Acoustic imprinting in guinea fowl chicks: age dependence of 2-deoxyglucose uptake in relevant forebrain areas

DevelopmentalBrain Research, 31 (1987) 15-27 Elsevier

15

BRD 50479

Acoustic imprinting in guinea fowl chicks" age dependence of 2-deoxyglucose uptake in relevant forebrain areas Verena Maier and Henning Scheich Institute of Zoology, Technical University Darmstadt, Darmstadt (F.R.G.) (Accepted 15 July 1986) Key words: Imprinting; Auditory system; Bird; 2-Deoxyglucose; Sensitive phase

In 7-day-old guinea chicks play back of an imprinted acoustic stimulus was previously found to correlate with increased uptake of 2-deoxyglucose (2DG) in 3 rostral forebrain areas (HAD, LNH and MNH). Subdivisions of these areas defined by 2DG labelling may be association fields. Auditory areas did not show labelling differences between imprinted and control animals. In the present study imprinted guinea chicks of different age and with different experience were used in the 2DG experiments. Seven-day-old chicks (beyond the sensitive phase), 4-day-old (at the decline of sensitive phase) and 1- and 3-day-old chicks (within the sensitive phase) were given a 2DG injection and afterwards heard the stimulus to which they had been imprinted previously (1.8 kHz, 3 tone pips per s). While the typical labelling pattern was weak or absent in l-4-day-oid chicks the older animals consistently had high 2DG uptake in these areas. Unsuccessfully imprinted 7-day-old chicks, having all the behavioral test experience, showed no or weak labelling. These results are related to current literature on reticular activation of the relevant areas and on morphological changes there with termination of the sensitive phase. It is argued that the reticular activation of these areas, e.g. attention mechanisms are instrumental in securing imprinting success and that subsequent reorganization of the areas leads to stronger metabolic activation after the sensitive phase.

INTRODUCTION Identification of brain structures and mechanisms responsible for learned behaviors has proven to be one of the most difficult endeavours in neurobiology. Analysis of learning mechanisms, so far, was promising at the synaptic level of single neurons and at the circuit level in isolated parts of brain like hippocampus, cortex and cerebellum 5'6'11'2°'38,4°,53,57'58. A more accessible form of behaviorally relevant learning may be filial imprinting. Here a particular visual or auditory stimulus is irreversibly preferred as a mother surrogate after it has been experienced briefly during an early stage of life 28. Hence imprinting differs most conspicuously from other forms of learning in that it takes place exclusively during a early and restricted life period ('sensitive phase') and in that the imprinting effect is resistant to other experi-

ences (for recent theories see Bateson 1, Hess 17, Immelmann and Suomi19). Although the p h e n o m e n o n was studied extensively on the behavioral level little is known of underlying brain substrates. Previous anatomical, lesion, and biochemical studies focussed on identifying brain structures involved in visual imprinting2-4.18,27.32.33,42-44. Imprinting presumably modifies the responsiveness of particular neuronal populations to the imprinting stimulus. To identify and further investigate such populations of neurons experiments with ~4Clabelled 2-deoxy-D-glucose (2DG) may be significant. Intracellular accumulations of this tracer represents a measure of glucose metabolism 5~ which in turn is chiefly determined by the activity of the sodium pump 31. Consequently after some time of stimulus exposure neuronal populations with enhanced responsiveness to that stimulus are recognized in auto-

* Present address: Institute de Physiologie, Rue du Mus6e 5, 1700-Fribourg, Switzerland. Correspondence: H. Scheich, Institute of Zoology, Technical University Darmstadt, Schnittspahnstrasse 3, 6100 Darmstadt, F.R.G. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

10

radiographs of brain sections. The usefulness of the 2DG method for revealing learning of specific stimuli in brain areas has been demonstrated already for classical auditory conditioning I2'j3. In previous 2DG experiments, areas relevant for auditory imprinting became apparent in the rostral forebrain of acoustically imprinted guinea fowl chicks with play-back of the imprinting stimulus after the sensitive phase 3°. The present longitudinal study emphasizes the question when during the sensitive phase imprinted chicks would first show the typical labelling pattern. Interestingly, there is a very low incidence of the pattern before the end of the sensitive phase suggesting a labile state of the mechanisms of metabolic activation as long as imprintability is not terminated. If brain mechanisms of imprinting are a matter of interest acoustic stimuli have several practical advantages over visual stimuli in freely moving animals 36. First, acoustic stimuli do not need a particular orientation of the animal to be received by the ear. Second, the perception of important characteristics of the stimulus like rhythm and frequency is largely invariable against background, and hence independent of the animals's motion. Third, in contrast to the distributed representation of visual patterns ~9, sounds are topically represented in the relevant areas of the bird telencephalon. As already demonstrated, isofrequeucy planes are differentially and specifically labelled by 2DG in response to tone stimulation in unrestrained animals 16"45 47 For the present imprinting study, this allows for an excellent control of whether the stimulus was processed in a similar fashion by the auditory system of experimental and control animals. MATERIALS AND METHODS

Subjects Subjects in the 2DG experiments were 76 young guinea chicks, 35 naive controls and 41 chicks with imprinting experience. They were bred in our laboratory in an incubator kept at 37.5 _+ 0.5 °C, where they hatched on the 28th day of incubation (+ 1/2 day), called day 0. Fluffy chicks (~- 4 h) were picked up, marked, and housed together in small groups of 4 - 8 animals to avoid social deprivation. In the home cage, temperature was kept at 25-30 °C, and the

day/night cycle was 12 h/12 I1. Chicks had [roe access to commercial chicken food and water,

Behavioral procedures Imprinting stimuli were synthetically produced rhythmic tone bursts of either 1.8 kHz or 2.5 kHz, with the rhythm corresponding to the maternal contact call ('iambus') 29. This means that they consisted of two syllables, 50 ms and 200 ms long, which were separated by a 80-ms pause. Intervals between individual double bursts were either 395 ms ( 1.8 kHz) or 450 ms (2.5 kHz). The 1.8-kHz frequency is prominent in the iambus, whereas the 2.5-kHz frequency occurs in several other guinea fowl calls, including most chick calls the frequency band of which is between 2 and 3.5 kHz. In thefamilarization session (FS) on day 0 or 1 cage mates (i.e. 4 - 8 chicks) were placed in a lighted box with hay, together with a stuffed guinea hen, while the imprinting stimulus was played for 1 h. Tests Three different tests were carried out in a Y-maze with loudspeakers behind two branches. These branches had a small goal box, where the stuffed guinea hen could be placed hidden from the view of the chicks unless they entered the box. Chicks started a trial run in the third branch. In the pilot test (PT) the stuffed hen was visible to the chick while behind her the imprinting stimulus was played. Two trial runs were performed with the stimulus presented once from the right, and once from the left branch. A run was stopped when a chick investigated the hen or straight entered the box disregarding the hen, or it was picked up after spending 10 min without a specific reaction. In the approach test (AT), the imprinting stimulus was played behind one goal box where the stuffed hen was sitting out of view of the chick. A trial was scored correct if the chick first entered this box within 10 rain. One test consisted of 3 - 4 trials, separated by 30-min intervals. The side of stimulation was changed after each trial. A chick was successful, if all 3, or 3 out of 4 trials were correct. In the discrimination test (DT) again the imprinting stimulus was played behind the box where the hen was sitting. In addition the alternative stimulus (i.e., the tone frequency not used as the imprinting stimu-

17 ence and one group of naive controls. The experiments on day 4 included one group of successfully imprinted chicks and one group of naive controls. The imprinted chicks had the 2DG session the same day they had completed their tests. The experiments on day 1 and 3 included 3 groups of successfully imprinted chicks and 3 groups of controls. Two of these groups were tested for 2DG incorporation in the dark. In one group (1C-2) 4 animals were stimulated together in the 2DG session. Chicks were injected i.p. and i.m. with 18/~Ci/40 g b.w. [U:4C]2DG (NEN) in sterile saline. For stimulation single injected chicks (except for group 1C-2) were placed in a small, padded cardboard box covered with a fine cloth, and put in a sound-proof chamber. Then, the 1.8 kHz stimulus was played for 1 h. In most experiments (64 out of 76) a diffuse light was put on during the tone stimulation. After 1 h of stimulation the chick was decapitated, the brain quickly removed, frozen and processed according to the procedure given by Scheich et al. 47. In short, at a tempera-

lus) was played simultaneously behind the second goal box. Due to the slightly different intervals between the 1.8-kHz and the 2.5-kHz stimuli the phase of the two stimuli changed which made them easier to locate. Scoring, the number of trials and right-left balancing were the same as for the approach test. Chicks which succeeded in the approach test as well as in the discrimination test were scored as successfully imprinted.

Experiments with 2 DG Three experiments were carried out with the 2DG session taking place on different days after hatching (Table I). (1) On day 7, i.e. definitely after the sensitive phase. At this age, previously imprinted chicks were still reactive to the imprinting stimulus. (2) On day 4, i.e. ,at the decline of the sensitive phase. (3) On day 1 or 3, i.e., during the sensitive phase. Each experiment covered several groups of chicks as summarized in Table I. The experiments on day 7 included 4 groups of chicks with imprinting experi-

TABLE I

Synopsis of experiments Code of groups in column 1: first n u m b e r , day of 2 D G experiment; n u m b e r after h y p h e n , subgroup of m o r e extensive behavioral study; I, successfully imprinted; U , unsuccessfully imprinted (experienced, but failing in tests); C, naive control; D, stimulated in the dark during 2 D G session; *, no familarization session; 1C-2, chicks were sitting together during 2 D G session. C o l u m n 2: ~ background labelling < 1 < 2 < 3 grade of labelling (see Materials and Methods). C o l u m n 3: FS, familiarization session (took place on the hatching day 0 or the next m o r n i n g w h e n chicks hatched late); PT, pilot test; A T , approach test; D T , discrimination test. O n days put in parenthesis no full tests but only one or two discrimination trials were made to confirm preference for the imprinting stimulus until the 2 D G session.

2DG Experiment N 71-1 7U-1 7I-1" 7I-2 7C

7 4 3 4 10 N = 28

41-1 4C N= 1I-1

3I II-D 1C-1 1C-2 1C-D

Grade of 2DG labeling

Imprinting experience on day

3N

2N

3 2 -

-

3

4

(3N

FS

PT

AT

DT

1 4 -

-

0/1 0/1 0/1 .

2 2 1 2

4(5,6,7) 4(5,6,7) 1/4(5,6,7) 4(5,6,7)

1.8 1.8 1.8 2.5

.

.

2/3 2/3 1/2/3 2/3 .

1.8 k H z

.

2/3 .

4

.

1.8 k H z 1.8 k H z 1.8 kHz

1 -

-

3

1

6

5

10

6

7

7

-

2

-

5

7 14

-

1

1

5

0/1 .

1

2

4

-

-

3

0

1

1

1

4 6 4 4 6 N = 28

-

-

-

4

0

1

1

1

-

-

-

6

0

-

-

-

4

.

.

-

3

.

.

--

6

.

.

--

-

1 --

2

Imprinting stimulus

IN

-

26

1

1 .

.

1 .

. .

.

kHz kHz kHz kHz

IX ture o f - 1 5 °C the brain was cut into 30-jzm serial sections which were thaw-mounted on microscope slides and immediately dried at 50 °C. Every third section was contacted with a Kodak NMBX-ray film and exposed in Kodak X-Omatic cassettes for 3 weeks. Reference sections were stained for Nissl substance. Out of the 76 brains prepared, 70 made high-quality series of sections which were further analyzed.

Densimetric analysis In brain areas of interest, 5-15 autoradiographs from each chick were analyzed densimetrically. The measuring system consisted of a TV camera mounted on a Wild microscope. The TV picture was A-D-converted into a 256 × 256 point matrix with 8-bit intensity resolution, and the signals stored in a HP 21 MX computer for further processing. As a reference in each autoradiograph first a background measurement corresponding to 1.2 mm 2 in the autoradiograph (65 x 65 picture points) was made in a neostriatal area ventromedial to the ectostriatum with consistently low and homogeneous labelling (Fig. 4 and Table I1). Since large areas of white matter are not found in the bird telencephalon this region not affected by the experiment appeared to be a compromise. The optical density of picture points in areas to be analyzed was then transformed into ratios relative to the background. Relative optical densities rather than absolute densities were used to allow comparison of labelling in the brains of different chicks 34'5°. Quantitative densimetric profiles were generated in the following way: the 256 horizontal lines of the computer matrix were divided into 32 horizontal rows with 8 lines each. Relative optical densities were integrated across the 8 lines within each row and plotted as a horizontal profile. Hence the complete picture matrix is represented by 32 profiles. The ordinate of each profile represents factors × the optical density of the background which is set to 1. Factors are plotted on a linear scale. Complete picture matrices with 32 mediolateral profiles are illustrated in Fig. 4. In contrast, Figs. 3, 5, and 6 are compiled from individual profiles which showed the largest peaks of optical density in a given matrix of relevant brain areas. Here the individual profiles take a dorsoventral course through H A D and MNH.

RESULTS

Imprintability The 76 guinea fowl chicks investigated here with 2DG were selected from more than 300 animals tested in a behavioral imprinting study designed to identify various parameters influencing acoustic imprinting (Maier and Scheich, in preparation). Acoustic imprintability with either 1.8-kHz or 2.5-kHz rhythmic tone bursts was found to be high until day 3 ( ~ 72 h) after hatching (day 0 = hatching day), then to decline sharply, and to be totally lost on day 53~. In the behavioral study 50% of the animals were imprinted if the first experience with the imprinting stimulus occurred during the first 3 days of life and if all tests (pilot, approach and discrimination test) were carried out together on one of the 3 days. If tests were spread over several days 75% of successfully imprinted chicks were obtained. Thus variables like motivation or exhaustion play a role for the imprinting success. It was also found that chicks could be imprinted during tests as well as during the familarization session. The use of the stuffed hen as a reinforcer was not crucial for obtaining acoustically imprinted chicks. All but 4 of the experienced chicks used in the 2DG experiments were imprinted as they passed all tests.

2DG labelling Autoradiographs of 70 chicks were analyzed. Structures are named according to references 24'54 and the assumed homologous structures in mammals given in parenthesis 23'26'37.

Labelling of defined sensory structures In all brains, the nuclei of the auditory pathway were intensely labelledg'21'22: nucleus angutaris and nucleus magnocellularis (n. cochlearis complex), nucleus mesencephalicus lateralis dorsalis (inferior colliculus), and nucleus ovoidalis (medial geniculate body). In the forebrain, tonotopic labelling was found in field L (a primary auditory projection area) and in the hyperstriatum ventrale dorsally adjacent to field L 31)(a higher-order auditory field which receives input from and sends connections to field LT). Fig. 1, illustrates tonotopic labelling of field L (with layers

19 L 1, L 2, L3) and of the hyperstriatum ventrale (HV) in selected brains covering all experiments as given in Table I. The first 3 cases in each column are exampies of imprinted chicks, the last 3 cases, of controls. In all chicks, the input layer L 2 of field L was strongly labelled throughout. This is mainly due to its high background activity which is present also in unstimulated chicks46. The stripe across the layers Lx, L2, and L 3 reaching into HV was produced by the 1.8 kHz tone stimulation. Due to the tonotopic organization of field L and HV stimulation with different frequencies results in stripes with the same orientation but located either ventromedial (higher frequencies) or dorsolateral (lower frequencies) to the 1.8-kHz band 8't6'45-48. A comparison of relative strength of tonotopic labelling across the layers of field L and HV between naive and imprinted chicks or animals stimulated in the light or in the dark showed variabili-

Day I

ty within each group but no consistent difference between the groups. In addition to auditory structures high-density labelling was found in two visual nuclei, nucleus rotundus (n. lateralis posterior thalami) and the ectostriatum, Fig. 2 (parastriate cortex) 25. This may partly be the result of diffuse light stimulation during the 2DG seession, although most chicks stimulated in the dark (i.e. 9 out of 12) showed similar labelling thus indicating high spontaneous activity in these areas. Interestingly the visual W u l s t 26'34 (striate cortex) was not labelled in control animals (Fig. 2, HA).

Labelling in the rostral forebrain In the experiment with 7-day-old chicks distinct labelling differences between imprinted chicks and controls were found in the rostral forebrain, Whereas most control chicks showed fairly homogeneous and

Day 4

Day 7

Fig. 1. Tonotopic labelling in 1-, 4- and 7-day-oldchicks. Autoradiographs of 2DG labellingin field L, the primary auditory projection area in the bird's neostriatum (transverse plane). L2, input layer; L l, L 3 dorsal and ventral layers of field L. Note in all chicks the strongly labelled perpendicular stripe in the 1.8-kHz isofrequency plane which is due to the stimulation during the 2DG experiment. Chicks from groups within each experiment were chosen (see Table I). Cases 11, 4I and 71, imprinted chicks; 1C, 4C, 7C, 7U, controls.

2() low-density labelling except for the ectostriatum,

brain roof (Wulst) including in dorso-vcntral se-

most successfully imprinted chicks showed increased

quence the hyperstriatum accessorium (HA), the nu-

2 D G uptake in 3 brain areas (Fig. 2). Since the areas

cleus intercalatus hyperstriatum accessorium ( I H A ) ,

defined by metabolic activation did not respect ana-

the hyperstriatum intercalatum supremum (HIS),

tomical boundaries of known structures we chose

and the hyperstriatum dorsale (HD). 1HA, HIS and

composite names. (1) H A D covers the rostral fore-

H D receive visual projections from the thalamus-"¢"5¢L

7 I- 1

U A ~

7 I-1"

7C

Fig. 2. Rostral forebrain labelling related to successful imprinting in 7-day-old chicks. Autoradiographs of 2DG labelling in 3 rostral forebrain areas HAD, LNH and MNH. Equidistant (0.4 mm) serial sections from individual imprinted chicks of groups 7I-1, 7I-2, 7I-1", a control of group 7 and an experienced but not imprinted control (7U-l, see Table I) illustrate the labelling differences. A-E: frontal-caudal direction. Anatomically defined structures: HA, hyperstriatum accessorium; HD, hyperstriatum dorsale; HV, hyperstriatum ventrale; LH, lamina hyperstriatica; N, neostriatum. Areas defined by labelling in imprinted chicks: HAD, hyperstriatum accessorium including some hyperstriatum dorsale; LNH, lateral neostriatum and hyperstriatum ventrale; MNH, medial neostriatum including some hyperstriatum ventrale. Dorsoventral lines through HAD and MNH in B indicate level and orientation of densimetric profiles shown in Figs. 3, 5 and 6.

21 (2) LNH includes part of the lateral neostriatum (N) and the lateral hyperstriatum ventrale (HV), anterior and dorsal to the visual ectostriatum (E). (3) MNH includes the most medial, magnocellular area of N and a strip of HV, just across the lamina hyperstriatica (LH). In Fig. 2, autoradiographs of 6 chicks representative for the imprinted groups 7J-l, 7J-l* and 7J-2 and for the control groups 7C and 7U-1 were chosen to illustrate these differences. In imprinted chicks most prominent 2DG labelling was found anterior or around the rostral pole of the ectostriatum (sections A - C in Fig. 2) whereas more caudally differences between imprinted chicks and controls eventually disappeared (section E and following). In order to quantify these results a densimetric analysis of autoradiographs was performed (see Materials and Methods). In each chick, the analysis was

Chicks were divided into 4 labelling classes: Grade 3 labelling is strongest 2DG uptake, i.e. in all 3 areas mentioned above local regions with peak optical density 2.5 x the optical density of the background were found. Grade 2 labelling means that in all 3 areas local regions with peak optical density 1.5 x the density of the background were found. Grade 1 labelling means that at least in one area the optical density was 1.25 x the density of background. Grade 0 labelling means that in all 3 areas the optical density was below 1.25 x the density of background. In strongly labelled brains, the rostrocaudal extent of labelling was always greatest in H A D and smallest but most precisely demarcated in the medial MNH. Individual groups of animals showed the following labelling characteristics which are summarized in Table I.

carried out in 5-15 sections between frontal planes A and E. Since optical density measurements were given as ratios relative to the background, plots of such measurements for different chicks were comparable 12'34'5°. As another confirmation of homogeneity the mean background density when comparing imprinted animals and controls in each age group did not vary more than 10% (Table II).

These chicks were beyond the sensitive phase when they were given a 2DG session. In group 71-1 (where chicks had a familarization session and tests with 1.8-kHz and thus heard the imprinting stimulus during the 2DG session) out of 7 chicks 3 showed grade-3 labelling (profiles 14, 17, 18 in Fig. 3, and Fig. 4-1) 3 beyond grade-2 labelling (profiles 20-22) and one showed grade-1 labelling (profile 23). In group 7J-1" (where chicks had no familarization session but were imprinted during pilot test and approach test with 1.8 kHz and thus heard the imprinting stimulus during the 2DG session) two chicks showed grade-3 labelling (profiles 16 and 19 in Fig. 3, and Fig. 4-2). One chick showed grade-0 labelling (profile 24). In group 71-2 (where chicks experienced a familarization session and tests with 2.5 kHz but heard the 'wrong' 1.8 kHz during the 2DG session) all 4 chicks showed grade-2 labelling (profiles 25-28 in Fig. 3). In group 7C (where the subjects were naive controis) 3 out of 10 chicks showed grade-2 labelling (profiles 1-3 in Fig. 3). In one chick grade-1 labelling was found (profile 4). The remaining 6 chicks had grade-0 labelling (profiles 5, 10, and Fig. 4-3). In group 7U-1 chicks had the same imprinting experience and experimental conditions as chicks of group 71-1 but had failed the discrimination test, thus constituting an experienced control group of not-imprinted chicks. In all 4 chicks grade-1 labelling was found (profiles 11-14 in Fig. 3).

TABLE II Densimetric values

Variation of densimetric values of background measurements in the reference area (see Fig. 4). A value of 255 means full light transmission of the source, a value of 0 no light transmission (i.e. the camera lens is closed), n, number of brains contributing to the measurements (from each brain the mean of 5 measured sections was taken). Group

Optical density of background mean +- S.D.

1I 3I lC-1 1c-2 II-D 1C-D 4I-1 4C 7I-1 71-1" 7U-I 71-2 7C

n

113 + 20

8

104 + 20

8

104 + 21 99 + 25 116 + 24 114 + 33

6 6 7 7

127 + 16 123 + 26 136 + 14 117 + 32

10 4 4 10

Experiments on day 7 (n = 28)

7C

71-1 7 I-1"

15 1

2

.

S

:

i

i

2o

> 8

•

~0

:

~

o

~

23 ~

24

71-2

7U-1

i

~

25

11

26

12

•

14

--

i

HAD

----

i

i

2r

•

~,-- M N H

--

MNH

--~

--

HAD

--i OSmm

dors.

vent.

vent.

I

dors.

[ii

Fig. 3. Densimetric profiles of 2DG labelling of the 28 chicks from experiments on day 7 (see Table I). Profiles correspond to average labelling in a dorsoventral window of 0.15 mm width through HAD and MNH from plane B of autoradiographs (see Fig. 2). On the left, profiles stem from naive control chicks (7C, numbers 1-10) and from experienced, but not imprinted chicks (7U-l, numbers 11-14). On the right, profiles are from successfully imprinted chicks which heard the imprinting stimulus during the 2DG session (7I-1, numbers 15, 17, 18, 20-23; 7I-1, numbers 16, 19, 24), and from successfully imprinted chicks which heard the alternative stimulus during the 2DG session (71-2, numbers 25-28). Background optical density defined as 1 is indicated by a point beside each profile. The ordinate scale (bottom right) represents optical density relative to the background.

Experiments on day 4 (n = 14)

Experiments on days 1 and 3 (n = 28)

These chicks had the 2DG session when imprintability sharply declines. Two imprinted chicks showed grade-2 labelling (profiles 8 and 9 in Fig. 5) and 5 grade-0. In the control group one chick was found to have grade-2 labelling (profile 1) and another chick grade 1 labelling (profile 2). The remaining profiles in Fig. 5 illustrate the weak and homogeneous labelling in the brains of the other chicks in these experiments whether imprinted or controls.

Among these chicks which had the 2DG session before 4 days of age and thus were still imprintable, only two showed grade-2 labelling, and the rest grade-0 labelling. One was an imprinted chick (profile 9 in Fig. 6). The other was a naive control chick (profile 1). In summary, differential labelling was positively correlated with successful imprinting and termination of sensitive phase since high-density labelling

71 - 1

7C

71-1 ~

t

2a

:l

f[

.11

'i i i

Fig. 4. Two-dimensional 2DG densitometry (a) and the corresponding autoradiographs (b) of relevant brain areas from two successfully imprinted chicks which heard the imprinting stimulus during the 2DG session (71-1 and 71-1") and from one naive control (7C). Individual profiles in the graphs correspond to a mediolateral window in the autoradiograph. The right hand ordinate represents optical density relative to background having a value of 1 on a linear scale. Note that the scale factor is 1/3 of that used in Fig. 3. Chicks are represented in Fig. 3 by the profiles 15, 17, and 9. Anatomical structures: HA, hyperstriatum accessorium; HD, hyperstriatum dorsale; HV, hyperstriatum ventrale; N, neostriatum, E, ectostriatum. The small squares in the autoradiographs represent the reference area of neutral labelling defined as background. Vertical pointed lines in panels 2 indicate position and orientation of dorsoventral profiles shown in Figs. 3, 5, and 6.

4C

41-1

o

i S

i

"

•

6

i

"

•

- -

E

HAD

--i

=

'

3x

~-MNH

---i

t.--

HAD

"-(LImm

d.

f 21

,--MNH~

v.

d.

Ix

Fig. 5. Densimetric profiles and 2DG labelling from 7 controls (4C, numbers 1-7) and 7 imprinted chicks (4I-1, numbers 8-14) of experiments on day 4 (see Table I). Profiles correspond to a dorsoventral window of 0.15 mm width through HAD and MNH from plane B of autoradiographs (see Fig. 2). Background optical density (= 1) is indicated by a point beside each profile. The ordinate (bottom right) represents optical density relative to the background on a linear scale.

24

1C-1+1C-2

11-1

i 3

~

•

S

i

i

-

i

i

'°

12,, o i

8

HAD

--!

i

!

i

T

~--

MNH

--

---

MNH

.

.

.

14

HAD

. ~S~m

d.

v.

[ii

Fig. 6. Densimetric profiles of 2DG labelling from 8 controls ( 1C- 1. numbers 1,2, 7, 8, and 1C-2, numbers 3-6) and 8 imprinted chicks .[ 1I-1, numbers 9-12, 3I, numbers 13-16) of experiments on days 1 and 3 (see Table I). Profiles correspond to a dorsoventral window of 0.15 mm width through HAD and MNH from plane B in autoradiographs (see Fig. 2). Background optical density (= 1) is indicated by a point beside each profile. The ordinate (bottom right) gives the optical density relative to the background on a linear scale•

was merely found in successfully imprinted, older chicks (i.e. group 71-1, 71-1" and 71-2). A Wilcoxon test which compared labelling in chicks of these successfully imprinted groups with the controls (7U-1 and 7C) was significant (P < 0.01). The 5 chicks with most extensive regions of strongest labelling belonged to the groups which experienced 1.8 kHz as the imprinting stimulus in the behavioral tests, and heard this same stimulus during the 2DG session. Interestingly, 7-day-old chicks which had failed in tests (7U-l) and consequently were not considered imprinted, all showed minor degrees of labelling. DISCUSSION

Activation of rostral areas Although chicks of all age groups were successfully imprinted as shown by the behavioral tests, high-density labelling was found primarily after the sensitive phase. To our knowledge this finding represents the first direct neural correlate of sensitive phase reported for filial imprinting. This late appearance of high metabolic activity is the most intriguing result of the present study. It may have a number of reasons, but the following ones can be excluded. First, labelling in older chicks is not due simply to a developmental process independent of imprinting experience since naive controls of the same age rare-

ly showed the labelling pattern. Note that all controls had the same social and other experience except for the imprinting stimulus. Second, merely auditory experience with the imprinting stimulus appears not to be the crucial point since older, experienced groups which failed in tests showed hardly any labelling (group 7U-l). Third, a longer time lapse to establish an 'engram' is obviously not crucial since also a chick which experienced the 2DG session immediately after tests showed labelling (group 11-1). Fourth, there is no reason to believe that 2DG uptake is generally age-dependent in the forebrain since auditory fields are labelled similarly in all age groups. Two other explanations are more plausible which are derived from other forms of learning, namely conditioning. Elements of conditioning are not considered mandatory in classical theories of imprinting but appear to emerge in the recent imprinting literature ~. The two proposed explanations focus on the possibility that under some conditions the typical labelling effect may be found in younger chicks, due to an assumed lability of brain mechanisms during the sensitive phase. The first concept suggests that imprinting is susceptible to modifications, i.e., neural effects of imprinting are not crystallized before the end of the sensitive phase. The considerable behavioral discrepancy between relatively short imprinting tests in the

25 maze and the 2DG session (including prevention to approach the stimulus and much longer-lasting separation from siblings and home cage in the latter case) could have modified the initial imprinting. In other words, there may be extinction during the sensitive phase whereas after the sensitive phase the imprinting result is assumed to be stable. Consequently neuronal responsiveness to the imprinting stimulus may decrease or may even become inhibited during the unrewarding 2DG session. An alternative explanation would be a different persistence of attention and behavioral motivation in the age classes. Although in tests successful chicks of all age classes performed alike, in pilot experiments naive chicks showed age dependent behaviors when placed in an unpleasant strange environment (i.e. the Y-maze without any stimulus). After 2 min, 9 out of 10 chicks observed during their sensitive phase (1-3 days of age) were drowsy, whereas all 10 chicks observed after the sensitive phase (4-7 days of age) quickly walked around and explored the environment, for at least 10 min. Similarly, younger chicks might have lost interest in the imprinting stimulus during the 2DG session whereas older chicks showed more persistence. This persistent-attention-hypothesis receives support from recent 2DG experiments which show that all 3 areas relevant for imprinting may be selectively labelled in older chicks with stimulation of the reticular formation as well as in social stress situations which provoke intense arousal (MOiler and Scheich35). Thus there is a strong access to these areas from the reticular formation, a pathway which awaits clarification. The rationale for the age-dependent effect of the imprinting stimulus on metabolic activation could be as follows. The 2DG method is based on a cumulative principle and requires prolonged increase of neuronal activity to produce intense labelling51. Consequently, labelling in younger chicks could be negligible if the activation of the described brain areas were not persistent in this age group. This would be no conceptual contradiction to successful performance in behavioral tests which require only a few min of attention from the animals. Weak or absent labelling in chicks during the sensitive phase could mean therefore that except for brief increases the average

neuronal activity is low. After the end of the sensitive phase activity may become more consistently dependent on hearing the imprinted stimulus. This currently favored idea should be seen in the context of a parallel Golgi study of neurons in MHV of domestic chicks which describes a change of anatomical organization after imprinting (Wallh~iusser and Scheich55). There, a substantial reduction of dendritic spines of a common neuron type is found after the end of the sensitive phase if chicks were acoustically imprinted. In non-imprinted controls spine density remains high. This finding suggests a reorganization of connectivities in this area after the sensitive phase which may lead to the postulated persistent activation by the imprinting stimulus (see Discussion in ref. 55). Mechanisms of a presumably different nature are reflected by the fact that some of the controls showed the typical labelling pattern. Medium intensity labelling occurred in older chicks which were successfully imprinted on 2.5 kHz but heard the 1.8-kHz stimulus during the 2DG session. Possibly these chicks recalled the 1.8 kHz stimulus as the alternative stimulus of the discrimination tests or, in the absence of the favorite stimulus their responsiveness was caused by generalization. Note that the rhythm of both stimuli was virtually the same. Similarly the rare labelling found in some naive controls (group 7C) might have been caused by generalization. It was previously assumed that chicks kept socially, as was the case here, can imprint to one another, i.e. to follow contact calls or distress calls 17'41'49. During the 2DG session these chicks could have generalized the sibling call sequences to our motherlike synthetic calls which have a similar rhythm, as was shown to occur in behavioral experiments with ducklings 14,15. In summary, a certain age appears to favor labelling in those chicks which were successfully imprinted even if the imprinting occurred to an acoustic pattern which had only some similarity to the play-back stimulus. Some of the mechanisms which may cause increased metabolic activity in the relevant brain areas after the sensitive phase are described in the following paper 55. The working hypothesis proposed there is capable of explaining stimulus-specific activation of the areas including some generalization.

26 ACKNOWLEDGEMENTS We thank Mrs. 1. R6der and Mrs. M. Hansel for

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