Auditory imprinting leads to differential 2-deoxyglucose uptake and dendritic spine loss in the chick rostral forebrain

Developmental Brain Research, 31 (1987) 29-44

29

Elsevier BRD 50480

Auditory imprinting leads to differential 2-deoxyglucose uptake and dendritic spine loss in the chick rostral forebrain Elisabeth Wallh/iusser and Henning Scheich Institute of Zoology, TechnicalUniversity Darmstadt, Darmstadt (F.R. G.) (Accepted 15 July 1986)

Key words: Imprinting; Learning; Auditory system; Bird; 2-Deoxyglucose; Dendritic spine

Newly hatched chicks of the domestic fowl (White Leghorn) were imprinted to an acoustic stimulus (Group I: 400 Hz, 3 bursts per s, Group II: 900 Hz, 2 bursts per s) and tested in a straight runway with loudspeakers behind two opposite goal boxes. Those chicks were considered imprinted which headed for the imprinting stimulus in an approach test and subsequently preferred it to a new stimulus (imprinting stimulus of the other group) in a simultaneous discrimination test. On day 7 after hatching (after the sensitive phase) imprinted chicks and naive controls were injected with 2-[14C]deoxyglucose (2DG) and exposed to the imprinting stimulus. Autoradiographic analysis of their brains revealed 3 well demarcated areas of increased 2DG accumulation in the rostral forebrain of imprinted chicks compared to controls: (1) HAD in the rostral Wulst; (2) MNH, an auditory area in the rostromedial neostriatum and hyperstriatum ventrale; (3) LNH in the rostrolateral neostriatum and hyperstriatum ventrale. Analysis of these brain areas in 7-day-old acoustically imprinted and control animals with the Golgi-Cox method revealed a highly significant reduction of spine frequency of a large neostriatal neuron type in MNH of imprinted chicks. An additional Golgi-Cox analysis was carried out with chicks imprinted on a broody hen, i.e. on the whole spectrum of natural stimuli. In that group spine frequency of the same neuron type was between that of acoustically imprinted and control animals. A hypothesis of filial imprinting is presented which consideres spine loss as a crucial mechanism. INTRODUCTION L o r e n z 53 characterized the p h e n o m e n o n of filial imprinting in nidifugous birds by a n u m b e r of criteria. Two of L o r e n z ' s criteria p r o v e d to be m o r e generally valid, n a m e l y that an a d e q u a t e stimulus presented during a sensitive phase has a permanent effect on behavior. This distinguishes imprinting from other learning processes where forgetting and revision of learning a p p e a r s to be n o r m a l and no sensitive phases are known 43. Some authors conceive the sensitive phase to be a genetically p r o g r a m m e d ontogenetic process (see ref. 37). Following this hypothesis there should be a g o o d chance to detect some reorganization of brain structures, which could be held responsible for d e t e r m i n i n g a sensitive phase 74. The p h e n o m e n o n of filial imprinting has b e e n thoroughly e x a m i n e d on the behavioral level 27'31'37'54

as to sensitive phase, type and p r e s e n t a t i o n of stimuli. Classical experiments on filial imprinting focus on following responses of nidifugous birds to a m o t h e r object 31. Most e x p e r i m e n t o r s used visual stimuli as a m o t h e r substitute. It has been known for a long time, however, that m a n y species of birds including domestic chicks are particularly susceptible to auditory imprinting stimuli (for review see ref. 37). Fischer 27 and M a i e r and Scheich 54'55 showed that domestic, respectively G u i n e a fowl chicks can successfully be imprinted on acoustic stimuli alone. M o r e recent investigations are concerned with physiological, biochemical and morphological changes after visual imprinting in chick brains 6'11-13' 38,50,58,70,71. Several forebrain areas were identified which showed postimprinting changes and m a y be involved in the imprinting process. N o n e of these methods applied could test for a direct response to

Correspondence: E. Wallh/iusser, Institute of Zoology, Technical University Darmstadt, Schuittspahnstrasse 3, 6100 Darmstadt, F.R.G. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

3~J the imprinting stimulus. Thus it was difficult to establish whether those brain areas indeed processed that imprinting stimulus or showed epiphenomenal changes as a result of stimulus learning elsewhere in the brain. This gap is closed by the 2-deoxyglucose (2DG) method 77 which clearly identifies stimulus-specific metabolic responses similar to electrophysiological analysis35'4°'72'76. One important consequence is that 2DG imprinting studies allow social rearing of chicks (prevention of deprivation effects) since other learned experiences do not interfere with the 2DG test for the imprinted stimulus, as shown by the auditory imprinting studies in Guinea fowl chicks by Maier and Scheich 5~'55. The 2DG method has also been applied to visually imprinted domestic chicks with partially deviating results 5°. In contrast to the visual imprinting study with 2DG 5° an internal comparison of auditory processing between imprinted and naive birds was obtained because tonotopic representation of the acoustic stimulus can be monitored in 2DG autoradiographs of the auditory nuclei 3s'54'72. The experiments in this study were undertaken with the aim to identify Golgi-morphological correlates in the forebrain areas of domestic chicks defined by 2DG labelling after acoustic imprinting ('°'~'1. A typing of Golgi-stained neurons in the relevant areas preceded this study 85.

MATERIALS AND METHODS

2DG experiments Subjects Eggs of White Leghorn chickens were obtained from a local hatchery and incubated in our laboratory at 37.5 + 0.5 °C. After hatching chicks of both sexes were reared in 8 groups of 4 - 8 animals each (room temperature 25-27 °C, day/night cycle 12 h/12 h). Commercial food and water were available ad lib.

Behavioral procedures A description of auditory imprinting in Guinea fowl chicks has been published 54. In the present study the experimental animals were divided into two groups. Group I was imprinted on a rhythmic tone burst of 400 Hz with a repetition rate of 3 bursts/s and

burst duration of 70 ms. This stimulus was prcviously found to be effective as an imprinting stimulus for domestic chicks :7. The imprinting stimulus presented to group II consisted of a rhythmic tone burst of 91)1/Hz with 2 bursts/s and 70 ms burst duration, Imprintability by this stimulus was determined through pretests in our laboratory. Low frequency sounds with those repetition rates are predominant in hens" clucking and roosting calls, whereas chicks' own vocalizations are above 2 kHz 17. The imprinting stimulus was presented continuously with an intensity of 80 dB SPL beginning at day 14 of incubation until hatching. This procedure was used to prime the imprinting process scheduled after hatching since prenatal auditory imprinting had been observed in chicks 1~'23"32. On the first two days after hatching cage mates were placed together with a stuffed hen emitting the stimulus through a built-in loudspeaker (65 dB) for two l-h sessions per day. On the following days approach and discrimination tests were performed to ensure that imprinting had been successful. Tests were conducted in a straight runway (1.5 x 0.5 m) with goal boxes at both ends. In one of these boxes the stuffed hen could be placed, invisible to the chicks unless they entered the box. Consequently, the hen could not serve as a visual cue to locate the correct sound source. It only served as reinforcement to keep the chick close to the sound source during repeated testing. In approach tests only the imprinting stimulus was presented by a loudspeaker inside the hen (about 65 dB). In discrimination tests the animals heard the imprinting stimulus from the loudspeaker inside the hen (65 dB) together with an unknown sound (imprinting stimulus of the other group) emitted by a second loudspeaker (65 dB) placed behind the opposite end of the runway. Tests scored correct, if a chick entered first the box from which the imprinting stimulus was played first and stayed with the hen for 30 s. At the end of each trial chicks were taken back to their cage mates. Each test consisted of 4 trials. In tests the occurrence of the imprinting stimulus was balanced between right and left box for each chick according to a random schedule. Lumping the results together a chick was considered imprinted, if at least 3 out of 4 trials in each test scored correct. The random incidence for a positive result (multiplication theorem for independent samples) was P = 0.0256.

31

2DG exposure On day 7 after hatching 2 D G experiments were conducted with 6 successfully imprinted chicks and 6 naive controls which had not been exposed to the imprinting stimulus before. At that age the sensitive phase for filial imprinting, lasting the first 4 days after hatching 33, was already over. Thus accidental imprinting during the experiment could be excluded for control animals. Imprinted chicks, however, still showed following behavior. Six successfully imprinted chicks (3 of group I, 3 of group II) and 6 naive controls were injected with 2fluoro-2-deoxy-D-[U-14C]glucose (Amersham), 18 pCi/animal in 0.2 ml sterile saline into the pectoral muscle. Subsequently each chick was placed in a cage inside a sound-proof chamber illuminated by diffuse light. There it heard its imprinting stimulus, respectively the unknown sound (controls), delivered by a loudspeaker at 65 dB. After 60 min it was decapitated, the brain removed and frozen on the freezing stage of a cryostat. Brains were cut at 30/~m in the transverse plane. Serial sections were collected on microscope slides, rapidly dried at 50 °C, pressed on Kodak NMBX-ray film in Kodak-X-Omatic cassettes and exposed for two weeks. Thereafter they were stained with Cresyl violet and structures were identified by superposition with the autoradiographs using stereotaxical atlases of Karten and Hodos 46 and Youngren and Phillips 88.

Densimetric analysis Three autoradiographs at the level of H A D , L N H and MNH in the rostral forebrain of each chick were analyzed with a two-dimensional image processor 30,35. In all autoradiographs, a reference density measurement was made in a comparable area of low and even background labelling not influenced by the experiment, in the most medial part of lobus parolfactorius (Fig. 1). Density ratios relative to background in the reference area were calculated in order to compensate for labelling differences between individual animals. For two-dimensional displays density ratios of picture points were integrated to a map of 32 horizontal profiles (Fig. 2). Peak values of density ratios from the 4 largest profiles over H A D , LNH and MNH each were obtained from the map of a given autoradiograph. Means of the 12 peak values of 3 au-

toradiographs are presented in Table I for each area in individual animals.

Golgi-Cox experiments In another group of chicks (White Leghorn) differences in neuron morphology between acoustically imprinted, naturally imprinted, and not imprinted animals were investigated with the Golgi-Cox method. Results obtained with the 2DG method in socially experienced chicks are not disturbed by incidental imprinting to additional stimuli, since only the response of a brain to the stimulus played back during the 2DG experiment is measured. This is different with the Golgi-Cox method which reflects a morphological state resulting from the sum of influences on the brain. Consequently, animals whose brains were examined with the Golgi-Cox method, were socially isolated in order to exclude stimuli which could lead to incidental imprinting. Since they were kept in a lighted environment with some noise level, they were less sensorily deprived than animals in visual imprinting studies 39'5°.

Behavioralprocedures Isolation started 3 days before hatching because acoustic communication between siblings was observed at least on the day before hatching 31'81. After hatching the animals were kept solitary in sound-protected chambers with a constant illumination at a temperature of 25-27 °C. White noise of 45 + 2 dB was used as a background to further mask auditory perception of outside stimuli. Additionally, experimental chicks were continuously exposed to the imprinting stimulus (400 Hz, 3 bursts/s, burst duration 70 ms) starting on the 14th day of incubation until hatching. They were stimulated on the first two days after hatching for 1 h twice a day. On the following 5 days chicks were removed from the isolation chambers for a short time to test imprinting success in approach and discrimination tests as described for the 2DG experiments. A third group of eggs was incubated in the laboratory for 13 days and then exchanged with the clutch of a broody hen. Chicks hatched and were adopted by the hen under natural conditions on a farm. Their responses to the hen and following behavior was observed and considered normal.

32

Histology

M N H w e r e m a d e at a m a g n i f i c a t i o n of × 15(I(~ in or-

Brains of 13 a n i m a l s , 7 days old (two successfully

der to classify n e u r o n s in t h o s e areas. N e u r o n s w e r e

i m p r i n t e d to the artificial acoustic stimulus, 5 natu-

typed c o n s i d e r i n g size (1) and shape (2) of perika-

rally i m p r i n t e d and 7 c o n t r o l s ) w e r e p r o c e s s e d ac-

ryon, d i a m e t e r (3), o r i e n t a t i o n (4) and branching

c o r d i n g to the G o l g i - C o x

m e t h o d as m o d i f i e d by

p a t t e r n (5) of d e n d r i t e s 85. D e n d r i t e s of the so d e f i n e d

Van d e r L o o s s4. C a m e r a lucida d r a w i n g s of a b o u t

n e u r o n types in the 3 groups of animals w e r e c o m -

100 n e u r o n s in h y p e r s t r i a t u m a c c e s s o r i u m ( H A ) and

p a r e d in a p r e l i m i n a r y fashion with respect to n u m -

Fig, 1. Autoradiographs of transverse forebrain sections of 7-day-old imprinted and control animals. Both imprinted groups (stimulus A: 400 Hz, 3 bursts/s, stimulus B: 900 Hz, 2 bursts/s) show a marked increase of 2DG accumulation after 60 min exposure to the imprinting stimulus in 3 clearly discernible rostral forebrain areas. These are area HAD in the rostral Wulst (1), area MNH in the rostromedial neostriatum and hyperstriatum ventrale (2) and LNH an area in the rostro-lateral neostriatum and hyperstriatum ventrale (3). In contrast, controls exhibit low and even background labelling in those areas. High 2DG accumulation was also observed in the ectostriatum, no matter whether the animals were imprinted or not (reference density measurements for densimetric analysis, see Fig. 2, covered the area in lobus parollactorius marked by the window). E, ectostriatum; HA. hyperstriatum accessorium: HD, hyperstriatum dorsale: HV, hyperstriatum ventrale: LH, lamina hyperstriatica; N, neostriatum.

33

Imprinted

656 from chicks imprinted to the hen and 1038 from control animals) were counted and spine frequency was determined as number of spines per 10/~m. All results were statistically analyzed using the onetailed M a n n - W h i t n e y U-test.

Control

RESULTS 2DG method

1ram

Fig. 2. Computer-generated densimetric profiles of autoradiographs as shown in Fig. 1, left hemisphere. Areas 1-3 are equivalent to areas 1-3 in Fig. 1. A reference density measurement in the medial part of Iobus parolfactorius (windows in Fig. 1) was set to 1. For two-dimensional display density ratios of picture points relative to reference were integrated to a map of 32 horizontal profiles. Scales on the right side of each inset represent the reference value of each profile. Upward deviations from reference levels represent increased 2DG uptake. The large scale shows the range of density ratios between 1 and 3 aligned with the bottom profile. ber of spines per 10/~m (spine frequency) (6). Magnification for determining spine frequency was x 2500. The survey of H A and M N H revealed one large neuron type in the neostriatal part of M N H with differences in spine frequency dependent on the state of imprinting. From this type systematic spine counts were taken if the individual neurons were well impregnated and most dendrites were met in the horizontal plane. Neurons were selected under low power to avoid a bias with respect to spine frequency. Dendrites were divided into the following segments: basal, behind first branching, middle and distal segment. Ten neurons from each of the 14 animals were analyzed. Spines of a total of 2044 dendritic segments (350 from chicks imprinted to the artificial stimulus,

Autoradiographic analysis of the brains of imprinted domestic chicks revealed essentially the same labelling pattern as seen in acoustically imprinted Guinea fowl chicks of the same age 54. Between imprinted chicks and controls no systematic difference was found in the tonotopically labelled telencephalic auditory areas such as field L and the overlying hyperstriatum ventrale 8,72,8°. High 2 D G accumulation was also observed in the ectostriatum no matter whether the animals were imprinted or not. Beside illumination of the test chamber this may be due to a high spontaneous activity in the ectostriatum, since high metabolic activity may also be found without visual or auditory stimulation 54. However, there were marked differences between imprinted animals and controls in the degree of 2 D G uptake in the rostral forebrain around the anterior pole of the ectostriatum (Fig. 1). In imprinted chicks 3 areas or regions (defined by the labelling pattern) showed enhanced metabolic activity. (1) The dorsal forebrain roof (Wulst), including the H A proper, nucleus intercalatus hyperstriatum accessorium, hyperstriatum intercalatum supremum, and lateral parts of hyperstriatum dorsale, together designated H A D . (2) A rostromedial area close to the ventricle including mostly neostriatum and a narrow strip of hyperstriatum ventrale across the lamina hyperstriatica, to-

TABLE I 2DG uptake in areas HAD, LNH, M N H in individual animals

Densimetric values represent factors x background intensity in the reference area which is set to 1. Numbers are the averages of peak values of profile analysis as shown in Fig. 2 (see Materials and Methods). Imprinted stimulus A

Control stimulus A

Imprinted stimulus B

Control stimulus B

HAD

LNH

MNH

HAD

LNH

MNH

HAD

LNH

MNH

HAD

LNH

MNH

1.89 2.41 1.67

1.64 3.19 1.68

2.48 2.86 1.73

1.07 1.11 1.15

1 1 1

1 1 1

1.39 1.25 2.09

1.71 1.52 1.88

1.56 1.25 2.69

1.15 1.16 1.18

1 1 1

1 1 1

34 gether designated M N H (medial neostriatum/hyperstriatum ventrale). (3) Part of the neostriatum and hyperstriatum ventrale anterior and dorsolateral to the ectostriatum, together designated L N H (lateral neostriatum/hyperstriatum ventrale). In control animals those areas showed merely background activity which resulted in a rather homogeneous labelling of the rostral forebrain except the visual ectostriatum (Figs. 1 and 2, and Table I).

Golgi-Cox analysis Our classification of the 70 neurons drawn from H A agreed in principle with that of Watanabe et al. 86 in the quail. By using morphological criterial 1-5

(Materials and Methods) 7 different neuron types could be distinguished 85. Preliminary analysis of this material did not reveal any obvious differences of spine frequency between imprinted and control animals which is in accordance with the findings of Bradley and Horn II in visually imprinted chicks. In the neostriatal part of the M N H 4 different neuron types could be distinguished sS. One neuron type was found which resembled migrating neurons 66. The 3 remaining types were isopolar and differed in soma size and several other respects. A small spineless neuron had a soma diameter of 11/~m, many short dendrites and was quite rare. The second type, a medium-sized spiny neuron with a soma of 12urn diame-

3

i

1500p m ,

,

ii

1250p m i

Fig. 3. Left area MNH in Nissl and Golgi-Cox stain. The Golgi preparation shows a group of large impregnated neurons (large arrowhead) in the neostriatal part of MNH. These neurons lose spines as a result of imprinting. A pair of smaller neurons below which do not reduce spines is indicated by a small arrowhead. Hv, hyperstriatum ventrale; LMD, lamina medullaris dorsalis; LPO, lobus parolfactorius; nMNH, neostriatal part of MNH; V, medial ventricle; m, medial; d, dorsal.

35

Fig. 4. Photographic m o u n t s of G o l g i - C o x - i m p r e g n a t e d large n e u r o n s from the neostriatal part of M N H of 7-day-old chicks. A: neuron from a chick imprinted to 400 Hz. B: from a chick imprinted u n d e r natural conditions to a hen. C: from a non-imprinted control.

ter had 4 - 6 dendrites longer than the former type with several branchings. It often occurred in pairs or triplets (see Fig. 3). The third type had a large soma with a d i a m e t e r of a b o u t 20ktm but a length of the spiny dendrites not longer than the second type. Den-

A

drites were rather thin with diameters of about i ltm and with up to 4 branchings per dendrite. Fig. 3 gives an overview of the M N H with several impregnated neurons of the second and third isopolar types. The large neurons of the third type showed a m a r k e d dif-

C

B

I

I

50 ;urn

Fig. 5. Camera lucida drawings of the same neuron conditions A, B and C as in Fig. 4. Note the different spine frequencies. Dorsal axons are indicated by arrowheads.

37 TABLE II Means and standard deviations of spine frequencies per lO ~m of the large nMNH neuron type in individual animals Animal

Basal

First branching

Middle segment

Distal segment

Imprintedwith~0Hz 1 2

5.4±3.9 3.9±3.1

9.3±3.6 9.0±3.4

11.6±3.5 9.0±2.9

13.2±3.8 11.4±3.9

Imprintedwithhen 1 2 3 4 5

3.7±2.1 8.6±4.4 3.8±2.7 4.7±3.6 4.4±3.1

13.8±2.6 15.0±4.7 11.7±3.2 11.5±3.9 10.4±3.5

15.3±2.9 17.4±3.5 14.5±2.7 15.3±3.8 15.0±3.0

14.9±2.7 17.9±3.0 15.3±3.3 16.7±3.4 16.4±3.2

9.2±4.2 8.2±3.6 10.6±4.3 9.9±6.0 11.0±6.1 7.2±5.8 8.4±6.3

14.8±4.7 16.1±4.0 17.8±5.2 18.8±5.0 17.1±5.8 16.5±5.0 19.0±6.5

18.0±3.1 18.0±3.1 21.5±3.3 19.9±3.7 21.8±3.9 17.9±4.2 19.9±4.6

17.8±2.9 18.1±3.7 21.5±3.5 20.4±3.9 22.5±4.4 18.6±4.2 21.0±5.2

Controls 1 2 3 4 5 6 7

Fig. 6. Photographs of dendrites from large neurons as in Figs. 4 and 5. A imprinted to 400 Hz, B imprinted to a hen, C non-imprinted control. Bar = 10/~m.

ference of dendritic spine frequency between imprinted animals and controls (Figs. 4 - 6 ) . The spine frequency of large neurons was analyzed in animals imprinted to 400 Hz, imprinted to the living hen and in controls. Since there were no significant differences in spine frequency between animals within each group (Table II), values of the animals in a group were pooled. Spine counts were not corrected for hidden spines as the diameter of dendrites was small compared to the length of spine-stalks and did not differ among the groups. Hence, an error originating from hidden spines should be the same in all groups. In all 3 groups spine frequencies are lowest in the basal and highest in the medial and distal segments. In contrast absolute spine frequencies varied between the 3 groups. On the average spine frequency in naturally imprinted chicks was smaller by about 27% compared to controls (Fig. 7, Table III). Chicks which were imprinted to the artificial acoustic stimulus showed an even further reduction of spine frequency which was about 45% compared to controls and about 22% compared to naturally imprinted chicks. With the exception of the basal dendritic segments in naturally and acoustically imprinted chicks, spine frequency differences in all other segments were found to be highly significant (P < 0.0001) between all 3 groups (one-tailed M a n n Whitney U-test).

38

~

25-

imprinted imprinted • control

E o

tion. There are aspects of early brain reorganization including degeneration which roughly correlate with a sensitive phase 79"~7, but the key issue is to identify effects which clearly d e p e n d on an imprinting stimulus. A n o t h e r area of possible confusion is deprivation. It is known that sensory deprivation, for instance prevention of the most e l e m e n t a r y visual information by keeping animals in the dark during dev e l o p m e n t , will have fundamental effects on behavioral, biochemical, physiological and morphological aspects of brain organization 3'19'24'25'29"~'46~'69"7~'s3.

nMNH

( 4 0 0 Hz) (hen)

15 o.

E

(D_

CJ~ 09 m

"5 E

n

79

148 224

basal

73 115 226

100 216 350

98 177 4C1

lStbranching

middle s e g

distal

Fig. 7. Histogram showing the average spine frequencies (number of spines per 10~m) of 10 large MNH neurons per animal. Data are from two chicks imprinted to 400 Hz, 5 animals imprinted to a live hen, and 7 controls. Numbers (n) at the bottom refer to dendritic segments included in each column. Thin bars represent + S.D. Since there were no pronounced differences within each group (Table III), values from animals in a group were pooled. Dendrites were divided into basal, behind 1st branching, middle and distal segments. Spine frequencies are highest in controls (black columns), lowest in artificiallyimprinted (white columns) with a reduction of about 45% and intermediate in naturally imprinted animals (hatched columns) with a reduction of 27% compared to controls. Except for the difference in the basal segment of the two imprinted groups all differences are highly significant (P < 0.0001) Mann-Whitney U-test. DISCUSSION

Implications of the 2 D G method for imprinting studies The identification of brain areas relevant for filial imprinting and the analysis of imprinting mechanisms critically d e p e n d on tests which are capable of separating imprinting effects on the brain from widespread non-specific correlates of perinatal matura-

T h e r e f o r e it is not entirely a p p r o p r i a t e to work with such animals in imprinting studies. The 2 D G m e t h o d , in principle, is suitable to detect sensory learning in the brain since it visualizes the parallel metabolic activity in the neuronal spaces reached by a particular stimulus before and after a learning process. The fundamental difference to most other histochemical techniques is that it allows a direct test for a changed responsiveness of neurons to a particular stimulus and does not simply reflect an alteration of a biochemical substrate as a consequence of learning. Thus, the 2 D G m e t h o d when visualizing stimuli identification by neurons is more closely related to the aspect of information transfer in the nervous system. 2 D G labelling was previously shown to be a g o o d correlate of local e v o k e d potentials 2 and also of e v o k e d spike activity8°. Since the m e m b r a n e sodium p u m p uses the bulk of the energy from glucose metabolism 56, synaptic neuropit with its high volume fraction of m e m b r a n e s from presynaptic terminals and dendrites and not p e r i k a r y a a p p e a r to be the focus zone of 2 D G uptake 36"~':. A p r e d o m i n a n t 2 D G uptake in synaptic neuropil reflecting local preand postsynaptic electrical activity favors the use of the m e t h o d for learning studies 3"'54.

TABLE III

Reduction of spine frequency in % Either the reduction is highly significant or not significant (n.s. ; P > 0.05), one-tailed Mann-Whitney U-test.

Basal segment Imprinted (400 Hz) compared to imprinted (hen)

8.3 (n.s.)

First branching

Middle segment

Distal segment

26.3*

31.6*

22.9*

Imprinted (hen) compared to control

45.3*

23.4*

20.8*

18.8"

Imprinted (400 Hz) compared to control

49.8*

43.5*

45.8*

37.4 ~

*P < 0.0001.

39

Functional inputs to the areas relevant for imprinting The present study confirms the results in Guinea fowl chicks54'55 for domestic chicks and for two imprinting stimuli, 400 Hz 3 bursts/s and 900 Hz 12 bursts/s. Areas labelled were the same as in Guinea fowl chicks. Several interesting features of areas HAD, MNH and LNH may be relevant for the assumed functional role in filial imprinting. Labelling of each of the areas, the dorsal HAD, the medial area MNH and the lateral LNH was sharply demarcated from surrounding tissue suggesting a confinement of relevant neuronal mechanisms to these neuronal spaces. This is intriguing in a functional anatomical sense since all 3 areas defined by high 2DG uptake, in spite of continuous labelling, reach across at least two of the major dorsoventral subdivisions of the bird telencephalon. Labelling of the H A D in the rostral Wulst covers in dorsoventral sequence the anatomical subdivisions HA, IHA, HIS and HD, confined ventrally by the lamina frontalis superior (LFS). It covers the projection of the thalamofugal visual pathway to the ventral Wulst layers IHA, HIS and HD 4'21' 48,29. The lateral area LNH involves rostral neostriarum and hyperstriatum ventrale separated by the lamina hyperstriatica. The neostriatal part includes the periectostriatal belt areas which are extensively interconnected with ectostriatum, the target of the tectofugal visual pathway 47. Labelling of LNH is continuous with that of prominent metabolic activity in the ectostriatum but has its peak rostral to the tip of the ectostriatum. MNH adjacent to the medial ventricle involves neostriatum and a strip of hyperstriatum ventrale dorsally just across the lamina hyperstriatica. Consequently, we may have functional networks defined by the 2DG labelling in which the dorsal subdivisions, i.e., hyperstriatum ventrale in the cases of MNH and LNH and hyperstriatum accessorium in the case of HAD, are known to receive little or no primary sensory input, but may receive relayed input from ventral subdivisions, i.e., from neostriatum or from the ventral Wulst layers. As indicated by their connectivities H A D and LNH are activated by visual input. This input must be of a nature to strongly capture the animal's attention, as shown by 2DG experiments 6°. Attention-dependent activation in H A D and LNH was also described in visually discriminating pigeons 7s. In contrast,

MNH alone is labelled with 2DG by play-back of chick vocalizations to socially reared chicks isolated in the dark (Miiller and Scheich, in preparation). This suggests relevant auditory input to the MNH, which is further substantiated by electrophysiological recordings in our laboratory (unpublished). There are previous reports on auditory responses in the rostral neostriatum of pigeon and starling 22"42'49. In summary, the two areas H A and LNH may be activated by behaviorally relevant visual and MNH by relevant auditory stimuli. The surprising result in the present play-back experiment after imprinting is that an auditory imprinting stimulus alone activates all 3 areas together. These features are suggestive of a common activating mechanism which functionally interconnects the 3 areas in terms of a system. The common behavioral denominator may be the wellknown fact that filial imprinting is optimally achieved when visual and auditory stimuli act together 37. A result supporting this hypothesis of a functional interconnectivity of the 3 areas is a concomitant increase of activity in a social stress situation which produces strong arousal 6°. Thus arousal and specific attention mediated by the reticular formation may coactivate the 3 areas and gear them together for simultaneous processing of visual and auditory cues.

Brain correlates of visual imprinting studies Other attempts to identify brain mechanisms of filial imprinting, to our knowledge, were restricted to the visual modality in domestic chicks. There, brain incorporation of uracil during imprinting 38, loss of retention of an imprinted stimulus after lesions 58, training by electrical brain stimulation 57 and some other experiments 39 led to the identification of an area in the intermediate hyperstriatum ventrale (IMHV). This area was not labelled in any of the described experiments from our laboratory54"55'6°. As judged from the published material, IMHV was not sharply defined in the anteroposterior direction but must be caudally adjacent to our area MNH, using the ectostriatum as a landmark. Due to the visual modality, the possible consequences of dark-rearing, and to the undefined role of acoustics the results of these experiments at present are difficult to reconcile with ours. Kohsaka et al. 5° used the 2DG method while exposing visually imprinted chicks to their imprinting

4(1 stimuli. Interestingly, they found increased labelling in large areas of the rostral forebrain covering our areas MNH and LNH, but apparently little effects in IMHV of the above authors, using the ectostriatum as a landmark in published figures. The two labelled areas were not sharply demarcated, however. The lateral area in the rostral neostriatum/hyperstriatum ventrale labelled in experiments of Koshaka et al. 5° was previously lesioned by Saizen et al. 7°'7j and implicated in acquisition and retention of visual imprinting stimuli, while McCabe et al. 5s could not replicate these lesion effects. In spite of these discrepancies with our results it appears entirely possible that other imprinting paradigms cause alterations in other brain areas since filial imprinting behavior covers so many different aspects of brain function that the assumption of one brain area involved is counter-intuitive.

Dendritic spines and synaptic selection hypothesis of imprinting Loss of neuronal elements among other signs of plasticity appears to be a rather widespread and characteristic phenomenon during postnatal ontogenesis of the brain 9"10"20'2s'41"45"51"52'65"73. More specifically, there is a rapid loss of synapses in the rat visual cortex after the sensitive phase for occular dominance organization is, and a loss of spines on granule cells of the olfactory bulb in the ferret around the end of the sensitive phase for food odor imprinting I. Rausch and Scheich 67 reported a 50% reduction of spine frequency on dendrites of the largest neuron type in the vocal-motor nucleus HVc of mynah birds. In that case spine reduction during the first year correlated with a reduction of many variable utterances to a small stable repertoire of imitated sounds, Functional development of visual cortex and vocal learning in song birds bear several features in common with filial imprinting including sensitive phase and irreversibility 7. Consequently we suspect reduction of synapses to be a central aspect of imprinting mechanisms. How could spine reduction be integrated into concepts of information storage? There appears to be some evidence that in adult animals various forms of learning are accompanied by growth of neurons including formation of new synapses rather than by a lOSS15"26'34'63"75'82. Imprinting on the other hand, has properties which makes the reverse process more

likely. As proposed by Bateson 5 imprinting can be thought of as narrowing pre-existing prelerences. First, there is the large body of evidence cited above that during postnatal ontogenesis, i.e., during the period of most imprinting phenomena, loss of neuronal elements is seen in many brain areas. Second, imprinting has characteristics in contrast to conventional learning which favors a selection model of neuronal connectivity. Arguments are provided not so much by the accepted chief characteristic of all forms of imprinting, i.e., the sensitive phase 44, but by some other features originally proposed by Lorenz s3 for filial imprinting, which may be crucial for an understanding of underlying brain mechanisms. For instance, imprinting appears to be restricted by a frame of supraindividual species-specific characters, i.e., a limited choice of stimulus patterns on which a given species may be imprinted. This aspect advocates a species-specific predisposition and limitation of the type of information which may be imprinted. Second, within the range of a predisposition animals may be imprinted on extremely simplified dummies, i.e. a few crucial properties make a dummy acceptable. This may relate to another of Lorenz's criteria namely that learning is completed before the behavior is actually performed. In contrast to other forms of learning there appears to be little feedback involved in testing the appropriateness of a stimulus. Thus, whether a stimulus is biologically meaningful, even if it is extremely impoverished or grotesque, is not relevant for the imprinting success. Third, after imprinting the preference for the imprinting stimulus is largely irreversible making corrections almost impossible. After considerable controversy over the years it appears to be clarified by double-choice tests that preference for an original imprinting stimulus is stable 16,43. These three characteristics together are compatible with the following hypothesis at the level of brain mechanisms. A neuronal network which selects for imprinting stimuli requires only a limited number of preliminary connections covering in some way the range of expected stimuli in a given species. The imprinting process consists of a selection for a subset of these options represented by some proportion of total connections. The chosen connections are stabilized and superfluous connections are dropped making the selection irreversible after the sensitive phase.

41 This working hypothesis allows several predictions some of which have been tested in the present work. First, a brain area involved in imprinting should respond to an imprinting stimulus by a reduction of preliminary connections. The reduction of dendritic spines may be taken as a correlate of reduction of synapses, as evidenced by Turner and Greenough 82 in a combined Golgi and EM study in normal cortex. Another expected effect is that without feedback control of biological relevance and little possibility of correction there should be a high tolerance for simplification of stimuli. Thus the number of connections kept after the end of the sensitive phase should simply reflect the complexity or simplicity of the imprinting stimulus, i.e., the amount of information necessary to define that stimulus. The fact that chicks imprinted on a living hen keep more spines than those imprinted on the simplified auditory stimulus appears to support this point. Third, if the area involved in stimulus selection is prevented from making this selection, preliminary connections, i.e. spines, should be kept for longer periods exceeding the sensitive phase. This test is presently under way. Preliminary data from chicks kept in isolation for 20 days show that these controls have spine frequencies comparable to corresponding controls at day 7. Most relevant is the finding from these studies that such socially isolated chicks remain imprintable after the sensitive phase. Synopsis o f 2 D G and Golgi data 2DG labelling after play-back of the imprinting stimulus to successfully imprinted chicks is seen in area M N H which obviously experienced spine reduction by the preceding imprinting process. These two phenomena together raise some additional issues of interpretation. How can a neuronal network which has lost a substantial amount of connections show increased metabolic activity during specific stimulus REFERENCES 1 Apfelbach, R. and Weiler, E., Olfactory deprivation enhances normal spine loss in the olfactory bulb of developing ferrets, Neurosci. Lett., 62 (1985) 169-173. 2 Auker, C.L., Meszler, B.M. and Carpenter, D.O., Apparent discrepancy between single-unit activity and 14C deoxyglucose labeling in optic tectum of the rattlesnake, J. Neurophysiol., 49 (1983) 1504-1516. 3 Bagnoli, P., Burkhalter, A., Vischer, A., Henke, H. and

presentations? There are at least two tentative explanations which are not mutually exclusive. The 2DG method is a cumulative method which requires a persistently high electrical activity in a large neuronal space to produce contrast. Consequently there could be temporal or spatial failure of 2DG labelling in an area which nevertheless may show electrical activity of neurons. In our case 2DG may be sufficiently accumulated over the exposure time of 60 min only after synapses have become stabilized by the selection process. This view is supported by a 2DG study of chicks during the sensitive phase which rarely show the characteristic labelling pattern even though they have passed the behavioral imprinting tests (see Maier and Scheich55). In other words, some of the initially abundant preliminary connections may first go through a functional stage which allows short periods of attention to the imprinting stimulus and performance of following behavior but not a degree of persistent electrical activity which would show up during the long time of 2DG exposure. This point is emphasized by a much lower persistence of chicks in the behavioral test arena during the sensitive phase than after it 55. Another idea is that synaptic stabilization and spine loss entail some reorganization of MNH in such a way that the imprinting stimulus can activate the whole of the area. This would require the assumption that before these events activation of MNH is limited to some neurons or that neurons within the local network cannot activate each other. These alternatives of temporal or spatial failure of 2DG labelling are testable and experiments are under way to resolve this issue.

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