Eyeblink conditioning deficits in the old cat

Neurobiologyof Aging, Vol. 4, pp. 45-51, 1983. ©Ankho International. Printed in the U.S.A.

Eyeblink Conditioning Deficits in the Old Cat JEAN HARRISON

AND JENNIFER

BUCHWALD

Brain Research Institute, Mental Retardation Research Center, Department o f Physiology UCLA Medical Center, Los Angeles, CA 90024 R e c e i v e d 29 D e c e m b e r 1982 HARRISON, J. AND J. BUCHWALD. Eyeblink conditioning deficits in the old cat. N EUROBIOL AGING 4(1) 45--51, 1983.mAcquisitinn of a conditioned eyeblink reflex was studied in aged cats 10-23 years old and young cats one to three years old. All ten young cats became conditioned within 1,000 trials, after a mean of 270 trials, with a stimulus protocol including a 1500 msec 4 kHz tone CS+ followed by a shock unconditioned stimulus, and click C S - . Nine of 15 aged cats failed to become conditioned with 1,000 trials. Six old cats became conditioned after a mean of 522 trials. Four aged cats tested with a simpler protocol involving a 400 msec CS+ and no click C S - became conditioned with less than 500 trials. All retained the conditioned response when clicks were added to the 400 msec CS+. Three aged animals which failed to become conditioned with the 1500 msec CS+ and clicks were trained with an 800 msec CS+ without clicks. Only one became conditioned within 500 trials. Thus duration of the tone CS+ was critically important for acquisition of a conditioned eyeblink response by aged cats. Conditioned eyeblink reflex

Aged cats

Young adult cats

CS-US interval

to the hippocampus, and lesioned animals exhibited electrophysiological changes in the hippocampus as well as impaired acquisition o f a maze task [19,28]. The foregoing studies indicate a variety o f learning deficits in aged mammals, and, taken together, suggest that learning deficits become more apparent as the difficulty of the task increases, e.g., with increasingly difficult discriminations or prolonged intervals between cue and reinforcement. In cases in which acquisition proceeds relatively normally, intermediate or long-term retention may appear markedly abnormal. The present study grew out o f an experiment in which auditory conditioning procedures were utifized in conjunction with recordings o f endogenous e v o k e d responses in aged and young cats [10]. In order to maintain the cat's attention on the auditory modality during recordings for endogenous responses to clicks, a tonal conditioning stimulus (CS) was paired with an eyelid shock unconditioned stimulus (US). To our surprise, many of the old cats appeared incapable o f acquiring the conditioned eyeblink responses which were shown by all the young cats. The present study evolved from these initial observations, therefore, as the first in what we hope to be a series o f investigations o f learning deficits shown by the aged Oat during classical conditioning procedures.

L E A R N I N G deficits have been reported in a number of mammalian species as a function of the aging process. These deficits have been described behaviorally in terms of abnormal acquisition and/or abnormal retention of the task to be acquired. Mice aged 6-15 months have slower acquisition of shuttlebox avoidance compared to mice aged two-five months [8,22], and aged mice have deficits in retention of a passive avoidance task [3]. Old rats have deficits both in acquisition and in retention o f visual discrimination and maze tasks [1,24] and other studies show normal acquisition but diminished retention in aged rats [9, 13, 23]. Old monkeys (18 years and older) can reproduce patterns after a short delay as well as middle aged (14 years) monkeys, but the old monkeys are much worse with long delays, even when deficits in motivation, attention, and stimulus processing ability a r e carefully controlled [4,16]. Aged monk e y s can learn visual discriminations o f varying difficulty as well as young controls, but are severely impaired in reversal learning [2]. Middle aged (14 years) and old (21 years) m o n k e y s learn concurrent discriminations equally well the first day, but the old monkeys retain much less after 24 hours [17]. A variety o f experiments to localize brain systems particularly involved in the acquisition and retention deficits of aging have been carried out utilizing electrophysiological recordings, brain stimulation and lesion procedures. Deficits in retention o f an avoidance task by aged rats have been correlated with deficits in frequency potentiation and posttetanic potentiation o f the hippocampus [13,14]. A similar correlation was found in hippocampal slices [14]. Deficient maze retention b y aged rats has been correlated with deficient hippocampai long-lasting enhancement [1]. The medial septal a r e a o f rats was lesioned to interrupt cholinergic input

METHOD Conditioned eyeblink responses were studied in 10 young adult cats o f documented age, ranging from one to three years, and in 15 old cats with ages ranging from 10 to 23 years, documented by veterinarian o r o w n e r records. In studies o f aged animals confounding variables such as dis-

45

46 ease, hormonal depletion (in neutered subjects), and environmental effects (home versus laboratory rearing) may result in interpretive difficulties. We have attempted to be sensitive to these difficulties. The old animals used in this study were routinely monitored by staff veterinarians of the U C L A vivarium facility. The old cats appeared healthy, generally alert, and showed exploratory behavior in the laboratory. All cats oriented to auditory stimuli, their external ear canals were clean, and the tympanic membranes appeared intact under otoscopic examination. During conditioning sessions, the cats were restrained in a canvas cat bag and placed at a constant location in a sound isolation chamber (Industrial Acoustics). EMGs were recorded at the onset of conditioning as well as when CRs began to appear and at the end of a particular training protocol. Bipolar recordings were made from subcutaneous E M G needle electrodes placed in the orbicularis oculi muscle just ventral to the left orbital margin. The EMG was amplified 50,000x with a filter setting of 0.1 Hz to 3 kHz, recorded on a Hewlett-Packard 3964A tape recorder and averaged off-line, after rectification and integration, by a PDP 11/10 computer. EMGs accompanying CS+ and C S - presentations were averaged separately in 25 trial blocks. In addition, the E M G for single trials was monitored on-line on a Tektronix 5BI2N storage oscilloscope tri~Jzered by each auditory stimulus. Presence or absence of EMG activity and latency were scored for each CS+ and C S - trial. Eyeblink responses were also monitored behaviorally through a one-way mirror. Acoustic stimuli were-delivered free-field through a speaker centered 33 cm from the ears. Sound intensity was measured at the external acoustic meatus with a calibrated Bruel and Kjaer 4144 condenser microphone. The CS+ was a tone burst of 4 kHz, 96 dB SPL with a duration of 1500 msec, 800 msec or 400 msec generated by a gated Wavetek 134 oscillator. The C S - were loud and soft rarefaction clicks generated by 0.1 msec square waves from two Grass S-88 stimulators. Intensity was adjusted by Hewlett-Packard 350D attenuators. Loud clicks, 30 dB above each individual c a t ' s threshold for auditory brainstem responses (ABRs) were randomly varied with soft clicks 15 dB above ABR threshold. A B R recordings were carried out utilizing EMG pins in the skin at the vertex, skin of the neck at the midline and at the left bulla for the active, reference and ground electrodes, respectively. E E G activity was amplified 50,000x by a Grass P511 amplifier with bandpass filter settings at 30 Hz to 3 kHz. Responses to C S + and C S - were averaged on-line by a PDP 11/10 computer, with an intersample interval of 50 /~sec over a 10 msec period, and plotted at the end of each training session. (Although clearly defined ABRs could be recorded from the scalp of cats with unrestrained heads, the waveform usually appeared somewhat abnormal compared to ABRs recorded from skull screws in restrained cats.) EEG was continuously monitored from the ABR vertex electrode. In response to the tonal C S + and click C S - , ABRs of average amplitude and waveform were recorded from all cats included in this study. The C S + (paired with the US) was delivered in four 25trial blocks with a randomly varying interstimulus interval averaging 30 sec. The cat was exposed to four blocks per day. The C S - click stimuli were randomly delivered at 1.5 sec intervals so that each 25-trial C S + block also contained 475 C S - d i c k s . The unconditioned stimulus (US) was a 50 msec burst of five 0.1 msec duration shocks delivered through a pair of

H A R R I S O N AND B U C H W A L D PROTOCOLS

FOR CONDITIONED

EYEBLINK

RESPONSE

A. 1500ms CS+ with Loud ¢md Soft CLICK CSCS+

us

CS+

I

!

B. 400ms CS+ with NO CSCS+

CS +

I C. 800ms

us

I

CS+ with NO CS-

I

I Isec

FIG. 1. Protocols A, B and C for establishing the conditioned eyeblink response. The CS+ was in all cases a 4 kHz tone, with the eyelid shock US at the CS termination. Protocol A utilized a 1500 msec CS+ with loud and soft click C S - . Protocol B utilized a 400 msec CS+, and Protocol C an 800 msec CS+.

subcutaneous EMG electrodes just dorsal to the left orbital margin. The voltage was adjusted for each cat so as to elicit a brisk unconditioned blink reflex (UR) without head withdrawal or other visible response. The US occurred at the termination of the C S + . Most o f the observations reported were made during the course o f one of the following training procedures (Fig. I): Protocol A. 1500 msec C S + / U S trials at random 20-30 sec intervals with interspersed loud and soft C S - clicks at 1.5 sec intervals. Protocol B. 400 msec C S + / U S trials at random 20-30 sec intervals without C S - clicks. Protocol C. 800 msec C S + / U S trials at 20-30 sec intervals without C S - clicks. The naive cats often showed a short-latency unconditioned acoustic blink reflex. As they became conditioned, a longer latency sustained EMG response developed during the C S + but did not occur in response to the click C S - . When CRs occurred during at least 80% of the 25 CS + trials per block for two successive blocks, criterion response level was achieved. Training was not extended beyond 1000 trials (10 days) even if CR criterion was not met. RESULTS Protocol A

The initial Protocol A used in this study, which employed a 1500 msec tonal C S + paired with eyelid shock US and randomly interspersed click C S - , resulted in a conditioned eyeblink response (CR) in all ten young cats (four female, six male, mean age two years) studied. The mean number of trials to criterion was 270 (Table l). In Fig. 2, the orbicularis oculi EMG o f one of these animals is illustrated before and after CR acquisition. Among the group of 15 old cats subjected to the same

47

C O N D I T I O N I N G DEFICITS IN T H E OLD CAT

CAT#24

TABLE 1 CONDITIONINGWITHPROTOCOLA (1500msec CS+ and click CS-) Young I0

Total N

N % Total Mean Trials Mean age Sex

2 YRS.

ABRs

Old 15

No CR

CR

No CR

CR

0 0% --

10 100% 270

9 60% 1,000+

6 40% 522

--

2

13

12

--

49,6d"

49 56"

39 36"

eyeblink conditioning procedure, nine (four female, five male, mean age 13 years) failed to develop CRs at criterion level within 1000 trials, when training was terminated. Lack of conditioning was determined by direct observation of the cat through a one-way mirror, observation of each single-trial EMG on a storage oscilloscope, and by observing the averaged EMG for a block of 25 stimuli. Typical orbicularis oculi EMG recordings from one of these old cats after 1000 training trials is shown in Fig. 3. The remaining six old cats (three female, three male, mean age 12 years) developed eyeblink CRs with a mean of 522 trials to criterion, which is a significantly greater number than the mean trials to criterion required by the young cats (p<0.025). There was no significant difference between the ages of the old cats which became conditioned and those which did not (p=0.34, 2-tailed t-test). Within the group of old cats which became conditioned, there was no significant correlation of age with number of trials (correlation=0.46, p =0.36). In all cases, the CS + produced clear auditory brainstem responses (ABRs) and usually elicited an unconditioned, short-latency acoustic blink reflex. The US intensity was adjusted so as to produce a brisk unconditioned blink response in each subject. The EEG remained desynchronized throughout the training sessions in both old and young subjects.

Protocol B Insofar as the old cats showed marked deficits in conditioned association with Protocol A, a subsequent, simpler training procedure was used. The tonal CS+ was shortened from 1500 msec to 400 msec and the click C S - was omitted (Fig. l). Two young cats (one female, one male, mean age two years) which had previously been subjected to Protocol A and had reached criterion as noted above, were trained with Protocol B. They remained conditioned and continued to blink during the 400 msec C S + . Four old cats, one naive and three that had shown no CRs after 1,000 trials of Protocol A, were trained with Protocol B. As illustrated in Fig. 4 (first trace), an unconditioned short-latency acoustic blink reflex was produced by the 400 msec CS initially. After 200 trials, a large sustained CR was elicited by the CS (second trace). All cats (three female, one male, mean age 12 years) attained CR criterion level in less than 500 trials, with a mean of 170 trials (Table 2). The development of CRs by old cats subjected to Protocol B indicated that the CS and US were received centrally and that, under the appropriate training situatiofi, conditioned

15OOms CS÷ CLICK CStrials 1-25

T

CS

US

• ~ & ~2OO-225 ~f l ~ O 1t~

r

i

a

l

s

T

T

cs

us

oo..

FIG. 2. Eyeblink CR acquisition typical of young cats. The top trace shows the rectified EMG recorded from orbicularis oculi averaged over the first 25 presentations of Protocol A. There is no response until the shock US. Auditory brainstem responses to the CS+ are shown in the inset. This cat became conditioned after 75 trials. The bottom trace shows conditioned EMG activity elicited by the CS+, averaged over 25 trials.

CAT # 4 9

13 YRS.

A~RI

1500 ms CS + CLICK C S after

I000 trials

t

CS

US

2OOms

FIG. 3. Deficit in CR acquisition to 1500 msec CS+ typical of many old cats. The rectified EMG is averaged over 25 trials in an old cat which had not become conditioned after 1,000 trials. The small short-latency unconditioned acoustic blink reflex and the ABRs (inset) evoked by the CS+ provide evidence that the cat could hear the CS+. The marked unconditioned response to the shock US indicates effectiveness of the US.

48

HARRISON A N D B U C H W A L D TABLE 2 CONDITIONING WITH PROTOCOL A, B, C

Young

Protocol A. 1500 msec

CS+ and click B. 400 msec CS+ C. 800 msec CS+

Total Cats (N)

CR % Total

10

2 2

Old

Mean Trials

Total Cats (N)

N

No CR % Total

Mean Trials

N

100%

270

15

9

60%

1,000

6

100% 100%

(<25)* (<25)*

4 3

0 2

0% 67%

-500

4 I

CR % Total

4(F~ 100% 33%

Mean Trials 522 170 400

*These cats were already conditioned with Protocol A.

associations could be made. In order to determine the relative influences of CS duration versus C S + / C S - discrimination on the learning deficits accompanying Protocol A, a 400 msec C S + in combination with the click C S - o f Protocol A was subsequently presented to young and old cats. Four young control cats, with conditioned discriminations to Protocol A, similarly showed criterion level CRs to the 400 msec C S + and no CRs to the C S - . Two naive young cats (female, age one year) became conditioned with this combined protocol after a mean of 163 trials. The four aged cats, which did not acquire CRs with Protocol A but showed criterion level CRs to Protocol B, also showed criterion level CRs to the 400 msec C S + and no CRs to the C S - .

AaRs

CAT #71

Lr'~/-~ TRMO0 6$

I0 YRS

400ms.,.CS+ }11

r R zoo-

/ ' 7"//% " US

O 0

Protocol C The preceding data suggested that the old cats' conditioned learning deficits were more related to the interval between onset of C S + and US (i.e., C S + duration) than to C S + / C S - discrimination. This hypothesis was further tested in experiments on three old animals which had previously shown no CRs with 1000 trials of Protocol A. These cats were next trained with Protocol C, which had an 800 msec C S + and no clicks. Two cats (one female, one male, age 10 years) failed to reach criterion in 500 trials. Thereafter, these cats did acquire CRs to Protocol B. The third cat (female, age 13) acquired CRs to Protocol C after 400 trials (Fig. 5, first trace). When CS duration was extended to 1500 msec, however, the CRs diminished (Fig. 5, second trace) and disappeared (Fig. 5, third trace). The three cats which failed to learn Protocol C are in the group which then learned Protocol B. One was then retested with Protocol C. The CS was lengthened to 800 msec, Protocol C, and training was continued (Fig. 4). A CR initially was elicited by the 800 msec CS at the latency of the Protocol B CR (Fig. 4, second and third traces). The CR gradually extinguished (Fig. 4, fourth and fifth traces) although it returned with a return to Protocol B (Fig. 4, bottom trace). DISCUSSION

Taken as a whole, these data indicate a deficiency on the part of old cats in utilizing conditioned stimuli which extend across time periods of 1500 msec, whereas no conditioning deficits occur across 400 msec CS periods. A temporal threshold for conditioned associations in the old cat appears around 800 msec, since old cats unable to acquire CRs with a 1500 msec CS in some but not all cases become conditioned

CS

US

TR1-20

B O O m s CS +

CS

TR.21-40

US

~$

TR.81-100

US

•S 400ms

'~--

\

US

CS +

t

.

.

.

.

~:s us FIG. 4. CR acquisition by aged cat to 400 msec tone CS+ (Protocol B). The top trace with an expanded time base shows the averaged EMG for the first 100 trials. An unconditioned short-latency acoustic blink reflex is followed by absence of activity until the US onset. The unconditioned acoustic reflex and the ABRs (inset) indicate that the CS+ was centrally transmitted. The second trace shows a strong sustained conditioned blink response to the CS+ which appeared after 120 trials. The next 3 traces show extinction of the CR when the CS+ was lengthened to 800 msec (Protocol C). The bottom trace shows rapid re-estabfishment of the CR with Protocol B.

C O N D I T I O N I N G D E F I C I T S IN T H E O L D C A T

CAT #/49 13 YRS.~

ABRs

q/-'~

1500msCS+

1'

1500 ms CS +

C

click.

49

~

US

I|

t.

S

-

T

T

CS

US

'20Ore,~

FIG. 5. CR acquisition by an aged cat to 800 msec CS+ (Protocol C). This cat failed to condition with Protocol A, although the CS+ evoked ABRs (inset), but acquired a conditioned response tO the 800 msec CS+ of Protocol C (first trace). When subsequently tested with a 1500 msec CS+, CRs diminished (middle trace) then disappeared. There were no CRs when the cat was retested with Protocol A (bottom trace).

with an 800 msec CS. Furthermore, CRs established with a 400 msec CS are sometimes extinguished when the CS is extended to 800 msec, and a CR to 800 msec is sometimes extinguished when the CS is extended to 1500 msec. These results contrast sharply with those in young cats, in which 1500 msec CS periods do not preclude conditioned associations and CRs are easily transferred across a range of CS durations. Although all of the old cats appeared alert and healthy and were monitored by staff veterinarians during the course of this study, an initial question with regard to their learning deficits concerns the transmission of the conditioning stimuli into the central nervous system. In the case of the US, .the stimulus intensity was adjusted for each cat so as to produce a brisk, strong blink reflex. This eyebrink reflex was usually displayed by both the stimulated eyelid and the contraiateral eyelid, but other head o r body movements were not induced. Thus, the US was adjusted to be equally effective in central reflex activation o f the eyeblink in the old and young cats. With regard to the acoustic CS, transmission from the periphery into the brainstem was monitored both by ABR and unconditioned acoustic blink reflex measures. In all animals, the conditioned stimuli were above ABR threshold and elicited clearly defined ABR waveforms. Although a significant increase in A B R threshold has been reported for old cats, the parametric response characteristics o f ABRs in old cats, e.g., to stimulus intensity or rate, do not differ from those o f ABRs in young cats [ l l ] . Thus, the presence of A B R s on all CS trials indicated adequate transmission of the stimuli from the periphery through the brainstem auditory pathway.

The unconditioned acoustic brink reflex appeared in the orbicularis oculi E M G recordings as a short latency, short duration response to the acoustic stimuli in the naive cat. While this reflex was not necessarily present on all trials, it was noted in all o f the old as well as in the young cats in response to the CS. The pathway of this reflex has not been well defined but involves neurons in the brainstem auditory pathway which project through one or more interneurons to output motoneurons in the facial nucleus [18,25]. It was present in a decerebrate child, further evidence that this blink response is a brainstem reflex [25]. Our data indicate that this system was functional in the old cat, i.e., the CS was not only transmitted through the brainstem auditory pathway but also through brainstem interneuron reflex connections. These data indicate that the deficient learning of the old cats was not due to inadequate brainstem transmission of either CS or US. Another question which arises is the relative " s t a t e " of the subject during conditioning. Behavioral observations were made continuously during the course o f each training session, which generally was completed within 50 minutes. The old animals did not sleep, i.e., their eyes remained open, and they vocalized and struggled periodically as did the young cats when they became restless. The old cats' E E G was monitored continuously on the oscilloscope, in conjunction with ABR recordings, and remained fast and desynchrunized. At a qualitative EEG and behavioral level, these measures of wakefulness and arousal did not indicate any difference between the old and young cats within the conditioning sessions. Taken together, these observations led us to conclude that the failure of the old cat to learn Protocol A was not a function of deficient CS or US transmission from periphery to brainstem, nor o f the animal "going to s l e e p . " The question then arose as to whether the deficit was one o f acquisition or retention. In order to address this question, an easier learning situation, Protocol B, was used with a CS duration (400 msec) in the optimal range for conditioned associations [12, 27, 29, 30] and without any distracting discrimination stimuli, i.e., the C S - clicks. Old cats which failed in Protocol A and Protocol C readily acquired criterion level CRs with Protocol B. The ability of all the cats presented with Protocol B to acquire a conditioned eyeblink established several points. First, it provided further indication that the CS and US were transmitted into the central nervous system at a level sufficient to support conditioned associations. Second, it supported the E E G and behavioral observations that the animals were awake and receptive. Third, the data indicated that under certain conditions, e.g., Protocol B, CR acquisition occurred, while under other conditions, i.e., Protocol A, it did not. Fourth, maintenance o f the CR from one day to the next indicated that CR retention over this time period was normal in the old cats. From these considerations we concluded that the old cats' inability to learn Protocol A reflected a deficiency in acquisition rather than in retention. In o r d e r to determine whether the long duration C S + or the frequent presentation o f C S - clicks was the cause of the Protocol A learning failure, the 400 msec C S + of Pr0tocol B was presented with the C S - clicks o f Protocol A. The old animals already conditioned with Protocol B, but previously unable to a c q u i r e a CR during 1,000 trials of Protocol A, maintained CRs with this C S + / C S - combination. This result indicated that the C S - click stimuli did not interfere with CR maintenance and suggested that the inability of the old cats to acquire CRs

50

HARRISON AND BUCHWALD

with Protocol A was primarily a function of C S + duration. This hypothesis was further tested by changing the CS duration in training situations involving old cats that had failed with Protocol A (1500 msec CS) and C (800 msec CS). As CS duration was shortened from 800 msec to 400 msec, the CRs appeared at criterion levels. Another aged cat, which had failed to acquire CRs with Protocol A but succeeded with Protocol C, failed to maintain CRs when the CS duration was extended to 1500 msec. The course of this phenomenon resembled extinction insofar as the CR to the new, longer duration CS did not change latency but, rather, disappeared as though no US reinforcement were occurring. In previous work it has been shown that the conditioned eyeblink response can be acquired and retained by cats following surgical removal of the cerebral hemispheres bilaterally, a procedure which includes deletion of the hippocampus as well as all neocortex [21]. A less stable and higher threshold form of the eyeblink CR was also reported to be acquired and maintained in cats chronically decerebrated at the pre-coUicular level [20]. Insofar as the CS durations used in these studies were confined to the 300-400 msec range, the

ability of these truncated preparations to condition to a long duration, e.g., 1500 msec, CS is unknown. Although auditory cortex is not necessary for conditioning, it may normally be involved [18], and there is evidence that the motor cortex is normally involved [26,31], although it is not necessary. However, in the truncated preparations as well as in other studies of eyelid conditioned responses [5, 6, 7], the reticular nuclei of the brainstem, midbrain and thalamus have been implicated as essential substrates for the eyeblink conditioned association. Based on these data, deficient acquisition of eyeblink CRs in the old cats suggests possible dysfunction in the reticular core of the brainstem and midbrain. On the other hand, the temporal threshold of the conditioning deficit, in terms of CS duration, suggests a possible dysfunction in old cats of other structures implicated in sequential and temporal processing. ACKNOWLEDGEMENTS This work was supported by USPHS Grants AG1754 and HD04612. The authors gratefully acknowledge the assistance of George Strecker and Anthony Valdez.

REFERENCES I. Barnes, C. A. and B. L. McNaughton. Spatial memory and hippocampal synaptic plasticity in senescent and middle-aged rats. In: The Psychobiology of Aging: Problems and Perspective, edited by D. G. Stein. Amsterdam: Elsevier/North Holland. 1980, pp. 253-272. 2. Bartus, R. T., R. L. Dean, III and D. L. Fleming. Aging in the rhesus monkey: effects on visual discrimination learning and reversal learning. J Gerontol 34: 209-219, 1979. 3. Bartus, R. T., R. L. Dean, J. A. Goas and A. S. Lippa. Age related changes in passive avoidance retention: modulation with dietary choline. Science 209: 301-303, 1980. 4. Bartus, R. T., D. Fleming and H. R. Johnson. Aging in the rhesus monkey: debilitating effects on short-term memory. J Gerontol 33: 858-871, 1978. 5. Buchwald, J. S., E. S. Halas and S. Schramm. A comparison of multiple-unit and EEG activity recorded from the same brain site in chronic cats during behavioral conditioning. Nature 205: 1012-1014, 1965. 6. Buchwald, J. S., E. S. Halas and S. Schramm. Changes in cortical and subcortical unit activity during behavioral conditioning. Physiol Behav 1: 11-22, 1966. 7. Disterhoft, J. F. and J. S. Buchwald, Mapping learning in the brain. In: Biology of Reinforcement: Facets of BrainStimulation Reward. edited by A. Routennberg. New York: Academic Press, 1980, pp. 53-80. 8. Freund, G. and D. W. Walker. The effect of aging on acquisition and retention of shuttle box avoidance in mice. Life Sci 10: 1343--1349, 1971. 9. Gold, P. E. and J. L. McGaugh. Changes in learning and memory during aging. In: Neurobiology of Aging, edited by J. M. Ordy and K. B. Brizzee. New York: Plenum Press, 1975, pp. 145-158. 10. Harrison, J. B. and J. S. Buchwald. Auditory brainstem and endogenous evoked potentials in aged cats. Soc Neurosci Abstr 7: 452, 1981. i 1. Harrison, J. and J. Buchwald. Auditory braJnstem responses in the aged cat. Neurobiol Aging 3: 163--171, 1982. 12. Kimble, G. A. and B. Reynolds. Eyelid conditioning as a function of the interval between conditioned and unconditioned stimuli. In: Foundations of Conditioning and Learning, edited by G. A. Kimble. New York:. Appleton. 1967, pp. 279-287.

13. Landfield, P. W. Neurobiological changes in hippocampus of aging rats: quantitative correlations with behavioral deficits and with endocrine mechanisms. In: Recent Advances in Gerontology: Proceedings of the XI International Congress of Gerontology. edited by H. Orimo, K. Shimada, M. Iriki and D. Maeda. Amsterdam: Excerpta Medica, 1978, pp. 495-496. 14. Landfield, P. W. and G. Lynch. Impaired monosynaptic potentiation in in vitro hippocampal slices from aged, memorydeficient rats. J Geromol 32: 523-533, 1977. 15. Landfield, P. W., J. L. McGaugh and G. Lynch. Impaired synaptic potentiation processes in the hippocampus of aged, memory-deficient rats. Brain Res 150: 85-101, 1978. 16. Medin, D. L. Form perception and pattern reproduction by monkeys. J Comp Physiol Psychol 68: 412--419, 1969. 17. Medin, D. L., P. O'Neil, E. Smeltz and R. T. Davis. Age differences in retention of concurrent discrimination problems in monkeys. J Gerontol 28: 63--67, 1973. 18. Meting, T. A. Conditioned reflexes in the auditory system. Soviet Research Reports, vol 3, edited by C. D. Woody. Los Angeles: Brain Information Service, UCLA, 1978. 19. Mitchell, S. J., J. N. P. Rawlins, O. Steward and D. S. Olton. Medial septal area lesions disrupt theta rhythm and cholinergic staining in medial entorhinal cortex and produce impaired radial arm maze behavior in rats. J Neurosci 2: 292-302, 1982. 20. Norman, R. J., J. S. Buchwald and J. R. Villablanca. Classical conditioning with auditory discrimination of the eye blink in decerebrate cats. Science 196: 551-553, 1977. 21. Norman, R. J., J. R. Villablanca, K. A. Brown, J. A. Schwafel and J. S. Buchwald. Classical eyeblink conditioning in the bilaterally hemispherectomized cat. Exp Neurol 44: 363--380, 1974. 22. Oliverio, A. and D. Bovet. Effects of age on maze learning and avoidance conditioning of mice. Life Sci 5: 1317-1324, 1966. 23. Ordy, J. M., K. R. Btizzee and W. J. Hansche. Life-span changes in short-term memory in relation to cell loss in the cortex of the rat determined by an automated, quantitative image-analyzing system. Soc Neurosci Abstr 2: 177, 1976. 24. Ray O. S. and R. J. Barrett. Interaction of learning and memory with age in the rat. In: Psychopharmacology and Aging, edited by C. Eisdorfer and W. E. Farm. New York: Plenum Press. 1973. pp. 17-39.

CONDITIONING DEFICITS IN THE OLD CAT 25. Rushworth, G. Observations on blink reflexes. J Neurol Neurosurg Psychiatry 25: 93-108, 1962. 26. Sakai, H. and C. D. Woody. Identification of auditory responsive cells in coronal-pericruciate cortex of awake cats. J Neurophysiol 44: 223-231, 1981. 27. Spooner, A. and W. N. Kellog. The backward conditioning curve. Am J Psychol 60: 321-334, 1947. 28. Winston, J. Loss of hippocampal theta rhythm results in spatial memory deficit in the rat. Science 201: 160-163, 1978.

51 29. Wolfe, H. M. Time factors in conditioned finger withdrawal. J Gen Psychol 4: 372-378, 1930. 30. Wolfe, H. M. Conditioning as a function of the interval between the conditioned and original stimulus. J Gen Psychol 7: 80-103, 1931. 31. Woody, C. D. Aspects of the electrophysiology of cortical processes related to the development and performance of learned motor responses. Physiologist 17: 49-69, 1974.