA1 neurons, 51% (21/41) from trained rats had smaller
AHP peak amplitudes as compared to 15% and 25% of CA1 neurons from
naïve (7/47) and pseudoconditioned (5/20) rats. Forty-four percent of CA1
neurons (18/41) from trained rats had smaller 1 sec AHP amplitude as compared to 6% and 10% from naïve (3/47) and pseudoconditioned (2/20) rats.
Finally, 42% of CA1 neurons from trained (17/41) rats had smaller AHP area as
compared to 9% and 10% from naïve (4/47) and pseudoconditioned (2/20)
rats. N, naïve; P, pseudoconditioned; T, trace EBC. Numbers in parentheses
represent the number of neurons recorded from the respective groups. Fisher’s PLSD *P % 0.05, **P % 0.001, ***P % 0.0001.
the AHP area (p’s #0.8). More importantly, H 89 prevented Iso
from reducing the AHP measures (p’s #0.11). The occluding
effect of learning on the action of Iso strongly suggests that the
PNAS ! February 3, 2009 ! vol. 106 ! no. 5 ! 1621
Table 1. Basic membrane properties of recorded CA1 neurons
Duration (s)
Latency (ms)
IR (M')
Sag (mV)
RMP (mV)
Vh (mV)
Naïve
(n " 47)
Pseudo
(n " 20)
Trace
(n " 41)
3.93 $ 0.13
112.5 $ 7.3
65.1 $ 2.8
5.87 $ 0.22
(68.0 $ 0.8
(68.3 $ 0.2
4.04 $ 0.20
145.7 $ 14.5
74.3 $ 4.1
6.27 $ 0.41
(67.2 $ 1.0
(68.1 $ 0.3
3.55 $ 0.19
119.0 $ 8.6
74.8 $ 3.6
5.48 $ 0.21
(69.0 $ 0.6
(68.0 $ 0.2
Note: Duration, AHP duration; Latency, latency to AHP peak amplitude; IR,
input resistance; RMP, resting membrane potential; Vh, membrane holding
potential.
PKA signaling cascade is involved in maintaining the learningrelated AHP reduction and is active for at least one day ex vivo
after the last training session.
Learning Did not Occlude PKC-Mediated AHP Reduction. The post-
burst AHP is reduced by various PKC activators (17, 19, 21). We
chose to use 0.2 "M (–) indolactam V (Indo), as Wheal and
colleagues effectively demonstrated that this concentration
would reduce &50% of the slow AHP current (21). Thus, after
the initial baseline measurements, Indo was added to the perfusate, and the biophysical properties were remeasured. Indo
significantly reduced the AHP in neurons from both the control
and trained groups (Fig. 4). Our analyses did not reveal a
significant drug-by-behavior interaction (repeated ANOVAs for
peak AHP, 1 sec AHP, and AHP area: F’s %2.001, P’s #0.17).
Indo did significantly reduce the peak AHP amplitude, 1 sec
AHP, and AHP area (see Fig. 4, repeated ANOVAs comparing
initial measures vs. post Indo) (F1,22 # 43, P’s %0.0001). Thus,
unlike the PKA activator Iso, PKC activation was not occluded
from further reducing the AHP in CA1 neurons from animals
that learned the trace EBC task.
Discussion
The main finding of the present study is that learning the
hippocampus-dependent trace EBC task occluded the PKAmediated AHP reduction in CA1 pyramidal neurons. Additionally, the learning-related AHP reduction in CA1 neurons was
again demonstrated ex vivo. This reproducible AHP reduction in
CA1 neurons after hippocampus-dependent learning establishes
it as a cellular hallmark of learning-related intrinsic plasticity.
Thus, the question remains, how is PKA recruited to cause or
maintain this cellular feature of learning?
PKA is a cAMP-dependent protein kinase that has been
extensively studied for its role in the late phase of long-term
potentiation (L-LTP) and long-term memory (6, 25, 44). PKA
has also been shown to effectively reduce the AHP in hippocampal pyramidal neurons (23, 45). Recently, Lin et al. (46) demonstrated that activated PKA induces internalization of the
apamin-sensitive AHP channels, SK2, in the dendritic spines of
hippocampal neurons. In addition, compounds that reduce the
AHP facilitate LTP induction (15, 47–49). Thus, an interesting
dynamic exists between the two: small AHP facilitates LTP, and
a large AHP impairs LTP. Therefore, it is very possible that PKA
activation bridges the AHP reduction and LTP facilitation.
Both L-LTP and long-term memory consolidation of tasks
including trace EBC have been shown to be protein synthesisdependent (6, 7, 25, 27). There are multiple ways of triggering
protein synthesis, including the cAMP/PKA pathway (Fig. 5) (6).
Furthermore, transgenic mice that express a dominant negativeform of the PKA regulatory subunit (50) or that have target
mutations of CREB (51) have significant long-term memory
deficits on the spatial water maze and contextual fear1622 ! www.pnas.org"cgi"doi"10.1073"pnas.0807708106
Fig. 3. Learning occluded the PKA mediated AHP reduction. Examples of 1
"M Iso’s effect on a CA1 neuron from a naïve (A) and a trace EBC (B) rat are
illustrated. Note that Iso reduced the postburst AHP without abolishing it in
the CA1 neuron from the naïve animal. The APs have been truncated for
illustration purposes. (C) Significant drug-by-behavior interactions were revealed with repeated ANOVAs for the peak AHP amplitude (F1,30 " 4.241, P "
0.0482), 1 sec AHP (F1,30 " 6.314, P " 0.0176), and AHP area (F1,30 " 7.092, P "
0.0123). Further analyses revealed that Iso (1 "M) reduced the peak AHP
(8.37 $ 0.61 to 6.67 $ 0.79 mV, P % 0.005), 1 sec AHP and AHP area measures
in CA1 neurons (n " 17) from control rats. Iso did not have an impact on the
peak (5.46 $ 0.52 to 5.01 $ 0.70mV, P # 0.12), 1 sec AHP (P # 0.60) and area
(P # 0.33) measures in CA1 neurons (n " 15) from trained rats. Thus, the
resulting post-Iso AHP measures were nearly identical between the 2 groups
(unpaired t test: p’s #0.12). The impact of Iso on AHP peak amplitude is similar
to that shown for the AHP area, and thus is not illustrated. Pre, initial baseline
measures; Iso, post isoproterenol measures. Unpaired t test: *P % 0.01, **P %
0.005. Paired t test: †P % 0.005, ††P % 0.001.
conditioning tasks. Interestingly, EBC is also significantly impaired by disruption of the cAMP/PKA pathway in the cerebellum (29), a structure known to be critically involved in learning
this task (30). In addition, extinction of delay EBC, which
depends on an intact hippocampus (52), is impaired by lesion of
the locus coeruleus (53). CREB activation and the AHP have
been linked in a study by Barco and colleagues, who demonstrated that activation of CREB causes a significant AHP
reduction in CA1 pyramidal neurons (28). Thus, it is possible
Oh et al.
Fig. 4. Learning did not occlude the PKC-mediated AHP reduction. Examples
of 0.2 "M (–) indolactam V’s (Indo) effect on CA1 neurons from naïve (A) and
trace EBC (B) rats are illustrated. (C) Indolactam significantly reduced the AHP
peak amplitude, amplitude measured at 1 sec (1s), and the integrated area
(Area) in CA1 neurons from both the control and trained rats. The impact of
Indo on AHP peak amplitude is similar to that shown for the AHP area, and
thus is not illustrated. Pre, initial baseline measures; Indo, post indolactam
measures. Unpaired t test: *P % 0.05, **P % 0.01. Paired t test: †P % 0.05, ††P %
0.01, †††P % 0.001.
that the activation of the PKA signaling pathway led to the
activation of CREB, which could help maintain the learningrelated AHP reduction in hippocampal pyramidal neurons after
the subject has learned a hippocampus-dependent task, such as
trace EBC.
In addition to having the same protein synthesis requirement,
LTP and trace EBC have another interesting parallel. DelgadoGarcia and colleagues have recently demonstrated that inducing
LTP in the CA3-CA1 synapse just before or concurrently with
training mice on trace EBC prevents learning; whereas, LTP
induced a month before training has no effect on learning (54).
Furthermore, learning trace EBC enhanced synaptic transmission between the CA3-CA1 synapses (55) and prevented subsequent LTP (54).
Our present results do not rule out the involvement of other
signaling cascades, such as those mediated by PKC. In the
present study, we measured the AHP after the rats had consolOh et al.
idated the memory for trace EBC (see Fig. 1) and found that the
PKC activator, (–) Indo, reduced the AHP similarly in CA1
neurons from both trained and control animals. The slow AHP
in neurons from both trained and control rats was reduced by
&50% with 0.2 "M Indo, as previously described by Wheal and
colleagues (21) (see Fig. 4). Notably, 1 "M Iso reduced the slow
AHP in neurons from control rats by 40% (see Fig. 3). The
similar slow AHP reduction by both compounds in neurons from
control rats further suggests that the differential involvement of
PKC and PKA that we observed is not a nonspecific consequence
of differentially effective doses of drugs used to activate the 2
pathways. However, this does not rule out PKC involvement in
the learning-related AHP reduction. It is possible that PKC
activity may have played a critical role in the early phase of
learning and the resulting AHP changes, similar to that observed
for LTP induction (5, 56).
These other signaling pathways could be involved in modulating intrinsic plasticity and memory formation in other regions
of the brain. For example, the AHP reduction in layer II piriform
cortical neurons after odor discrimination learning (57) has been
shown to be mediated by PKC (37). It is possible that PKA is not
a viable option for maintaining the learning-related AHP reduction after odor discrimination tasks, as the adenylyl cyclase
(AC) critically involved for odor learning has been shown to be
tightly temporally regulated for successfully learning this task
(58). Cui et al. (59) recently demonstrated that a temporally
specific increase and decrease in cAMP levels in the olfactory
bulb are important for successfully learning odor preference.
These temporally regulated cAMP levels could lead to a tight
regulation of PKA activity, making PKC activity a better regulator of long-lasting memory for odor-discrimination tasks.
Learning may not engage all kinase cascades in every cell in
the hippocampus. Indeed, a detailed study of the cAMP/PKA
and ERK/MAP kinase signal cascade revealed that only about
10% of cells in CA1 show coactivation of AC, PKA, MAPK, and
the CREB kinase MSK1 after learning a contextual fearPNAS ! February 3, 2009 ! vol. 106 ! no. 5 ! 1623
NEUROSCIENCE
Fig. 5. Highly simplified schematic of PKA involvement in the AHP reduction
after learning. Under normal condition, the Ca2! influx into the cytosol, via
the L-type Ca2! channel and internal Ca2! stores, activates the AHP. During
learning, activation of monoamine receptors (e.g., noradrenaline, dopamine,
serotonin) along with a rise in internal Ca2! levels activate adenylyl cyclase,
causing an increase in cAMP levels leading to PKA activation (reviewed in (5,
6, 25, 27). Activated PKA leads to MAPK/ERK activation of the CREB signaling
pathway, ultimately leading to gene transcription and translation. The activated CREB and PKA also lead to a reduction in the AHP. This reduced AHP
channel activity, in turn, would lead to a longer duration depolarization,
resulting in a longer time window for the continuation of the cAMP/PKA
signaling cascade.
conditioning task (60). Similarly, not all hippocampal pyramidal
neurons undergo the dramatic AHP reduction after learning, as
reported here (see Fig. 2 B–D) and previously (40, 41).
In addition to reducing the AHP, PKA activity has also been
implicated in reducing another potassium current, the Kv4.2mediated A-type potassium current (61, 62). Recently, Hoffman and colleagues demonstrated that PKA activation led to
Kv4.2 channels being removed from the surface of the cell
membrane (62), similar to the SK2 apamin-sensitive AHP
channels (46). The internalization of the Kv4.2 via PKA
activity results in a reduced A-type potassium current (62).
Given the similar PKA action on the SK2 and Kv4.2 channels,
it is possible that the enhanced neuronal excitability observed
in hippocampal pyramidal neurons ex vivo after learning (via
reductions in both the medium and slow AHPs) is a result of
internalization of both the apamin-sensitive SK2 and channels
that underlie the slow AHP by PKA activity. However, it will
not be possible to confirm this until the molecular identity of
the slow AHP is discovered.
Age-related enlargement of the postburst AHP in hippocampal pyramidal neurons have been demonstrated and postulated
to contribute to the normal aging-related learning impairments
(63, 64). Age-related spatial learning impairments have been
linked to reduced levels of AC type 1 in the hippocampus of aged
mice (65) and can be ameliorated by compounds that enhance
cAMP/PKA signaling (66). Interestingly, !-amyloid has been
shown to inactivate PKA in hippocampal neurons (67). AC type
1 has also been shown to be reduced in hippocampus of
Alzheimer’s disease patients (68). Taken together, the normal
age- and Alzheimer disease-related learning impairments may
result from not only the enlarged AHP with aging, but also from
the lack of proper AHP modulation by PKA.
In summary, the present results strongly suggest that PKA
activity is one mechanism by which the learning-related AHP
reduction is maintained in CA1 pyramidal neurons. It is noteworthy that L-LTP, protein synthesis, long-term memory, and
now maintenance of learning-related AHP reduction all have
PKA activity as a common substrate. Given this and the inverse
relationship between AHP and LTP, the molecular mechanisms
and signaling-cascades that underlie the learning-related AHP
reduction may be similar to that found for LTP.
Materials and Methods
training session consisted of 30 CS-US pairings with an intertrial interval of 20
to 40 sec (30 sec average). A pseudoconditioning session consisted of explicitly
unpaired presentation of 30 CS and 30 US with a 10 to 20 sec intertrial interval
(15 sec average).
Whole-cell current clamp recordings were made from visually identified
CA1 pyramidal neurons using an Axoclamp 2A amplifier, as previously described (69). Briefly, 1 day after the last training session, hippocampal slices
(300 "m) were cut using a Leica vibratome in an ice-cold ACSF (124 NaCl, 26
NaHCO3,3 KCl, 2.4 CaCl2, 2 MgSO4, 1.25 NaH2PO4, and 25 D-glucose, gassed
with 95% O2-5% CO2). The slices were incubated for &30 min at 34 °C and
allowed to cool down to room temperature for at least another 30 min before
being transferred to a submersion chamber on an upright Leica DMLFS
microscope. The patch electrodes (3– 6M') were filled with (in mM) 120
KMeSO4, 10 KCl, 10 Hepes, 4 Mg2ATP, 0.4 NaGTP, 10 Na2 phosphocreatine,
0.5% neurobiotin, pH adjusted to 7.3 with KOH, 280 $ 5 mOsm. No correction
was made for a &10-mV liquid junction potential. All measurements were
made #5 min after membrane rupture to allow for adequate solution equilibration with the neuron held near – 67 mV (65 ( 70 mV) at 34 to 35 °C. A
potential error in membrane potential recording may occur if the current
injection and membrane potential recording are performed with the same
patch electrode (70). Thus, AHP measurements were obtained by a train of 8
antidromic pulses (50 Hz) using a concentric bipolar stimulating electrode
placed on the alveus near the recorded CA1 neuron. Similar AHP values were
observed with a train of 8 APs (50 Hz) evoked by direct somatic current
injections (data not shown). More importantly, the observed AHP correlated
well with learning irrespective of the mode used to trigger the AHP. Thus, a
train of 8 APs at 50 Hz was chosen to ensure a sufficiently large AHP to easily
observe learning-related AHP changes, as previous study illustrated that the
size of the AHP is dependent on the frequency and number of APs used to
evoke it (71). Included in the aCSF were 50 "M D-AP5, 10 "M NBQX, and 12.5
"M gabazine to eliminate NMDA-, AMPA-, and GABAA-mediated synaptic
responses. Biophysical measures were repeated after a 10-min bath application of drug compounds used throughout the experiments. A few experiments were carried out to ensure that neither switching the drug perfusate
line nor the duration of the biophysical recordings significantly impacted the
AHP measures (n " 5; paired t-tests of peak AHP, 1 sec AHP, AHP area, AHP
duration, sag, and holding potential; p’s #0.13). Notably, the input resistance
did increase over the duration of the recordings (P % 0.05), similar to that
previously reported for internal pipette solutions containing KMeSO4 (72).
However, no significant drug-by-group interaction was observed for the input
resistance measures with any of the compounds used (F’s %4.2, P’s #0.05).
Thus, it is unlikely that the effects of Iso or Indo on the AHP measures were
significantly impacted by the increase in input resistance over time. D-AP5,
NBQX, gabazine, Iso, and H 89 were obtained from Tocris Bioscience. KMeSO4
was obtained from ICN. (–) Indo was obtained from Axxora LLC. All other
compounds were obtained from Sigma.
Subjects were young adult (2–3 months old) male F1 hybrid Fischer 344 )
Brown Norway rats that were group housed (3 per cage) in a climatecontrolled vivarium on a 12:12 light:dark cycle with ad libitum access to food
and water. Animal care and experimental protocols were done following
National Institutes of Health guidelines and approved by the Northwestern
University Institutional Animal Care and Use Committee.
Trace EBC was performed as previously described (39). Briefly, using sterile
surgical techniques and under isofluorane inhalation anesthesia, the rats were
implanted with a headgear to deliver the airpuff and to record EMG activity
from the upper right eyelid, and allowed to recover for a minimum of 3 days
before training. Fig. 1 illustrates the training schedule. Rats were trained
individually in a sound-attenuating chamber with a tone conditioned stimulus
(CS; 250 ms, 8 kHz, 85 dB free field) and a corneal airpuff unconditioned
stimulus (US; 100 ms, 4.5 psi) separated by a 250-ms blank trace interval. A
Data Acquisition and Analysis. The biophysical recordings and analyses were
performed blind to the behavioral status of the animal. The behavioral and
biophysical data were digitized and analyzed on-line using custom software routines written in LabVIEW (National Instruments). Complete analyses were performed off-line using procedures developed with LabVIEW.
Statistical analyses were performed using StatView. Significant main effects were evaluated using Fisher’s PLSD post hoc tests. All data are
reported as the means $ SE.
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1624 ! www.pnas.org"cgi"doi"10.1073"pnas.0807708106
ACKNOWLEDGMENTS. The authors thank John Linardakis and Elizabeth
Matthews for help with the behavioral experiments, and Elizabeth Matthews
for critical reading and helpful discussion of the manuscript. This work was
supported by National Institutes of Health Grants AG08796 and AG20506
(to J.F.D.).
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