Modulation of memory processing by neuropeptide Y varies with brain injection site

Brain Research, 503 (1989) 73-82

73

Elsevier BRES 14995

Modulation of memory processing by neuropeptide Y varies with brain injection site James E Flood !"3, Margaret L. Baker t, Ernesto N. Hernandez t and John E. Morley ~'4 1Psychobiology Research Laboratory and 2GeriatricResearch, Education and Clinical Center, VeteransAdministration Hospital, Sepulveda, CA 91343 (U.S.A.) and Departments of 3Psychiatryand Biobehavioral Sciences and 4Medicine, School of Medicine, University of California, Los Angeles, CA 90024 (U.S.A.) (Accepted 2 May 1989)

Key words: Neuropeptide; Neuropeptide Y; Memory; Mouse; Recall; Retention; Localization; Forebrain

Neuropeptide Y (NPY) i.~a 36 amino acid peptide which was shown to enhance memory retention, recall and prevent amnesia induced by either scopolamine or anisomycin. In this study, we examined the effects of NPY administration into 6 areas of the mouse brain on memory retention for footshock avoidance training in a T-maze. NPY was injected into the rostrai and caudal hippocampus, amygdala, caudate, septum and thalamus shortly after training. NPY improved retention when injected into the rostrai portion of the hippocampus and septum, impaired retention in the caudal portion of the hippocampas and amygdala and had no effect in the thalamus and caudate. NPY was ineffectiveat either improving or impairing retention when injected 24 h after training, thus demonstrating that the effects of NPY on retention were time-dependent and not due to proactive effects on retention test performance per so. In addition, NPY had no effect on retention when injected into overlying cortical areas. NPY antibody impaired retention when administered into the rostral hippocampus and septum; it improved retention in the caudal hippocampus and amygdala. Thus NPY antibody had the opposite effect to that of NPY on memory retention suggesting that NPY has a physiological role as a modulator of memory processing within specific anatomical areas of the central nervous system.

INTRODUCTION I n 1982, Tatemoto so isolated and sequenced neuropeptide Y (NPY) from extracts of pig brain using a chemical assay for the C-terminal amide fragments. NPY occurs in very high concentrations in the central nervous system L 8,25. Recently, we demonstrated that NPY administered into the third ventricle of the brain modulated memory retention for T-maze foot-shock avoidance and stepdown passive avoidance training in a dose- and timed e p e n d e n t m a n n e r ~7. NPY was also found to improve recall without affecting acquisition. NPY prevented amnesia induced by an anticholinergic, scopolamine, and a protein synthesis inhibitor, anisomycin. High concentrations of NPY immunoreactive cell bodies occur in the amygdala and hippocampus25. The highest concentrations of NPY binding sites are in the hippocampus 5. Both the hippocampus and the amygdala are implicated as part of the anatomical sabstrate for learning and memory by studies in wldch low levels of electrical stimulation, lesions or drug administration altered retention t2.2°-e4,4t'43"54. In the studies reported here, we have characterized the effects o n memory processing of local injections of NPY into the hippocam-

pus, amygdala, septum, caudate and thalamus. In addition, we examined the effect of local injections of NPY antibodies on retention. These studies suggest a physiological role for NPY in the modulation of memory processing within specific areas of the central nervous system. MATERIALS AND METHODS

Subjects mid drugs After 1 week in the laboratory, CD-I male mice obtained at 6 weeks of age from Charles River Breeding Laboratories, Wilmington, MA, were individually caged 24-48 h prior to training and remained singly housed until retention was tested 1 week later. The median body weight was 35 g, with a range of 33-38 g. Animal rooms were maintained on a 12 h light-dark cycle with light on at 06.00 h. The mice were trained between 07.00 and 15.00 h. Mice were assigned randomly to groups of 15 unless otherwise indicated. Neuropeptide Y, porcine (NPY, F.W. 4254.21 80% peptide by weight) was purchased from Peninsula Laboratories, Inc., Belmont CA. NPY was either freshly prepared in saline or used after 1 freezing of no longer than 1 week. Rabbit anti-neuropeptide Y serum (aNPY, Lot 009026-4, 57.5 rig/bottle) was obtained from Peninsula Laboratories, Inc. The antibody had a cross reactivity to NPY of 100%, to human pancreatic polypeptide of 0.02% and tc peptide YY of 0.003%. NPY was dissolved into l0 ml of distilled water as a stock solution (5.75 ng/ml) which was frozen. The kso of this rabbit anti-neuropeptide Y serum was 90 pM. The antibody, normal rabbit serum or saline was injected at a volume of 0.5 #! per injection site or 1.44 pg per site.

Correspondence: J.E flood. Present address: 151/JC, VA Medical Center, 915 N. Grand Blvd., St. Louis, MO 63106, U.S.A. 0006-8993/89/$03.50 ~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

74 T-maze apparatus and trai, ing The T-maze consisted of a black plastic start alley (46 cm long) with a start box at one end and two goal boxes (17.5 em long) at the other. The maze was 12.5 cm deep and 9.8 cm wide. The floor consisted of stainless steel rods. A guillotine door prevented the mouse from leaving the start box until the training started, The intertrial interval was 30 s. A muffled doorbell-type buzzer (55 dB) served as the conditioned stimulus. The unconditioned stimulus was 0.30 mA of footshock (Coulbourn Instruments scrambled grid floor shocker model E3-08). A training trial started by placing a mouse into the start box. The guillotine door was raised, the buzzer sounded simultaneously, and then footshock was applied 5 s later. The goal box that the mouse first entered on this trial was designated as 'incorrect' and the footshock was continued until the mouse entered the other goal box, which on all subsequent trials was designated 'correct' for the particular mouse. At the end of each trial, the mouse was removed from the goal box and returned to its home cage. Entry into the correct goal box terminated the buzzer and footshock. The mice received 4 training trials, as this reliably yields poor retention (20-30% recall score) in control mice. As training proceeded, a mouse made one of two types of responses. A response latency longer than 5 s was classed as an escape from the footshock. A response latency less than or equal to 5 s was considered an avoidance, since the mouse avoided receiving a footshock. Two exclusion criteria were applied to reduce learning variability among mice, as follows. On the first training trials, mice with escape latencies greater than 20 s were discarded, as these mice typically do not reach the training criterion and those that do show poor retention, whether in a control group or in a group receiving drug. Mice not having at least one errorless escape latency between 1.5 and 3.5 s on training trials 3 or 4 were excluded because those with iatencies less than 1.5 s usually make an avoidance within the first 3 test trials whether they are in a control or drug group; including them would reduce oar ability to detect a drug effect on retention. Mice with latencies of longer than 3.5 s were excluded because these mice show little evidence of learning and usually have poor retention whether they are in a control or drug group. The exclusion of mice from the study by the above criterion has been used by this laboratory for the past 8 years and was not selected specifically for the experiments reported here. The total number of mice excluded from the study was less than 15% and most were excluded because of poor learning.

Measures of retention for T-maze footshock avoidance training One week after training and drug administration, the T-maze training was resumed until the mice made 5 avoidance responses in 6 consecutive training trials. The mean number of trials to reach the 5 out of 6 criterion was used as a measure of retention. The overall significance of the drug treatment effect was determined by a one-way or two-way analysis of variance''4"5-s. Dunnett's t-test was used to make multiple comparisons between each drug group and the control group -s-s. Tukey's t-test was used to make statistical comparisons among experimental groups34. A non-parametric measure of retention was derived to better visualize the effects of drug treatments on retention test performance. For this, the number of trials to the first avoidance response was dichotomized to yield a percent recall score as follows: those mice making their first avoidance on test trials 1, 2 or 3 were classed as remembering the original training. This criterion was adopted because it has provided optimal separation between the retention test performance of naive mice (with no T-maze training) and well-trained mice ~-s.The correlation between mean trials to the 5 out of 6 avoidance criterion and recall score is typically +0.95 or greater.

Drug administration The surgical procedures for injecting 0.5 Itl of saline or NPY bilaterally into the rostral hippocampus, caudal hippocampus, amygdala, thalamus, caudate and unilaterally into the septum were previously described "1. The injection coordinates are given in Table I.

TABLE !

Coordinatesfor administration of NPY into the mouseforebrain I The coordinates were confirmed in B.M. SIotnick and C.M Leonard, A stereotaxic atlas of the albino mouse forebrain, U.S Government Printing Office, Washington, D.C., 1975.

Structure

Anterior/ posterior*

Lateral**

Depth

Amygdala Caudate Hippocampus (rostra/) Hippocampus (caudal) Septum Thalamus

- 1.6 +0.5 - 1.6 -2.6 +0.5 - 1.6

3.3 2.0 1.6 2.6 0.2 1.6

4.5 3.2 1.6 2.5 3.4 3.2

* Placement relative to Bregma. ** All injections were given bilaterally ~xcept for the septum fm which administration was from the right of the central suture witl~ 1° angle of penetration toward the midline. T h e hippocampus, amygdala, septum and caudate were selected as injection sites because these structures have been implicated in memory processing and have cell bodies or fibers which contain NPY. The thalamus, in the same coronal section as the amygdale and the rostral hippocampus, was chosen as one of the injection sit~ because it was centrally located to other injection sites and the thalamus does not show substantial NPY immunoreactivity, ln~ection of NPY into the thalamus made it possible to test for localized effects of NPY on memory processing by having at least one structure where NPY should not alter memory retention. In brief, mice were anesthetized with methoxyflurane, placed in a stereotaxic instrument and a hole was drilled through the skull over each injection site after deflecting the scalp. Mice were trained 24-48 h after surgery. Immediately after training mice were again placed in the stereotaxie apparatus under enflurane anesthesia. Within 3 min after training, a 0.5/~1 solution of saline or NPY was injected into the target structure over a period of 60 s by a Sage Syringe Pump (Model 341A). The solution was administered through a 31 gauge needle attached to a 10/tl syringe by PE-1G tubing. This method of injection results in reliable admmistt~ation into the desired target structures. The reliability of the injections was determined by injecting dye into the sites and determining the location of dye in frozen brain sections. In ~c~dition, 31 gauge tubing was implanted and the mice were sacrificed and frozen brain sections examined to determine placement. After technicians practiced on between 50 and 200 mice depending on the brain structure, the accuracy of the injection was determined by histological verification of the location of dye or the cannula tip. Accuracy was 100% over 20-30 tests. Injections into the rostra/ hippocampus used a blunted needle to avoid penetrating the floor of the structure, other injections were given with a beveled needle. Typical injection sites for each structure are given in Fig. la-g, Due to some inconsistency in hippocampal nomenclature, we refer to our injections sites as rostral (Fig. le) and caudal (Fig. If,g) portions of the hippocampus. Fig. lg shows the location of the caudal hippocampus in a sagittal section.

RESULTS

Experiment 1. Improvement o f memory retention with N P Y administration into various forebrain structures W e t e s t e d if N P Y m o d u l a t i o n o f m e m o r y p r o c e s s i n g w o u l d s h o w s t r u c t u r a l specificity. M i c e w e r e g i v e n 4 t r a i n i n g trials as d e s c r i b e d a b o v e . T h e n p e r g r o u p w a s

75

g~2 '~x

Fig. 1. Representative brain section showing target structures. In many cases, the tract of the 31 gauge needle used for injecting solutions could not be found 1 week after training. To confirm the accuracy of the injection coordinates, the injection site was marked by a 26 gauge stainless steel tubing implant. Where implants damaged the tissue so that sections would not remain intact, 1 I~1 of absolute alcohol was injected to lesion the area where injections were given. Examples of frozen brain sections, cut at 30 ~tm and stained wilth thionin, are as follows: a, amygdala marked by alcohol lesion: b. caudate with cannula implant; c. septum with cannula implant: d, thalamus with alcohol lesion: e, rostral portion of the hippocampus; f, coronal section of caudal portion of the hippocampus with 31 gauge needle tract: g, sagittal section of caudal hippocampus with needle tract.

76 --'IANYGOALA

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NPY

TREkTNENT GROUPS

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Fig. 2. Dose-dependent enhancement of retention for T-maze footshock active avoidance. Of the 6 areas of the brain into which NPY was administered only the rostral portion of the hippocampus and the septum showed inverted U-shaped dose-response curves typical of compounds improving memory retention ~'. Administration of NPY at 0.05 pg yielded the highest recall score in both structures. See Table II for corresponding means and statistical evaluation.

15. Within 3 min after training, mice received a total dose of 0, (I.05, 0.10, 0.50 and 1.0 #g of NPY per brain structure. Retention was tested a week after training. Improved recall scores were evident only for the groups receiving NPY into the rostral portion of the hippocampus and septum (Fig. 2). Separate one-way ANOVAs run on mean trials to criterion indicated that

Fig. 3. Amnestic effect of NPY. In those brain structures not showing improvement of retention, NPY (1 ,ug/brain) was found to cause amnesia when injected into the amygdala and caudal portion of the hippocampus. See Table III for corresponding means and statistical evaluation.

only injections of NPY into the rostral portion of the hippocampus (F4,Tn = 7.16, P < 0.001) and septum (F4.70 = 7.42, P < 0.001) significantly reduced mean trials to criterion relative to the saline-injected control (Table 11).

Experiment 2. A test for the amnestic effect of NPY in forebrain structures

The structures in Expt. 1 that did not show enhanced memory retention in response to NPY administration might have been causing amnesia which could not be

TABLE II

Ej]['ct of localized administration of NPY on memory retention for T-maze footshock avoidance training The wdues are mean trials to make 5 avoidances in 6 consecutive trials + S.E.M. The recall score is the percent mice making their first avoidance response in 3 test trials or less.

Total dose Otg) 0 Amygdala Recall score Caudate Recall score Hippocampus (rostral) Recall score Hippoeampus (caudal) Recall score Septum Recall score Thalamus Recall score

8.67 + 0.48 27 9.20 + 0.41 20 9.80 ± (I.33 13 9.13 4-11.41 20 9.4(I + 0.38 20 9.27 + 0.38 13

0.05

8.33 +_0.45* 40 9.27 4- 0.45 27 9.27 4- 0.26 13 -

0.10

0.50

1.00

8.47 + 0.52 33 9,13 _+0.54 27 7.33 ___0.52** 67 8.40 + 0.56 4(1 7.53 4- 0.35** :,7 9.07 4- 0.56 33

9.00 4- 0.42 27 9.07 + 0.40 27 7.00 4- 0.29** 80 8.73 + 0.49 33 7,13 4- 0.43** 80 9.27 + 0.41 20

9.27 27 8.27 33 8,67 33 9.00 27 8.53 47 8.47 27

Significant differences in means from the 01,g dose groups: *P < 0.(15; **P < 0.01,

4- 0.47 4- 0.36 4- 0.41 + 0.51 + 0.48 + 0.44

T/

tOO

M SALINE [~]SERUW

m OOSE aNPY0 (pq/bratn)

~ o N P Y (2.88 pg/brotn)

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q: 40

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ROSTRALNIPP0rANPUS SEPTUN BRAIN REGION

Fig. 4. Modulating effect of NPY antibody (aNPY) on retention for T-maze footshock avoidance training. In the rostral portion of the hippocampus and septum, NPY enhanced retention test performance measured 1 week after training (Fig. 2). In this figure, aNPY impaired retention when injected into either the rostral hippocampus or the septum under training conditions where the groups receiving saline had good recall. The group receiving normal rabbit serum, the aNPY vehicle, di not have impaired retention. See top portion of Table VI for corresponding means and statistical evaluation.

detected in that paradigm. To determine if NPY caused amnesia in the amygdala, caudate, caudal hippocampus or thalamus 1/zg of NPY or saline was administered as above after training. The conditions for T-maze training were altered to insure that saline injected control mice (0 #g) would have high recall scores. This was accomplished by giving 5 training trials instead of 4. The footshock

TABLE III

Test for the amnestic effect of NPY (I.0 pg/brain) in Jbrebrain structures not previously found to enhance retention test performance after N P Y administration Mean trials to criterion

+ S.E.M. Amygdala Saline NPY Caudatc Saline NPY Hippocampus (caudal) Saline NPY Thalamus Saline NPY

7.40 + 0.33 9.07 + (I.51' 7.(17 __0.34 7.33 + 0.37 6.80 + 0.25 9.33 + 0.411"* 7.73 +__0.40 6.87 + 0.38

Statistical significance between mean trials to criterion for saline and NPY treated mice within each structure was evaluated by Student's t-test. *P < (I.01 ; **P < 0.0l)l.

20

-1 CAUDAL HIPPOCANPU8

ANYGDALA

BRAIN REGION

Fig. 5. Modulation of retention by aNPY. Retention test performance measured I week after training was impaired when NPY was

administered into the caudal portion of the hippocampus and amygdala (Fig. 3). This figure showes that under training conditions where the saline controls show poor retention, administration of aNPY in the caudal portion of the hippocampus resulted in an inverted U-shaped dose-response curve. Over the same dose range, aNPY injected into the amygdala resulted in an improvement of retention as the dose of aNPY increased. Normal rabbit serum had no effect on retenti,r, See the lower portion of Table VI for corresponding means and statistical evaluation.

intensity was increased from 0.30 to 0.35 mA. The buzzer intensity was 65 dB rather than 55 dB. The intertrial interval was increased from 30 to 45 s. The n per group was 15. Retention was tested 1 week later. Impaired retention test performance occurred only when NPY was administered into the amygdala and caudal portion of the hippocampus (Fig. 3). Separate Student's t-tests, run on mean trials to criterion, showed that NPY administered into the caudal hippocampus (t = 5.35, P < 0.001) and amygdala (t = 2.74, P < 0.01) significantly increased mean trials to the avoidance criterion c o m p a r e d to mice receiving 0 / t g (Table Ill).

Experiment 3. Time-dependent modulation o f memory retention by N P Y I m p r o v e m e n t of retention test performance with NPY administration into the septum and rostral hippocampus and amnesia with administration into the amygdala and caudal hippocampus may have been due to long lasting changes in brain function not specifically related to m e m o r y processing. If the altered retention test performance was due to lesions or other brain damage, it would

78 TABLE IV

TABLE VI

Failure of i.c.v, administration of NPY given 24 h post-traininl, to modulate retention demonstrates time-dependence

Effect of N P Y antibody (uNPY) administration of retention for T-maze footshock avoidance training

Test for enhancement Septum Saline NPY 0.5pg Hippocampus (rostral) Saline NPY 0.5pg Test for amnesia Hippocampas (caudal) Saline NPY l.(}pg Amygdala Saline NPY 1.0/~g

Mean trials to criterion + S. E.M.

Recall score (%)

9.60 + 0.40 9.33 + 0.28

13 20

9.40 + 0.41 9.53 + 0.41

20 13

6.80 + 0.31 6.80 + 0.29

80 87

7.27 + 0.36 7.13 - 0.35

80 80

Mean trials to criterion +-S.E.M.

also alter performance when NPY was administered 24 h after training. Compounds which modulate m e m o r y processing show a decreased effectiveness as the time of administration is delayed beyond the end of training 4°. In this experiment using 24 h post-training administration of NPY, a lack of altered re,ention would indicate that the modulation of retention seen in Expts. 1 and 2 was not due to non-specific proactive influences of NPY of retention test performance per se. To test for a proactive effect of NPY on retention test performance, NPY was administered 24 h after training at a total dose of 0.50 # g into the septum and rostral hippocampus as in Expt. 1 and at a total dose of 1,0pg into the amygdala and caudal hippocampus as in Expt. 2. Retention was tested 1 week after training. Separate Student's t-tests indicated that the NPY

TABLE

The table shows that aNPY had the opposite effect on retention for T-maze footshock avoidance conditioning as the administration of NPY.

NPY enhances retention Hippocampus (rostral) Saline Serum aNPY (2.88 pg) Septum Saline Serum aN Y (2.88 pg) NPY imvairs retention Hippocampus (caudal) Saline aNPY (pg) 2.88* 1.44 0.72 0.29 Serum 0 Amygdala Saline aNPY (pg) 2.88 1.44 0.72 0.29 Serum 0

7.20 + 0.39 7.13 + 0.38 9.00 4- 0.44** 7.07 + 0.40 7.07 + 0.31 9.40 + 0.44** 9.07 + 0.52 8.50 + 0.40 7.27 +_0.36** 8.67 +_0.46 8.73 _ 0.34 9.73 _+0.31 9.13 + 0.40 7.20 _+0.31"* 8.97 _+0.50 8.40 +_0.44 8.93 + 0.38 9.33 +_0.40

* Of 21 mice run in this group all showed some seizures; 9 died after tonic/clonic convulsions; such seizure activity w a s never observed when 2.88 pg of aNPY was injected into the rostrai portion of the hippocampus. Therefore even after replacing mice that died, we only had 12 mice in this group. The P-values indicate a significant difference in mean trials to criterion between mice treated with aNPY versus saline (Dunnett'~ t-test) and between aNPY versus rabbit serum (Tukey's t-test). A single value indicated both differences were significant at the same level, otherwise, the P-value with respect to the saline control group is given first. **P < 0.01.

V

Effect of administering NPY into the cortex on memory retention Mean trials to criterion +_S.E.M.

Cortical control for memory enhancement Septum Saline NPY 0.5pg H ippoeampus (rostral) Saline NPY0.5pg Memory impairment Hippocampus (caudal) Saline NPY l.Opg Amygdala Saline NPY 1.0pg

Recall score (%)

9.07 +_.0.45 9.07 _ 0.46

20 20

9.40 + 0.33 9.73 + 0.36

20 13

7.13 +- 0,20 7.27 + 0,33

87 73

7.53 + 0.33 7.33 _.+0,32

73 87

injections did not significantly (all t-values < 1 ) affect the mean trials to criterion c o m p a r e d to the saline-injected controls (Table IV). Experiment 4. Effect o f cortical N P Y administration on retention

The purpose of this experiment was to determine if NPY modulated retention in Expts. 1 and 2 by diffusion into the cortex above the corpus callosum. As a control for the sites where NPY improved retention, saline or 0.50 p g NPY (the highest dose improving retention in Expt. 1) was injected into the cortex above the septum and the rostral hippocampus. The training conditions were as in Expt. 1. As a control for the sites where NPY impaired retention, saline or 1.0/~g of NPY ~the dose

79 used Go induce amnesia in Expt. 2) was injected into the cortex above the immunoreactivity and the caudal hippocampus. The training conditions were as in Expt. 2. The n per group was 15. Retention was tested 1 week later. Student's t-tests indicated that none of the cortical injections of NPY significantly (all t-values <1) changed the mean trials to the avoidance criterion relative to the saline control (Table V).

of aNPY required significantly more trials to reach criterion than the group receiving 1.44 pg (P < 0.01, Tukey's t-test), aNPY administered into the amygdala improved retention as the dose of aNPY increased (Fig. 3). When aNPY was injected at 2.88 pg, it significantly reduced mean trials to criterion (Table VI) relative to groups receiving either saline (P < 0.01) or rabbit serum (e < o.ol).

Experiment 5. The effect of NPY antibody (aNPY) on memory processing The purpose of this experiment was to determine if the effects of NPY on memory retention were related to physiological processes. Since the administration of aNPY would bind endogenous NPY, it should have the opposite effect of NPY on retention. To test for an amnestic effect of aNPY administered into the septum or the rostral hippocampus, where NPY improved retention, mice were trained as in Expt. 2 so that the controls would have good retention scores. Separate groups of mice were injected after training with saline, normal rabbit serum or aNPY (2.88 pg/brain). To test for improvement of retention when aNPY was administered after training into the caudal hippocampus or the amygdala, mice were trained as in Expt. 1 where control groups had poor retention. Because administration of aNPY might yield an inverted U-shaped dose-response curve, typical of memory enhancing compounds, separate groups of mice received saline, rabbit serum, 0.29, 0.72, 1.44 or 2.88 pgtbrain of aNPY. The n per group was 15. Retention was tested 1 week after training. In the septum or the rostral hippocampus, aNPY administration clearly impaired retention test performance (Fig. 4). In the septum, a one-way ANOVA indicated that aNPY resulted in a significant treatment effect (F2.42 = 14.07, P < 0.001) with an increased mean number of trials needed to reach the footshoek avoidance criterion in aNPY treated mice compared to the means for mice treated with saline (P < 0.01) or rabbit serum (P < 0.01). In the rostral hippocampus, a similar statistical analysis indicated that aNPY significantly (F2.42 = 7.35, P < 0.005) increased mean trials to criterion compared to either control group (P < 0.01). aNPY modulated retention test performance in a dose dependent manner (Fig. 5). In separate one-way ANOVAs, a significant effect of aNPY on mean trials to criterion occurred when administered into the caudal hippocampus (Fs.sl = 4.47, P < 0.005) or amygdala (F5,84 = 4.06, P < 0.005). The dose-response curve had an inverted U-shape when aNPY was administered into the caudal hippocampus with the group receiving 1.44 pg of aNPY yielding the lowest mean trials to criterion (Table VI) and recall score (Fig. 3). The group receiving 2.88 pg

DISCUSSION NPY enhanced memory processing when injected into the septum and rostral portion of the hippocampus (Expt. 1) and impaired memory processing when injected into the amygdala and caudal hippocampus (Expt. 2). No effect of NPY on retention was seen when it was injected into the thalamus. Modulation o, memory processing by NPY was found to be time dependent (Expt. 3). Thus changes in retention were not due to some lingering effect, direct or indirect, which could have influenced retention test performance per se. Though NPY is found in the cortex, diffusion of NPY up the needle tract into the cortex did not account for either improved or impaired retention test performance since cortical administration of NPY did not affect retention (Expt. 4). The administration of aNPY had the opposite effect of administering NPY (Expt. 5). aNPY resulted in improved retention when injected into the caudal hippoeampus and septum. Since the antibody specifically binds endogenous NPY, the result indicates that endogenous NPY can modulate memory processing. The literature on the anatomical difference in the different CA regions of the hippocampus is considerable, but we were unable to find any studies which might help explain the improvement of retention when NPY was administered into the rostral portion of the hippocampus and impairment when administered into the caudal area. Given the likely diffusion pattern of less than 2.0 ram, NPY probably diffused into all segments of the rostral portion of the hippocampus, but only into the CA1 and CA4 regions (area about the dentate gyrus) in the caudal portion of the hippocampus. The possibility that difference in diffusion of NPY in the two areas of the hippocampus may account for different effects on memory retention is being studied. Another possibility is that NPY is acting on a different chemical substrate in the two regions of the hippocampus. We have found that neuropeptide K improved retention when injected into either the rostral or the caudal hippocampus (unpublished observations) indicating that differences in neural circuitry alone between the two hippocampal regions do not account for the effect of NPY on retention. Evidence indicates that NPY is co-localized with neurons contain-

80 ing acetylcholine, norepinephrine and somatostatin 7' 9.13.37.45 It is possible that NPY is co-localized with different neurotransmitters in the rostral and caudal portions of the hippocampus. When examining the areas in which NPY improved, impaired or had no effect on retention, it is clear that NPY did not diffuse far from the injection site. The two injection sites of the hippocampus were 1.8 mm apart and NPY enhanced retention when given in the rostral portion and impaired retention when given in the caudal portion. The thalamic area, in which administration of NPY had no effect on retention, was 1.6 mm below the rostral portion of the hippocampus in which NPY administration improved retention. When NPY was injected in the septum it improved retention but in the caudate, 1.8 mm lateral to the septum, it had no effect on retention. The coordinates of the injection sites and the differential effects on NPY on retention at these sites suggests that NPY diffused less than 2.0 mm, thus providing good localization. Radioimmunoassay studies of NPY (NPY-ir) are in general agreement concerning its distribution in the brain 4'13'19'46. NPY-ir occurred in the rat brain in quantities of pmol/g of tissue. The highest concentrations of [~2Sl]NPY binding sites are in the rostral portion of hippocampus (425 fmol/g tissue compared to 44 fmol/g in the cerebellum) with a more diffuse representation in the caudal ~:ippocampuss,46. All amygdaloid nuclei contain NPY-ir terminals. An especially high density of fibers is located within the medial and central nucleus of the amygdala. The medial and particularly lateral portions of tile septum contain a high number of NPY terminals. The caudate shows a ctiffuse pattern of NPY containing cell bodies, while the thalamus had not been found to contain immunoreactive NPY 46. The importance of the hippocampus and amygdala for normal memory processing is indicated by several types of studies. Localized drug administration, autoradiography and lesion studies suggest that the hippocampus is involved in learning and memory. Amnestic syndromes in humans are frequently linked to hippocampal damage 52. sa. Low levels of electrical stimulation in the amygdala, caudate and substantia nigra cause amnesia for passive and active avoidance 23.39.s~. Low levels of electrical stimulation in the hippocampus, reticular formation and its thalamic projections are associated with facilitated retention; high levels of stimulation disrupt retention of appetitive and aw,~rsive training tasks 39,s7. Ablation of specific brain areas generally impairs learning and, in some designs, memory retention. Hippocampal lesions impair retention for passive avoidance, brightness discrimination, successive discrimination, one-way active avoidance conditioning, maze tasks, reversal training and

shuttlebox conditioning 22"31'32'3s,36. The involvement of the septum in learning and memory is largely based on lesion studies which result generally in impaired acquisition s~. More recently, a loss of acetylcholine in the basal forebrain was demonstrated in a high percentage of patients who were diagnosed as having Alzheimer's disease 2~. Basal forebrain lesions including septal lesions and those of the nucleus basalis magnocellularis impaired retention for passive avoidance, radial maze and footshock avoidance in a T-maze 26. Others have reported modulation of memory processing with localized injections of a variety of substances. Naloxone, an opioid antagonist, improved retention when injected into the amygdala but not in the caudate zL 30.38,42. Intracaudal administration of the anticholinergic, atropine, impaired retention for passive avoidance47. Cholecystokinin octapeptide facilitated retention when administered into the amygdala, but impaired retention when it was injected into the nucleus accumbens ~4. Substance P, which in some regions of the brain is coexistent with NPY, was found to disrupt retention when applied to the substantia nigra and amygdala 2s'29 but facilitated retention in the septum 49 and nucleus basalis magnocellularis33. In this study, we report that NPY disrupted retention when injected into the amygdala and impaired retention in the septum. The differential effects of NPY across brain regions suggests that NPY acted on a physiological substrate specifically related to memory processing rather than through non-specific effects such as general arousal. The finding that NPY improved retention when given into the rostral portion of the hippocampus but impaired retention ik~ th¢ caudal portion of the hippocampus indicates a highly specific substrate is mediating the effect. This is further indicated by the occurrence of convulsions when NPY was given at 1.0 #g in the caudal but not in the rostrai portion of the hippocampus. The differential circuitry of these areas of the hippocampus clearly needs further study. The ability of aNPY to induce the opposite effects of NPY administration further suggests that NPY has effects on the physiological substrate modulating memory processing. Evidence that NPY may be involved in memory processing in humans is indi~ated by pathological changes occurring in Alzheimer's disease. In Alzheimer's type dementia, hippocampal neurons and axons containing immunoreactive NPY are involved in plaque formation 6. NPY-like immunoreactivity is present in neuronal plaques of Alzheimer's patients l°. NPY co-exists in the same cell bodies as somatostatin and these cell bodies are altered in the brains of patients with Alzheimer's disease 4s. Biochemical studies suggest that in Alzheimer's type dementia NPY may be reduced in the cerebral

81 cortical cells 4 and increased in the substantia innominata 2. Other studies have failed to show an effect of AIzheimer's disease on immunoreactive N P Y I~'w, suggesting that changes in N P Y may only occur late in the disease or only in some forms of the disease. In support of this possibility, an 18% reduction in NPY was found in the cerebrospinal fluid of patients with dementia of the AIzheimer type with extrapyramidal signs, but not in patients without extrapyramidal signs 3. Overall these studies are consistent with the hypothesis that NPY modulates normal m e m o r y processing in humans as well as mice. Previous studies showed that i.c.v, administration of NPY m e e t the criteria for a m e m o r y modulator by enhancing retention and improving recall in a dose- and

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