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The Journal of Neuroscience, July 15, 2000, 20(14):5476–5482 Increased Excitability of Aged Rabbit CA1 Neurons after Trace
Eyeblink Conditioning
James R. Moyer Jr, John M. Power, Lucien T. Thompson, and John F. Disterhoft
Department of Cell and Molecular Biology and the Institute for Neurosciences, Northwestern University Medical School,Chicago, Illinois 60611-3008 Cellular properties of CA1 neurons were studied in hippocampal tential, membrane time constant, neuron input resistance, or slices 24 hr after acquisition of trace eyeblink conditioning in action potential characteristics. Furthermore, comparisons be- young adult and aging rabbits. Aging rabbits required signifi- tween CA1 neurons from trace-conditioned aging and trace- cantly more trials than young rabbits to reach a behavioral crite- conditioned young adult rabbits revealed no statistically signifi- rion of 60% conditioned responses in an 80 trial session. Intra- cant differences in postburst AHPs or accommodation, indicating cellular recordings revealed that CA1 neurons from aging control that similar levels of postsynaptic excitability were attained dur- rabbits had significantly larger, longer lasting postburst afterhy- ing successful acquisition of trace eyeblink conditioning, regard- perpolarizations (AHPs) and greater spike frequency adaptation less of rabbit age. These data represent the first in vitro demon- (accommodation) relative to those from young adult control rab- stration of learning-related excitability changes in aging rabbit bits. After learning, both young and aging CA1 neurons exhibited CA1 neurons and provide additional evidence for involvement of increased postsynaptic excitability compared with their respec- changes in postsynaptic excitability of CA1 neurons in both tive age-matched control rabbits (naive and rabbits that failed to learn). Thus, after learning, CA1 neurons from both age groupshad reduced postburst AHPs and reduced accommodation. No Key words: aging; afterhyperpolarization; spike frequency ad- learning-related differences were seen in resting membrane po- aptation; associative learning; hippocampus; in vitro; intracellular Aged animals, including humans, are impaired in a variety of Landfield, 1990). In addition, calcium-dependent synaptic plasticity learning tasks (Zyzak et al., 1995; Thompson et al., 1996). We is altered in aging hippocampal neurons (Norris et al., 1996, 1998; adopted the rabbit eyeblink preparation as a model system in which Shankar et al., 1998), suggesting that one of the consequences of to study neurobiological correlates of aging and associative learning brain aging in mammals may be an impaired ability to modulate (Disterhoft et al., 1977; Akase et al., 1989; Moyer et al., 1990; intracellular calcium (Landfield, 1987; Disterhoft et al., 1994a; Thompson et al., 1992, 1996a; McEchron and Disterhoft, 1999).
Khachaturian, 1994; Thibault and Landfield, 1996).
Aging rabbits require significantly more trials to learn trace eye- Blockade of excess calcium influx has been shown to ameliorate blink conditioning than young adult rabbits (Graves and Solomon, age-related learning deficits. For example, the dihydropyridine 1985; Thompson et al., 1996a). Unlike standard delay conditioning calcium channel antagonist nimodipine facilitates acquisition of (Akase et al., 1989), trace eyeblink conditioning depends not only trace eyeblink conditioning in aging rabbits (Deyo et al., 1989; on brainstem–cerebellar circuitry but also on an intact hippocam- Straube et al., 1990; Kowalska and Disterhoft, 1994). Intravenous pus for successful acquisition (Moyer et al., 1990; Kim et al., 1995).
administration of the same dose of nimodipine that facilitates Trace eyeblink conditioning deficits exhibited by aging rabbits learning also increases spontaneous firing rates of aging rabbit CA1 parallel the deficits observed in hippocampectomized adult rabbits; neurons in vivo (Thompson et al., 1990). Subsequent in vitro studies both groups are profoundly impaired and show inappropriate tim- showed that postsynaptic excitability of aging CA1 neurons can be ing of the few conditioned responses (CRs) elicited during training restored to levels more closely resembling young adult neurons by (Moyer et al., 1990; Thompson et al., 1996a). These data suggest bath application of nanomolar concentrations of nimodipine that at least part of the deficit exhibited by aging rabbits involves (Moyer et al., 1992; Moyer and Disterhoft, 1994). Together, these data suggest that reducing calcium influx in aging CA1 neurons not Hippocampal slices are a valuable tool for studying various only alters their electrophysiological properties but also facilitates aspects of cellular neurophysiology. Using intracellular recordings, the ability of aged animals to learn.
we previously demonstrated that aging rabbit CA1 neurons had Previous studies have demonstrated that ion channels can be both larger postburst afterhyperpolarizations (AHPs) (Moyer et modulated by associative learning (Alkon, 1984; Disterhoft et al., al., 1992) and prolonged calcium action potentials (APs) (Moyer 1986; de Jonge et al., 1990; Woody et al., 1991; Moyer et al., 1996; and Disterhoft, 1994) than young adult neurons. These differences Thompson et al., 1996b; Saar et al., 1998). For example, learning- in calcium-mediated processes are similar to those observed in specific reductions of the calcium-dependent slow AHP have been aging rat CA1 neurons (Landfield and Pitler, 1984; Pitler and observed in CA1 and CA3 neurons after acquisition of hippocam- pally dependent trace eyeblink conditioning (Moyer et al., 1996; Received March 6, 2000; revised April 18, 2000; accepted April 25, 2000.
Thompson et al., 1996b). Furthermore, the reduced AHPs ob- This work was supported by National Institutes of Health Grants RO1 MH47340, served after learning are transient, decaying back to baseline within RO1 AG08796, and RO1 DA07633 to J.F.D. We thank F. Cutting and J. Hauser for 5–7 d, a time period appropriate for memory consolidation (Moyer Correspondence should be addressed to Dr. James R. Moyer, Jr., Department of et al., 1996; Thompson et al., 1996b). Similarly, reduced AHPs Psychology, Yale University, P.O. Box 208205, New Haven, CT 06520-8205. E-mail: were observed in layer II pyramidal neurons of rat piriform cortex after acquisition of an odor discrimination task (Saar et al., 1998).
Dr. Power’s present address: Department of Neuroscience, Australian National To date, no studies have used intracellular recordings in vitro to University, John Curtin School of Medical Research, Canberra, Australia 2601.
Dr. Thompson’s present address: School of Human Development, GR 4.1, Univer- evaluate learning-related changes in postsynaptic excitability of sity of Texas at Dallas, Richardson, TX 75083.
CA1 neurons in aging animals. To evaluate whether learning- Copyright 2000 Society for Neuroscience 0270-6474/00/205476-07$15.00/0 related changes also occur in aging animals, intracellular current- Moyer et al. • Increased Excitability of Aged CA1 after Learning J. Neurosci., July 15, 2000, 20(14):5476–5482 5477
Figure 1. Aging rabbits are significantly impaired in their ability to learn hippocampally dependent trace eyeblink conditioning. A, A plot of trial to criterion among rabbits that learned illustrates that aging rabbits required significantly more trials than young adult rabbits ( p Ͻ 0.05). B, A plot of percent CRs shows that, among rabbits that learned, young and aging rabbits were not significantly different from each other on either the first or last day of training.
C, Learning curves of rabbits that learned ( filled symbols) and rabbits that did not learn (open symbols). The dashed line represents the behavioral criterion of 60% CRs. The aging rabbits that eventually learned ( filled circles; n ϭ 7) had an average learning curve that was shifted far to the right of young adult rabbits that learned ( filled triangles; n ϭ 6). Notice the relatively poor performance of the slow-learning young adult (open triangles; n ϭ 4) and aging (open circles; n ϭ 5) rabbits.
clamp recordings were made from CA1 pyramidal neurons in slices For recording, slices were individually transferred to a submersion cham- taken from aging rabbits after acquisition of trace eyeblink condi- ber (Medical Systems, Greenvale, NY) and continuously perfused with tioning. These data were compared with data obtained from aging Electrophysiological recordings and data analysis. Intracellular recordings control rabbits that did not learn, from aging naive rabbits, and were made from 97 CA1 pyramidal neurons (50 young, 47 aging) using an similar data from young adult rabbits.
Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) and thin- Parts of this paper have been published previously in abstract walled microelectrodes filled with 3 M KCl (20 –50 M⍀) as described previously (Moyer et al., 1996). All CA1 pyramidal neurons included in this study exhibited little spontaneous activity at rest, had action potential amplitudes Ͼ80 mV from threshold, had action potential durations Ͼ1.2 MATERIALS AND METHODS
msec from rise threshold to recrossing of the resting potential, had input Behavioral training. New Zealand albino rabbits (Oryctolagus cuniculus) resistances Ն25 M⍀, and had stable resting membrane potentials more were purchased from Hazelton Rabbitry (Denver, PA) and maintained in negative than Ϫ60 mV. Cells were studied at membrane potentials near accordance with guidelines established by the United States Department of Ϫ65 mV (using Յ0.2 nA constant current injection, if necessary) to Agriculture and approved and managed by the Animal Care Committee of minimize the influence of voltage-dependent changes on membrane con- Northwestern University. Rabbits received 500 msec trace eyeblink con- ductances. The protocols used to study the properties of CA1 neurons and ditioning as described previously (Moyer et al., 1990; Thompson et al., the analyses of all intracellular data were identical with previously pub- 1996a). Briefly, rabbits were fitted with restraining head bolts and trained lished methods (Moyer et al., 1996). Briefly, current–voltage relationships in pairs in individual sound-attenuated chambers for daily 80 trial sessions were constructed using 400 msec current injections (range, Ϫ1.0 to ϩ0.2 (mean intertrial interval, 45 sec). The CS was a 100 msec, 85 dB, 6 kHz nA). Postburst AHPs were evaluated using a 100 msec depolarizing cur- tone presented via stereo headphones. The unconditioned stimulus (US) rent injection sufficient to elicit a burst of four action potentials. Spike was a 150 msec, 3.5 psi corneal air puff sufficient to elicit reliable extension frequency adaptation (referred to as accommodation in the present study) of the nictitating membrane (NM) (or third eyelid) as the unconditioned was evaluated by injecting the same amount of depolarizing current used response. Because aging rabbits typically fail to acquire this trace eyeblink to study the AHP but for an 800 msec duration. The number of action conditioning task to our usual criterion of 80% CRs in a training session potentials were counted and recorded. To evaluate the distribution of (Thompson et al., 1996a), a behavioral criterion of 60% CRs in a training changes within a population, individual cells were classified as having been session was used for both age groups (all references to learning in the text “changed by conditioning” if its data fell beyond 2 SDs from the mean of refer to acquisition of 60% CRs unless explicitly indicated otherwise). An the population of naive neurons studied in the particular age group (for NM extension was counted as a CR if it occurred after CS onset but before US onset. Slow-learning rabbits (Ͻ30% CRs after 15 sessions) served as an All reported values are the mean Ϯ SEM. Statistical analyses were done additional control population (Disterhoft et al., 1988a; Moyer et al., 1996; using unpaired t tests or ANOVA with the significance level set at 0.05.
Thompson et al., 1996b). Only one young adult rabbit was slow-learning Post hoc comparisons were made using Fisher’s PLSD only if a significant (compared with five aging slow-learning rabbits), so three additional slow- learning young adult rabbits were taken from a simultaneous cohort of behavioral studies and included in the present study. Learning curves for young and aged rabbits were normalized to the mean number of trials required to reach criterion for that group using linear interpolation algo- Aging rabbits are impaired in acquisition of trace
rithms (IgorPro; WaveMetrics, Lake Oswego, OR) (Thompson et al., 1996). Behavioral experiments were controlled by an IBM-PC clone com- eyeblink conditioning
puter using custom hardware and software as described previously (Akase Aging rabbits required 1405 Ϯ 246 trials to reach the behavioral et al., 1994; Thompson et al., 1994).
criterion of 60% CRs within an 80 trial trace conditioning session Slice preparation. Twenty-four hours after the last training session, rabbits were deeply anesthetized with halothane, and 400 ␮m hippocampal compared with 733 Ϯ 138 trials for young adult rabbits (t11 ϭ 2.276; slices were cut on a vibratome as described previously (Moyer et al., 1996; p Ͻ 0.05) (Fig. 1A). Comparisons between trace-conditioned Thompson et al., 1996a). For this study, 17 young adult rabbits (mean age, young and aging rabbits revealed no statistically significant differ- 2.2 Ϯ 0.1 months) and 19 aging rabbits (mean age, 42.3 Ϯ 1.3 months) were ences in percent CRs on the first (young, 7.5 Ϯ 1.9; aging, 4.1 Ϯ 1.9; used. Hippocampal slices were maintained in a holding chamber filled with 11 ϭ Ϫ1.27; p ϭ 0.23) or the last ( young, 69.8 Ϯ 2.6; aging, 68.6 Ϯ 2PO4, 2.4 CaCl2, 26 NaHC O3, and 10 D-glucose, gassed with 95% 11 ϭ Ϫ0.439; p ϭ 0.67) day of training. Learning curves O2–5% CO2 at pH 7.4) at room temperature (ϳ23°C) for at least 45 min.
constructed from the slow-learning (Ͻ30% CRs after 15 sessions) 5478 J. Neurosci., July 15, 2000, 20(14):5476–5482
Moyer et al. • Increased Excitability of Aged CA1 after Learning Table 1. Summary of learning-related changes in CA1 neurons from young and aged rabbits
Postburst after hyperpolarization (n) aNeurons from aged experimentally naive rabbits were significantly different from those of young naive rabbits (AHP amplitude, p Ͻ 0.005; AHP area, p Ͻ 0.01; AHP duration, p Ͻ 0.05; number of spikes, p Ͻ 0.01).
Significantly different from neurons of both age-matched control groups (naive and slow learners): *p Ͻ 0.05, ‡p Ͻ 0.01, §p Ͻ 0.001. Numbers in parentheses (n) indicate the ratio of individual cells with reduced AHPs or accommodation to the number of cells studied in that group.
Table 2. Properties of CA1 neurons from young and aged rabbits that do not change after acquisition of trace eyeblink conditioning
Action potential characteristics (n)a aAntidromic action potentials were used to allow for accurate width measurements without interference from the underlying depolarizing potential that occurs using current injection-evoked or orthodromically driven action potentials.
and trace-conditioned rabbits clearly illustrate the poor perfor- al., 1996b). CA1 neurons from experimentally naive aging rabbits mance of the slow-learning rabbits from both groups (Fig. 1C, open fired significantly fewer action potentials in response to an 800 msec symbols) compared with rabbits that learned the task (Fig. 1C, filled depolarizing current injection (t26 ϭ Ϫ2.988; p Ͻ 0.01) (Table 1, symbols). Previous studies have shown that slow-learning rabbits naive data) than did young control neurons. These data are con- serve as an excellent control group indistinguishable from sistent with previous observations of greater accommodation in pseudoconditioning rabbits (Disterhoft et al., 1988b; Moyer et al., aging rabbit CA1 neurons compared with young adult neurons 1996). The learning curves clearly show that, although the aging (Moyer et al., 1992; Oh et al., 1999).
rabbits were ultimately able to achieve a similar level of perfor- No statistically significant differences were observed between mance, throughout training they showed fewer CRs, and they took young and aging neurons in resting membrane potential, input nearly twice as long to reach the behavioral criterion of 60% CRs resistance, time constant, or action potential characteristics (Table 2, naive data). In addition, the amount of current injection required to elicit a burst of four action potentials (used to study the postburst Aging CA1 neurons exhibit decreased postsynaptic
AHP) did not vary as a function of age (aging, 0.70 Ϯ 0.05 nA; excitability compared with young adult CA1 neurons
Postburst AHPs of CA1 neurons from experimentally naive aged 26 ϭ 0.454; p ϭ 0.65). Analyses of within- burst firing also indicated no statistically significant differences rabbits were significantly larger than those from young adult neu- between aging and young adult neurons. Latencies from current rons. Aging CA1 pyramidal neurons had AHPs that were signifi- onset to each of the four APs elicited during the 100 msec current cantly larger in amplitude (t26 ϭ Ϫ3.199; p Ͻ 0.005), integrated step used to study the postburst AHP were calculated. No statisti- area (t26 ϭ Ϫ2.871; p Ͻ 0.01), and duration (t26 ϭ 2.068; p Ͻ 0.05) cally significant differences were observed in each of the following: than young adult CA1 neurons (Table 1, naive data). The enhanced (1) latency to the first AP (mean, ϳ5.4 msec; t AHPs observed in aging neurons were similar to previous reports 0.93); (2) latency to the second AP (mean, ϳ18 msec; t of age-related changes in the AHP (Landfield and Pitler, 1984; p ϭ 0.63); (3) latency to the third AP (mean, ϳ40 msec; t Moyer et al., 1992). The postburst AHP is primarily comprised of Ϫ0.901; p ϭ 0.38); or (4) latency to the fourth AP (mean, ϳ71 an outward, calcium-activated Kϩ current that modulates postsyn- aptic excitability of many cell types, including hippocampal and cortical pyramidal neurons (Hotson and Prince, 1980; Gustafsson Acquisition of trace eyeblink conditioning increased
and Wigstro¨m, 1981; Lancaster and Adams, 1986; Schwindt et al., excitability of aging and young adult CA1 neurons
Postburst AHPs were significantly reduced in both young and aging Spike frequency adaptation or accommodation is another mea- CA1 neurons after acquisition of hippocampally dependent trace sure of postsynaptic excitability (Madison and Nicoll, 1984; Hed- eyeblink conditioning. ANOVA indicated that the effects of learn- lund and Andersen, 1989; Moyer et al., 1992, 1996; Thompson et ing were statistically significant for amplitude (young, F(3,46) ϭ Moyer et al. • Increased Excitability of Aged CA1 after Learning J. Neurosci., July 15, 2000, 20(14):5476–5482 5479
Figure 2. Acquisition of hippocampally dependent trace eyeblink conditioning increased excitability of aging rabbit hippocampal CA1 pyramidal neurons.
A, Effects of trace conditioning on the size of the postburst AHP. 1, Overlay of voltage recordings of the postburst AHP in CA1 neurons from an aging naive rabbit (Naive), an aging rabbit that showed Ͻ15% CRs after 15 sessions (Slow), and an aging trace-conditioned rabbit (Trace). The resting membrane potentials of these cells were approximately Ϫ65 mV, with action potentials truncated for visualization of the AHP. The AHP was measured for 5 sec beginning after a 100 msec depolarizing current injection (solid black line), with minimal current (ϳ0.6 nA) required to reliably evoke a burst of four action potentials. 2, Mean effects of trace eyeblink conditioning on postburst AHP amplitude in aging rabbit CA1 neurons. Notice that, after learning, the AHP was significantly reduced compared with naive and slow-learning aging controls. B, Typical examples of accommodation responses in CA1 pyramidal cells from aging naive (Naive), aging slow-learning (Slow), and aging trace-conditioned (Trace) rabbits. Although the cell from the trace-conditioned rabbit fired more action potentials, accommodation was not abolished, as evidenced by the increase in interspike interval with time during the 800 msec depolarizing stimulus (solid black line), but rather was significantly reduced after learning. The resting potentials of these cells were approximately Ϫ67 mV.
21.100, p Ͻ 0.0001; aging, F(3,43) ϭ 35.382, p Ͻ 0.0001), integrated p ϭ 0.45; aging, F(3,43) ϭ 0.752, p ϭ 0.53), third (young, F(3,46) ϭ area (young, F(3,46) ϭ 11.561, p Ͻ 0.0001; aging, F(3,43) ϭ 10.780, 0.854, p ϭ 0.47; aging, F(3,43) ϭ 0.73, p ϭ 0.54), or fourth (young, p Ͻ 0.0001), and duration (young, F(3,46) ϭ 3.427, p Ͻ 0.05; aging, F(3,46) ϭ 1.09, p ϭ 0.36; aging, F(3,43) ϭ 1.296, p ϭ 0.3) action F(3,43) ϭ 4.127, p Ͻ 0.01) of the AHP (Table 1). An examination of potential within each burst were not significantly different after individual neurons indicated that, after learning, 6 of 15 (40%) acquisition of trace eyeblink conditioning.
young adult and 17 of 19 (89%) aging neurons had significantly After learning, CA1 neurons from both young adult and aging reduced AHP amplitudes relative to data from age-matched naive rabbits showed less accommodation than their age-matched control controls (Table 1). Previous reports of learning-specific AHP re- groups (Table 1). ANOVA revealed that significantly more action ductions in CA1 neurons involved only the use of young adult potentials were elicited after acquisition of trace eyeblink condi- rabbits (Disterhoft et al., 1986; Coulter et al., 1989; de Jonge et al., tioning (young, F(3,46) ϭ 7.232, p Ͻ 0.001; aging, F(3,43) ϭ 10.606, 1990; Moyer et al., 1996). The present study, however, evaluated p Ͻ 0.001). Figure 2B clearly shows this effect in aging neurons.
whether learning-related changes in postsynaptic excitability of Notice that neurons from experimentally naive and slow-learning CA1 neurons are also observed in aging rabbits. Figure 2 shows the rabbits exhibited robust accommodation, whereas neurons from effects of trace eyeblink conditioning on measures of postsynaptic trace-conditioned rabbits fired more action potentials.
excitability of aging CA1 pyramidal neurons. The voltage traces The learning-related changes in postsynaptic excitability (AHP shown in Figure 2A clearly illustrate the reduced size and duration and accommodation) were present in the absence of any statisti- of the AHP in CA1 neurons from aging rabbits that reached the cally significant changes in resting membrane potential, time con- criterion of 60% CRs in a session relative to CA1 neurons from stant, input resistance, or action potential characteristics (Table 2).
aging control rabbits (naive and slow-learning).
This was true for CA1 neurons from both young adult and aging The learning-related changes in size and duration of the AHP did not result from differences in current required to elicit the burst of four action potentials used to study the postburst AHP. ANOVA DISCUSSION
indicated that, for both young adult and aging neurons, the current Aging rabbits were significantly slower than young adult rabbits in required to elicit four action potentials did not vary as a function of acquiring the trace eyeblink conditioning task. CA1 neurons from training condition (young, F(3,46) ϭ 0.633, p ϭ 0.6; aging, F(3,43) ϭ aging naive rabbits had larger AHPs and exhibited more accom- 0.404, p ϭ 0.75). Also, there were no differences in within- modation relative to neurons from young naive rabbits. After burst firing in either age group as a function of training condi- learning, both the postburst AHP and spike frequency accommo- tion. Latencies to the first ( young, F(3,46) ϭ 0.228, p ϭ 0.88; dation were significantly reduced in a learning-specific manner in aging, F(3,43) ϭ 1.126, p ϭ 0.35), second ( young, F(3,46) ϭ 0.903, CA1 neurons from young adult and aging rabbits. These data 5480 J. Neurosci., July 15, 2000, 20(14):5476–5482
Moyer et al. • Increased Excitability of Aged CA1 after Learning represent the first evaluation of learning-related changes in aging rabbits had AHP amplitudes that were 39.6% smaller after acqui- rabbit CA1 neurons using intracellular recordings in vitro and sition of trace eyeblink conditioning than control neurons with 40% implicate changes in postsynaptic excitability of hippocampal neu- of the neurons exhibiting reduced AHPs (Table 1). When given a rons in both aging and associative learning.
long depolarizing current injection, young adult CA1 neurons fired Aging rabbits were clearly impaired in their ability to acquire the 41.7% more action potentials after learning than did control neu- trace eyeblink conditioning task (Fig. 1), consistent with previous rons. Such changes were not observed in CA1 neurons from aging observations of impaired learning ability in aging rabbits (Graves or young rabbits that showed fewer than 30% CRs after 15 training and Solomon, 1985; Deyo et al., 1989; Solomon and Groccia- sessions (Table 1, Slow learners), suggesting that the effects were Ellison, 1996; Thompson et al., 1996a). The aging rabbits required learning-specific, as previously observed in CA1 and CA3 neurons nearly twice as many training trials than did young adult rabbits to from young adult rabbits (Moyer et al., 1996; Thompson et al., reach a behavioral criterion of 60% CRs in a session. Of the 13 aging rabbits that received trace eyeblink conditioning, only eight Previous in vitro studies only evaluated the electrophysiological were able to reach criterion, whereas the other five remained below properties of aging rabbit CA1 neurons in experimentally naive 30%, even after 15 training sessions. Of the eight aging rabbits that animals (Moyer et al., 1992; Moyer and Disterhoft, 1994; Oh et al., were able to learn, only two did so at a rate similar to that seen in 1999). The present data show that, although CA1 neurons from young rabbits. These observations are consistent with previous data aging control rabbits had larger AHPs and stronger accommoda- indicating substantial heterogeneity of learning ability among pop- tion than neurons from young adult controls, aging rabbits that ulations of aging rabbits receiving trace eyeblink conditioning learned the trace eyeblink conditioning task had AHPs that were (Thompson et al., 1996a). In addition, the inability of the aging significantly reduced relative to aging controls. In fact, after acqui- rabbits probably did not result from an inability to process CS and sition of trace conditioning, AHPs from aging rabbit CA1 neurons US information because animals switched to the delay conditioning were reduced to a size that was similar to that observed in the task learn within several training sessions (Thompson et al., 1996a).
young adult rabbits after learning (Table 1). This latter point is Hippocampal CA1 neurons recorded from experimentally naive quite interesting because it suggests that a similar level of postsyn- aging rabbits had significantly larger, longer lasting postburst AHPs aptic excitability must be attained for successful acquisition of trace (Table 1), consistent with previous reports of decreased postsyn- conditioning, independent of the age of the animal. The actual aptic excitability of aging rabbit and rat CA1 neurons (Landfield differences between aging control and aging conditioned neurons and Pitler, 1984; Moyer et al., 1992). After a burst of action were much greater than those between young control and young potentials, the larger, longer lasting AHPs of aging CA1 neurons conditioned neurons (Table 1). That is, the aging neurons had to could act to dampen the impact of excitatory inputs for several change more than the young adult neurons to achieve the same seconds (the duration of the AHP). Thus, a barrage of excitatory level of postsynaptic excitability (e.g., similarly sized AHPs). The inputs onto an aging CA1 neuron during the AHP would be less greater change required for an aging neuron to reach the condi- likely to drive the cell to threshold than if the cell was at or near its tioned state may partly underlie the need for aging rabbits to resting membrane potential. The larger AHPs observed in aging receive significantly more training trials to successfully learn the CA1 neurons could result from an excess influx of calcium during trace eyeblink conditioning task. These data provide strong support depolarization because bath application of nanomolar concentra- for a correlation between changes in postsynaptic excitability, tions of the L-type calcium channel antagonist nimodipine effec- learning, and aging-related learning deficits.
tively eliminates the aging-related increase (Moyer et al., 1992).
In the present study, rabbits were trained to a behavioral crite- Additional evidence implicating calcium or calcium-dependent rion of 60% CRs in an 80 trial session. When rabbits were trained processes in aging comes from work demonstrating that aging CA1 to the more difficult criterion of 80% CRs, there were little addi- neurons have prolonged calcium action potentials (Moyer and tional increases in CA1 postsynaptic excitability (Table 1). Al- Disterhoft, 1994) and larger calcium currents (Landfield, 1996) though only one aging rabbit was able to reach 80% CRs, three of compared with young adult neurons. Preliminary data from whole- the four cells recorded from this aging rabbit had reduced AHPs, cell voltage-clamp experiments also suggest that the calcium- and the mean amplitude was basically the same as those trained to activated potassium current underlying the slow AHP is enhanced a criteria of 60% CRs (Ϫ3.06 Ϯ 0.5 vs Ϫ3.01 Ϯ 0.2 mV, respec- in aging rabbit CA1 neurons (Power et al., 1999).
tively). The data from young adult rabbits indicated that, when In addition to the enhanced AHPs, CA1 neurons from aging trained to a criterion of 80% CRs, their CA1 neurons had a mean control rabbits also exhibited more robust accommodation during a AHP amplitude that was only slightly smaller than those trained to long depolarizing current injection than young adult neurons (Ta- a criteria of 60% CRs (Ϫ2.5 Ϯ 0.3 compared with Ϫ3.02 Ϯ 0.2 mV, ble 1) (Moyer et al., 1992). This latter observation suggests that, respectively). Similarly, there was a slight change in accommoda- even when aging CA1 neurons reach threshold, they are less likely tion as a result of using a behavioral criterion of 80% CRs. On to exhibit a sustained firing pattern in response to a continuous average, in both age groups, training to a criterion of 80% CRs stream of inputs. That CA1 neurons from experimentally naive resulted in an increase of approximately one action potential dur- aging rabbits exhibited both larger AHPs and more robust accom- ing accommodation versus that seen when rabbits were trained to a modation than neurons from young adult rabbits is not surprising 60% criterion (Table 1). Interestingly, there were no statistically because modulation of the AHP by intracellular calcium or neu- significant differences in AHP amplitude, AHP area, AHP dura- rotransmitters typically alters accommodation (Schwartzkroin and tion, or accommodation between young or aging CA1 neurons from Stafstrom, 1980; Cole and Nicoll, 1983; Haas and Greene, 1984; either trace-conditioned group. When compared with data from a Hedlund and Andersen, 1989; Oh et al., 1999; Weiss et al., 2000).
previous study in which young adult rabbits were trained to a After learning, CA1 neurons from aging rabbits had postburst criterion of 80% CRs, the AHP and accommodation data observed AHPs that were 52.5% smaller in amplitude than those from aging 24 hr after acquisition (Moyer et al., 1996) were similar to the data control rabbits (Table 1). Inspection of individual neurons indi- obtained in young adult CA1 neurons after conditioning to 80% cated that, after acquisition, 89% of the aging CA1 neurons exhib- CRs in the present study. These data suggest that the AHP reduc- ited reduced AHPs. These effects were observed on the amplitude, tions were nearly maximal when aged rabbits were trained to a the integrated area, and the duration of the AHP. In response to behavioral criterion of 60% CRs. However, in young adult rabbits, long depolarizing current steps, CA1 neurons from aging rabbits further reductions of the AHP occurred with additional training to fired 76.9% more action potentials after learning than did neurons from aging control rabbits (Table 1). These data indicate that Additional support for involvement of changes in postsynaptic acquisition of trace eyeblink conditioning in aging rabbits was excitability of hippocampal CA1 neurons with learning and aging accompanied by increased postsynaptic excitability of CA1 pyrami- comes from studies in which compounds that reduce both the AHP dal cells. In addition, CA1 pyramidal neurons from young adult and accommodation were given to young adult or aging animals.
Moyer et al. • Increased Excitability of Aged CA1 after Learning J. Neurosci., July 15, 2000, 20(14):5476–5482 5481
For example, administration of the L-type calcium channel Haas H, Greene R (1984) Adenosine enhances AHP and accommodation antagonist nimodipine facilitates acquisition of trace eyeblink in hippocampal pyramidal cells. Pflu¨gers Arch 402:244–247.
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tions of nimodipine reduce both the AHP and accommodation in Hotson JR, Prince DA (1980) A calcium-activated hyperpolarization fol- aging rabbit CA1 neurons in vitro (Moyer et al., 1992). Similar lows repetitive firing in hippocampal neurons. J Neurophysiol effects have also been observed in aging rabbits treated with cho- Khachaturian ZS (1994) Calcium hypothesis of Alzheimer’s disease and linesterase inhibitors and muscarinic agonists (Kronforst-Collins et brain aging. Ann NY Acad Sci 747:1–11.
al., 1997; Oh et al., 1999; Weiss et al., 2000). Compounds that Kim JJ, Clark RE, Thompson RF (1995) Hippocampectomy impairs the directly enhance the postburst AHP have not been tested in eye- memory of recently, but not remotely, acquired trace eyeblink condi- blink conditioning, but the aforementioned data suggest that such tioned responses. Behav Neurosci 109:195–203.
Kowalska M, Disterhoft JF (1994) Relation of nimodipine dose and serum concentration to learning enhancement in aging rabbits. Exp Neurol Increased postsynaptic excitability appears to be one mechanism used by hippocampal neurons in both young and aging animals for Kronforst-Collins MA, Moriearty PL, Schmidt B, Disterhoft JF (1997) acquisition of trace eyeblink conditioning. Previous data from Metrifonate improves associative learning and retention in aging rabbits.
young rabbits demonstrated that changes in hippocampal excitabil- Lancaster B, Adams PR (1986) Calcium-dependent current generating the ity were transient, lasting 5–7 d after acquisition to a criterion of afterhyperpolarization of hippocampal neurons. J Neurophysiol 80% CRs in a session (Moyer et al., 1996; Thompson et al., 1996b).
Although in the present study postsynaptic excitability of aging Landfield PW (1987) “Increased calcium current” hypothesis of brain ag- rabbit CA1 neurons was similar to young neurons after learning, it Landfield PW (1996) Aging-related increase in hippocampal calcium is unknown whether the increased excitability seen in aging neu- rons would last as long as those seen in young adult neurons. The Landfield PW, Pitler TA (1984) Prolonged Ca 2ϩ-dependent afterhyper- current study was not designed to address this issue, but data from polarizations in hippocampal neurons of aged rats. Science aging rats suggest that memory consolidation (Oler and Markus, Madison DV, Nicoll RA (1984) Control of the repetitive discharge of rat 1998) and information processing (Barnes et al., 1997; Tanila et al., CA1 pyramidal neurones in vitro. J Physiol (Lond) 354:319–331.
1997) are significantly impaired in aging rats.
McEchron MM, Disterhoft JF (1999) Hippocampal encoding of nonspa- In conclusion, the present study is the first to report learning- tial trace conditioning. Hippocampus 9:385–396.
related excitability changes in aging CA1 neurons. These data Moyer Jr JR, Disterhoft JF (1994) Nimodipine decreases calcium action potentials in an age- and concentration-dependent manner. Hippocam- provide additional support for the hypothesis that alterations in postsynaptic excitability are involved in both aging and associative Moyer Jr JR, Deyo RA, Disterhoft JF (1990) Hippocampectomy disrupts trace eye-blink conditioning in rabbits. Behav Neurosci 104:243–252.
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