Development of the time coding pathways in the auditory brainstem of the barn owl

THE JOURNAL OF COMPARATIVE NEUROLOGY 373:467-483 ( 1996)

Development of the Time Coding Pathways in the Auditory Brainstem of the Barn Owl

C.E. CARR AND R.E. BOUDREAU Department of Zoology, University of Maryland, College Park, Maryland 20742-4415

ABSTRACT The barn owl’s head grows after hatching, causing interaural distances to more than

double in the first 3 weeks posthatch. These changes expose the bird to a constantly increasing range of interaural time cues. We have used Golgi and ultrastructural techniques to analyze the development of the connections and cell types of the nucleus magnocellularis (NM) and the nucleus laminaris (NL) with reference to the growth of the head. The time coding circuit is formed but immature at the time of hatching. In the month posthatch, the auditory nerve projection to the NM matures, and appears adult-like by posthatch day (P)21. NM neurons show a late growth of permanent dendrites starting at P6. Over the first month, these dendrites change in length and number, depending upon rostrocaudal position, to establish the adult pattern in which high best frequency neurons have few or no dendrites. These changes are not complete by P21, when NM neurons still have more dendrites than in the adult owl. The neurons of NL have many short dendrites before hatching. Their number is greatly reduced by P6, and then does not change during later development. Like NM neurons, NL neurons and dendrites grow in the first month posthatch, and at P21, NL dendrites are longer than those in the adult owl. Thus, the auditory brainstem circuits grow in the first month after hatching, but are not yet mature a t the time the head reaches its adult size. , I W ~ h’ i~ey-~iss , ~ n c .

Indexing terms: cochlear nucleus, magnocellularis, laminaris, auditory nerve

Barn owls use interaural time differences to determine the azimuthal location of a sound (Knudsen and Konishi, 1979a,b; Moiseff, 1989; Olsen 1989). These time cues are processed in a specialized pathway in the central auditory system in which the timing of the auditory stimulus is preserved in the phase-locked firing of the auditory nerve. The auditory nerve enters the brain and innervates the two cochlear nuclei (Boord and Rasmussen, 1963; Takahashi 1984; Takahashi and Konishi, 1988a). The cochlear nucleus magnocellularis (NM) is specialized for the processing and encoding of phase information, and projects bilaterally to the nucleus laminaris (NL) (Sullivan and Konishi, 1984; Takahashi and Konishi, 1988a,b; Carr and Konishi, 1990). Sensitivity to interaural time differences first appears in NL, a brainstem nucleus that is homologous to the mammalian medial superior olive (Boord, 1968; Carr and Konishi, 1988; Carr and Konishi, 1990). The projection from NM to NL forms a circuit that conforms to the model proposed by Jeffress to explain sound localization by detection of interaural time differences (Konishi et al., 1988). In the Jeffress model, delay lines project to coincidence detector neurons that require simultaneous arrival of spikes from the two sides to elicit a maximal discharge. In the owl brainstem, the NM projections to NL function as delay lines to form maps of interaural phase difference in NL (Carr and Konishi, 1988, 1990). These maps of interaural phase

difference are tapped by coincidence detector neurons in NL (Sullivan and Konishi, 1986; Carr and Konishi, 1990).

The delay line circuit must adjust to increasing interaural time cues when the head grows during development. Skull width more than doubles in the first month after hatching (Haresign and Moiseff, 1988; Knudsen, 19841. The head reaches adult width a t about posthatch day (P)21. or 3 weeks after hatching, and auditory cues stabilize at P60, or 2 months after hatching, when the facial ruff’ reaches its adult dimensions (Haresign and Moiseff, 1988 Knudsen, 1984). Since the connections between the auditory nerve, NM and NL are formed before hatching (Can. and Boudreau, 1989), the developing map must be subjeci, to constantly changing sensory cues as the distance between the two ears increases during the first month posthatch. We have analyzed the development of the cell types of the auditory brainstem and the assembly of the delay line circuit in NL with reference to these changes in head size. This paper describes the development of the cell types and connections of the circuit responsible for th? detection of interaural time differences in the brainstem of

Accepted March 5 . 1996 Address reprint requests to Dr. Catherine Carr. Department of ZOO~O~:,!,

University of Maryland. College Park. MD 20742-4415. E-mail: carriu zool.urnd.edu

i 19% WILEY-LISS, IYC.

468 C.E. CARR AND R.E. BOUDREAV

50

40 -

30 -

20 -

10 -

0

TABLE 1. Development of Nucleus Magnocellularis (NM) and Nucleus Laminaris (NL1 Showing Changes in Numbers of Synapses, Cell Size, and Dendritit Length From Both Golgi Data and Ultrastructural Material1

HATCH I I I

Nucleus magnocellularis Nucleus laminaris

Age (days)

E28 E30

2 fi 8

I2 15 20 21 45 adult

Area (&1

270 1237, 26) 336 1148, 251 342 1243,211 470 1t61.291 552 1+73. 521 638(2109.481 634 12 136, 35) 985 1 2 193, 171 956 12 189.201 950 ( ? 142.301 945 ( t 129,301

Synapses/ cellZ

348:’ nd 357 nd 772 972 nd

1169 nd

1120 1175

Total dendrite length (pm)

nd 0 nd

16 (-t 10, 2216 135 (595, 3516 104 (282.48i 282 ( 2 148,351 470 ( 2 181,201 327(2158, 17)

nd 166 l t72 .56)

Mean dend length ( p m )

nd 0

nd nd 75 49

117 nd 172 nd 83

Dendrite number Area ( wn2) Length ( b m ) Dendrite number

nd 0 nd

0.3‘ 1.8 2. I 2.4 nd

I .9 nd

2 ‘I

154 (125, 251 387 (t63, 101

nd 342 ( i 5 0 . 3 2 ) 370 (256 , 15) 436(259,37) 4901+119,47i 5021r72, 151 527 1?52, 151

nd 648 i i60,201

nd nd

nd nd 2.3 (k0.4, 91

3 .2 (21.3, 241

40 (25, I01

15 (24.7, 24) 16 ( t 4 . 7 , 101 4.0 (10.7, 91 14.6 (23.5. 33) 4.5 (21.2. 301

7.9 i22.1,291 17 ( 1 3 . 7 . 311 18.4 12.5, 151

nd nd nd nd

10.2 (L.8, 151

18.0 124.3, 15) 5 (21.9.301

‘Data were pooled from the three rostrocaudal divisions of each nucleus because few significant differences were obtained from comparisons within age groups (Students t-test). Adul: data were obtained Srum Carr and Boudreau (1991, 19931. Golgi measurements were of totdl dendritic length per NM neuron with dendrites. Since dendritic number and branching decreased with age, this method underestimated the increase in the length of individual dendrites. The mean dendritic length (length of individual dendrites, w d S calculated by dividini: the total len@h by the number ddendrltes. “he table shows the estimated mean number of synapses per NM neurun. with the assumptions that the cell was spherical, and that the synapses were evenly distrihuted over thiy soma. Calculations of total number based on profile counts have an inherent error (Coggeshall and Lekan, 19961 hut have been provided here because they indicate a trend In Increasinl: synapse numbers. ’Counts were made from only central and rostra1 regions of the NM only, where immature endbulbs were tbund. ‘Cuunts were from the rostra1 repon of NM only, since other regions had not yet produced adult dendrites. ‘In all cases except the adult, the mean number of dendrites per cell were calculated for all cells. Far the adult, two means were calculated. For only those cells with dendrites the meail was 2 i 0 94, n = 26 For all cells, including those without dendrites, the mean was 0.9 2 1.4, n = 56.

the barn owl. Since discrimination of small time differences requires accurate processing of the stimulus, we have concentrated on the development of a number of specializa- tion’s for coding timing information in the auditory system. These include changes in the number and location of synapses, growth, and regression of dendrites and changes in cell size in the neurons of the auditory brainstem. Preliminary accounts of some of these findings have been published (Carr, 1989).

MATERIALS AND METHODS This study is based on results from 18 barn owl chicks

and 2 barn owl embryos (Tyto alba) of both sexes. Table 1 summarizes the age of each case. Since the owls were hand raised in the laboratory (US Department of Fish and Game permit PRT 7357161, in most cases only one animal was used to construct each time point. All owls have been or will be used in parallel studies, and their care and use con- formed to NIH guidelines. Head growth was measured in all owls and embryos, using the methods of Haresign and Moiseff (1988). Data points from six other embryos were also used to construct Figure 1.

Golgi material The Rapid Golgi technique (Valverde, 1970) was used on

seven owls and one embryo. Owls were anesthetized with Ketamine, followed by a lethal dose of Pentobarbital (20 mgikg IM; Abbott Laboratories). After intracardiac injec- tion of heparin, owls were perfused transcardially with normal saline, and followed immediately by 200 ml-1 liter of 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Brains were then blocked transversely and placed in Golgi fixative. Impregnated NM and NL neurons were found in all brains.

Electron microscopy For normal ultrastructural studies, 11 owls and 1 embryo

were anesthetized with Ketamine (30 mg/kg IM) followed by an overdose of Pentobarbital (20 mgikg IV) and perfused transcardially with either normal saline or avian Tyrodes

-20 0 AGE 20 (days) 40 6 0

Fig. 1. Changes in head width with development: The head grows slowly around the time of hatching (arrow), followed by a period of rapid growth between P8-20. The head reaches i ts adult width by about, P25.

solution (139 mM NaCl, 17 mM NaHC03, 3 mM KCl, 1 mM MgCl, 3 mM CaCl, and 12.2 mM glucose, pH 7.3; Jackson and Parks, 19821, and followed immediately by 1 liter of 2.5% EM grade glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2. The brain was postfixed overnight at 4°C. The brainstem was then sectioned using the vibratome and sections washed in buffer and postfixed for 1 hour at 4°C in a 1.0% solution of osmium tetroxide. Following postfixation, the tissue was again washed in several changes (10 minutes each) of buffer, dehydrated through ethanol, passed through propylene oxide, and infiltrated and embedded in Araldite resin. Thick sections were mounted on glass slides and stained with toluidine blue. Thin sections taken from areas immediately adjacent to the thick sections were placed on formvar-coated wide slot grids and stained with uranyl acetate and lead. Embry- onic tissue was prepared according to the protocol of‘ Jhaveri and Morest (1982a). The embryo was cooled and then removed from the shell and perfused with saline

DEVELOPMENT OF AUDITORY BRAINSTEM 469

followed by 1.5% paraformaldehyde and then cold 2% paraformaldehyde/2.5% glutaraldehyde.

Data analysis Drawings were made of Golgi impregnated material with

the aid of a camera lucida attachment on an Olympus microscope. In order to measure dendritic lengths and cell sizes, drawings of impregnated cells and dendrites were measured with the aid of two image analysis programs (PC3D from Jandel and NIH Image version 1.57).

The ultrastructure of the auditory brainstem was studied by first making a camera lucida drawing of one of the adjacent semithin sections. The entire thin section was then viewed on the electron microscope in scanning mode in order to identify major landmarks and any profiles to be examined in serial sections. Terminals were surveyed in both NM and NL, and were categorized by size, morphology, and vesicle type. In order to measure profiles in and around NL, a detailed morphometric analysis was per- formed. All cells, terminals and axons were measured from electron micrographs with the aid of an image analysis program tPC3D from Jandel).

RESULTS The head grows after hatching, subjecting the bird to

constantly increasing interaural time cues. We have analyzed the development of the connections and cell types of NM and NL with reference to this growth of the head.

Growth Barn owl eggs took 32 days to hatch, and at hatching, the

head was about 18 mm wide. During the last 2 weeks of embryogenesis and in the first week posthatch, the head grew a t a rate of 0.45 mmiday (Fig. 1). The head began a period of more rapid growth (1.3 mmiday) between P8 and P25 and reached its adult width (about 45 mm) by about 25 days after hatching (Knudsen, 1984; Haresign and Moiseff, 1988) (Fig. 1 ). The external auditory meatus became visible about 3 weeks before hatching. I t grew slowly until P8, when it began a period of rapid growth which has already been documented (Knudsen, 1984; Haresign and Moiseff, 1988).

The brain grew rapidly in the first month after hatching, although, with the exception of the granule cells in the cerebellum, no new neurons were born in the brainstem or midbrain (Cohen and Carr, unpublished observations). Growth in the auditory brainstem was not uniform, but followed the tonotopic axes of these nuclei. Rostrolateral regions of high best frequency developed first and appeared to lead the most caudal low best frequency portions of each nucleus by 2-4 days. This brief description of the spatial gradient has been used to introduce the developmental study of the auditory brainstem because the time points described below must either be approximate or refer to specific regions of each nucleus. In general, regions of each nucleus have been specified in descriptions of developmental events.

Development of auditory nerve endings in the nucleus magnocellularis

In the adult, the medial branch of the auditory nerve conveys phase information to the cells of NM via large axosomatic endings or endbulbs of Held (Carr and Bou- dreau, 1991). The development of these endbulbs, and the

accompanying changes in the number and distribution of the synapses on the NM neurons, has been described using both ultrastructural and Golgi techniques.

Development of endbulb morphology. Endbulb auditory nerve terminals developed from immature terminals before the embryo was exposed to airborne sound. End- bulbs were observed a t the ultrastructural level at embryonic day (E)28 in the rostral and central regions of the NM, and, with the Golgi technique, in all but the most caudal regions of the nucleus by E30. E30 is 2 days before hatching, and is the time that the embryo normally pipped and is exposed to airborne sound. Before this time, the auditory nerve endings formed fine branching terminal arborizations which were similar to those described for the chicken by Jhaveri and Morest (1982a). They will not be described in detail here, since our material does not cover the first embryonic stages of auditory nerve growth. The transition between the immature terminal arbors and the acquisition of the endbulb morphology was rapid (Figs. 2 and 3), and coincided with the disappearance of the immature NM dendrites (see below).

Ryugo and Fe!rete (1982) described a three-stage development of endbulbs in the cat auditory nerve. Since the development of the owl endbulbs followed a similar pattern, we have adhered to their nomenclature in this study. In the cat, the transformation from growth cone to endbulb began with a solid ending with filipodia (type I), was followed by a fenestrated ending with a t least one broad solid region (type II), and ended with the reticulated ending of the adult (type 111). In the E30 owl embryo, Golgi impregnated auditory nerve terminals in the most caudal regions of the nucleus formed branching terminal arborizations which had not yet condensed into endbulbs (not shown). The caudal and central regions of the nucleus contained endings which resembled growth cones or the type I endings of Ryugo and Fekete (1982; Fig. 2). These type I endings were about 10-15 Fm in length and consisted of a large lamellate expansion tipped with short filipodia (Fig. 2, top). In the rostral regions, more mature, fenestrated type I1 endings were observed (Fig. 2). This rostral to caudal developmental axis could also be observed in the P6 case, where some type I endings remained in the caudal region, type I1 fenestrated endings with at least one broad solid region were found in central regions and some type I11 or adult form endbulbs were observed in the rostral part of the nucleus. NM grew rapidly between P6 and P12 (see below), and by P12, most of the endbulbs observed were type 111. These type I11 endbulbs were also larger than the endbulbs found in the younger animals. The increase in endbulb size occurred at about the same time as the NM neurons began to increase in size, towards the end of the first week posthatch (Table 1 ).

At the ultrastructural level, immature auditory nerve endings differed from endbulbs, and immature endbulbs differed from mature endbulbs (Fig. 3). Immature non-endbulb auditory nerve terminals from the caudal regions of E28 embryos had expanded foliate shapes, and a filiform appearance. Typical endings may be seen in cross section in Figure 3A, surrounding an immature NM neuron. These terminals formed few synapses on the cell body, and more synapses on the profiles of immature NM dendrites. Once the endbulbs formed, many more synapses were found on the cell bodies (Fig. 3B).

Developmental changes in the number and distribution of synapses were quantified as follows: the numbers of

Distribution of auditory nerve synapses.

E30

P 1

P21

Fig. 2. Projections of the auditory nerve into nucleus magnocellularis (NM). Drawings of selected Golgi impregnated endbulbs a t four ages. embryonic day E30, P6, P12, and P21. Terminals are arranged in a caudorostral eradient. with those from the most caudal replon on

the left, and cells from rostra1 regions a t right. Note the changes in endbulb form between E30 and P6, and the increases in size between P6 and P12. Scale bar = 100 km.


Fig. 3. The auditory nerve forms both embryonic terminals and endbulbs in NM in an E28 owl chick. A: The caudal region of the nucleus contains the embryonic form of NM neurons with many dendrites, surrounded by filiform auditory nerve endings. B. Neurons in the central region of the nucleus receive Type I endbulb terminals

(*). Note the many immature synapses with large synaptic densities on the cell body (arrowheads). Synapses were also located on the large somatic spines of the NM neuron. Astrocytic processes surround the endbulb. Scale bars = 5 *m in A, 1 km in B.

472 C.E. CARR AND R.E. BOUDREAU

synapses were counted in a shell of 20 ym radius around the cell body in cross sections through cells in the caudal and central regions of NM in an E28 embryo. In sections through the caudal region of NM containing immature NM neurons with many embryonic dendrites, the mean number of synapses on the cell body was 0.521.2 (n=6) with more synapses observed in the surrounding neuropil (512.7, n=6; Table 1). In the adjacent central portion of the NM, endbulbs were observed on NM neurons which appeared to have lost their immature dendrites. These neurons received many more synapses on their cell bodies. The mean number of synapses per section through the cell body of neurons in the central region was 16.517 (n=6), while few synapses were observed in the surrounding neuropil(1.5*1.1, n=6). Thus the change to endbulbs from immature auditory nerve terminals was accompanied by both an increase in the total number of synapses and by a change in the location of those synapses from dendrites to the cell body.

In addition to the change in the number and distribution of auditory nerve synapses that occurred coincident with the formation of the endbulbs, the total number of synapses also increased with the transition from type I to type I1 and I11 endbulbs. The total number of auditory nerve synapses per NM cross-section was counted in thin sections throughout NM a t different ages (Table 1). For E28 NM, about 19 synapses per section were found (18.8k5, n=8). Assuming a random distribution of synapses, and a soma area of 270 pmZ, it was estimated that each NM neuron received about 350 synapses. Similar calculations for other ages showed a steady increase in number of synapses per cell until P20, which accompanied an increase in cell size (Table 1). From P21 on, synapse numbers remained fairly constant.

Developmental changes in spines and synapse morphology. Between E28 and P12, the increase in the numbers of synapses per NM neuron was followed by an apparent shift in the location of synapses from the cell body to the somatic spines. Until P12, more synapses were located on the soma membrane than upon the spines. At P12, there was an equal distribution of synapses on soma and spines, and from P20 on, about 65% of the synapses were located on the spines. The endbulb also changed shape and increased in complexity during this time (Fig. 2). The spines indenting into immature endbulbs appeared to be both shorter and blunter than those in adult type I11 endbulbs (Fig. 4A). In the adult, fine 0.3 km diameter (mean = 0.2920.08, n=22) somatic spines indent into the endbulb; similar spines were observed indenting into all immature endbulbs. The mean spine width for all neurons examined for E28-P20 owls was 0.57 km (20.24, n=58). Although only one animal was examined for each age, there appeared to be a decrease in spine width with age. The mean spine width for P30 endbulbs was 0.35 pm (10. 12, n = 121, similar to adult spine widths (Fig. 4B). In other respects, by P12, the type I11 endbulbs were ultrastructurally similar to the adult, receiv- ing many somatic spines and forming numerous synapses.

The individual auditory nerve synapses in the immature type 1 endbulbs resembled those described in the adult (Fig. 4). The synapses were asymmetric, with a prominent dense undercoating on the cytoplasmic face of the NM neuron, and a 30 nm synaptic cleft. Although these observations were not quantified, there appeared to be fewer vesicles, membranous cisterns, and clathrin coated vesicles in immature endbulbs than in the adult endbulbs. In the adult, small clusters of 45 nm clear vesicles were found at the

Fig. 4. Maturation of endbulb synapses (arrowheads) within the NM. A Synapses were about equally located on the soma and the spines at P8 (see also Table 1 ). Although spines were thicker than in the adult, the synapses were morphologically similar, with clusters of vesicles and prominent postsynaptic densities. B: The somatic spines of the P30 NM neurons were narrower than those of the more immature NM neurons. Scale bar = 1Fm in A.

synapse, with other vesicles of the same size distributed throughout the endbulb. In E28 endbulbs from the central and rostra1 regions of the nucleus, 45 nm clear vesicles were distributed throughout the endbulb, but few clusters were seen at the synapses. In P2 endbulbs, vesicle clusters were observed apposed to most synapses. Most auditory nerve synapses onto NM appeared to take on their adult form by P2, or before the time that all synapses have been formed.

Development of the nucleus magnocellularis NM is located on the floor of the fourth ventricle, caudal

to NL. I t is an oval nucleus which is widest caudally where it extends across the dorsal surface of the brainstem. Rostrally it narrows and lies medial and dorsal to NL. The nucleus receives auditory nerve input and projects bilaterally to NL. In the adult, the caudal low best frequency regions of the NM receive non-endbulb auditory nerve terminals (Koppl, 1994) and contain distinct small cells (Koppl and Carr, in press). We have not described the development of this caudal region in detail, but have restricted our observations to the main body of NM.

Peak dendritic growth of NM occurs in the month after hatching. NM neurons also go through a period of dendritic

DEVELOPMENT OF AUDITORY BRAINSTEM 4 73

growth and retraction in the egg, which resembles that seen in the chick (Jhaveri and Morest, 1982b), and which will not be described in detail here. NM neurons have almost all lost their embryonic dendrites by the time of hatching. In the first week posthatch, they begin to sprout adult dendrites which change in both number and length during the first month posthatch. We used the Golgi technique to examine changes in the number of these dendrites, their dendritic morphology and dendritic length.

Growth and retraction of magnocellular dendrites. At 2 days before hatching (E30), all NM neurons were aden- dritic, except in the most caudal regions of the nucleus where a very few cells remained with embryonic dendrites (Fig. 5). It should be noted that NM also possesses a distinct population of bipolar low best frequency cells which do not show the same patterns of dendritic loss and regrowth (Koppl and Carr, in press; Fig. 5 far left column). In NM proper, short (5-10 pm) processes emerged from neurons in the rostral third of NM by P2. By P6, nearly half the cells in the rostral and central regions of the nucleus had one or two of these fine short dendrites. By P8, some cells in the caudal regon had not yet grown adult dendrites, while cells in the central and rostral regions had many short and some long dendrites (Fig. 5). By P12 most cells in the nucleus had 1 or more dendrites, and the gradient of dendritic number had begun to emerge, with growth of dendrites in the caudal region outpacing growth in rostral regions (Figs. 5 and 6). By P15 almost all neurons had 2-3 dendrites (not shown in Fig. 5, but see Fig. 6 for summary; Table 1). The trend in increasing dendritic number was counteracted by a decrease in dendritic number in the rostral and central regions of the nucleus which begins between P15 and P21 (Fig. 6; Table 1). These two opposing trends form the adult rostrocaudal gradient in which the number of dendrites decreases from low to high best frequency regions of the nucleus (Fig. 6; see Carr and Boudreau, 1993).

By P21, the total number of dendrites per cell had begun to decline (Fig. 6). The dendrites changed in number and length during the first month, depending upon their rostrocaudal position. The changes were not complete by P21, since adult NM neurons had many fewer dendrites than the P21 cells (Fig. 6B). Thus NM neurons showed both late growth and regression of permanent dendrites.

Neuro- nal morphology and dendritic length changed during the first month posthatch. At the end of the first week posthatch (P6), the new dendrites of the cells in the rostral portions of the nucleus were so short (about 15 pm) that the cell body appears to be covered with thick spines (Table 1). Some of these short dendrites appeared to grow rapidly, and by P8 some neurons had several long ( > 100 pm) dendrites (Fig. 5, Table 1). It is possible that dendritic growth in NM neurons began by sprouting short dendrites, some of which elongated, and others of which retracted. Dendrites grew in length over the first month posthatch. Total dendritic length increased from a P8 mean of 135pm to P21 mean of about 300 pm (Table 1). Further changes in dendritic length and number take place after P21, since adult dendrites were both fewer in number and shorter (about 150 pm; Table 1).

The rostrocaudal differences in dendritic number of Figure 6 had no parallels in the measurements of dendritic length until P21, when the mean dendritic lengths were observed to differ significantly between rostral and caudal regions of NM (Student’s t-test; df = 69, t = -2.73,

Changes in morphology and dendritie length.

P>.OO7). At this time, the dendrites remaining in the rostral regions of NM tended to be shorter than those in caudal low best frequency regions. Thus decreases in the number of dendritesicell began about P15, while decreases in dendritic length began a few days later.

The cell bodies of NM also grew during the period of dendritic extension. Cell size was measured from both Golgi material and toluidine blue stained 2 pm sections, since Rapid Golg material and plastic sections showed comparable shrinkage. Cell size increased fairly regularly until adult size was reached by P21 (Table 1). NM cells therefore go through a period of both somatic and dendritic growth during the first 3 weeks posthatch. The period of dendritic regression began after the second week posthatch.

Development of the nucleus laminaris NL is located on the floor of the fourth ventricle, rostral

to the NM. It is an oval nucleus except at its most caudal extent where it forms a “2” or “S” fold at its lateral edge. Like the caudal low best frequency region of the NM, this caudolateral region contains unique cell types (Koppl and Carr, in press). The nucleus is encapsulated by a glial border and surrounded by fibers from the NM, with afferents from the ipsilateral NM forming the dorsal border of the nucleus and fibers from the contralateral NM forming the ventral border of the nucleus. These two fiber layers sandwich NL proper, which contains three neural elements; a single neuronal cell type, afferents from the two NM nuclei, and a GABAergic input (Carr and Boudreau, 1993). Golgi and ultrastructural techniques were used to describe the development of the neurons of NL.

Four variables were examined with the Golgi technique: the number of dendrites, dendritic length and thickness, and cell size. Unlike the cells of the NM, NL neurons changed very little except in size after the first week posthatch. At and before hatching, NL neurons were covered with many short dendrites. Since the dendrites were short and distributed evenly over the cell body, their total number could not be counted, because in any cross section, some dendrites were orthogonal to the plane of section or hidden behind the cell body. Therefore the only dendrites counted were those viewed in silhouette, and their number should be regarded as an index of the total. Visible dendrites were drawn with the aid of a camera lucida, focusing through the section. Counts of dendritic number were made from these drawings (Table 1).

In the days before hatching, NL neurons were covered with many fine ( < 1 pm) dendrites which presented a thatch-like appearance (Figs. 7 and 10). Many short (2-3 pm) fine (0.5 km) dendrites surrounded the cell body and received synapses from the growth cones that formed the NL neuropil (Fig. 8). In Golgi preparations, generally more than 40 of these fine dendrites haloed the soma of E30 NL neurons (Table 1, Fig. 7). Typically, the dendrites were evenly distributed over the soma surface and did not show any dorsoventral polarity. By the end of the first week posthatch (P6) the number of dendrites had decreased by more than 50% and those that remained in the rostral and central regions had thickened. Many cells in the caudal region retained longer thin dendrites (Fig. 7). After the first week posthatch, counts of the numbers of the dendrites around each neuron showed little change in dendrite number between P6 and adulthood (Table 1). By P12, all cells, including those in the central and caudal regions of the nucleus, had thick short dendrites studded with fine

Dendritie growth in NL.

E3

P

P

- Fig. 5. Composite drawing of selected Golgi impregnated NM

neurons at four ages, E30, P8, P12, and P21. Cells are arranged in a caudorostral gradient, with the cells from the most caudal region on the left, and the cells from rostral regions at far right. The neurons in the column at far left (stars) are a distinct cell type confined to the most caudal low best frequency regions of the nucleus. They do not follow the pattern of embryonic dendrite loss and regrowth observed for the NM neurons, and were not included in our analyses. At E30, an occasional cell in most rostral regions might display a short dendrite (one shown,

arrow), while some cells in the most caudal regions still retain some embryonic dendrites (arrowhead). Almost all P8 cells had begun to produce dendrites except for cells in caudal regon which had lost their embryonic dendrites (arrowheads). A newly formed dendrite was tipped by a growth cone ( * ) . By P12, the gradient in dendritic number had begun to emerge, with more dendrites on cells from low best frequency regions. By P21 NM neurons have fewer and longer dendrites, and the period ofdendritic loss (arrow) in rostral regions had begun. Scale bar = 20 Fm.


R 6 12 15 21 Adult

100 caudal

7 5

n

@ 5 0 W

4 25

6 \ o \

6 12 15 21 Adult cn 100

E-( W central

7 5

a

8

z 5 0 w a 25

- - 0 Y2 6 12 15 21 Adult

rostral

5 7 5

3 z 5 0

25

0 6 12 15 21 Adult

AGE (days)

processes (Fig. 7). These dendrites grew in length between P12 and P20, and by P20 the mean dendritic length was 10 pm, longer than the mean adult length of 5 pm (Carr and Boudreau, 1993). Therefore, some decrease in length oc-. curred between P20 and adulthood. NL cell bodies also grew in size during the first month posthatch (Table 1).

The caudolateral region of NL contains distinct cell types (far left, Fig. 7). These cells show a similar growth and thickening of NL dendrites. This regon adjoins a caudal, low best-frequency region of NL. In the adult, dendrites are longer in this region than in the rest of the nucleus tCarr and Boudreau, 1993a; Koppl and Carr, in press). Adult NL dendrites varied in length from about 2 pm to 20 pm (mean length = 5k1.9 pm), with the exception of cells in the caudolateral low best frequency region of the nucleus. The dendrites of these cells have a mean length of 12k4 pm. Differences in dendritic length were apparent in even the youngest material examined. From E30 on, dendrites in the caudal regions of NL were about 50% longer than those in the rostral regions (Fig. 7 ) . For example, at E30, caudal dendritic lengths were 5.153.4 (n=10) and rostral lengths were 2.0220.2 (n=10). At P15, caudal lengths were 10?1.2 (n= 10) and rostral lengths 5 .7t0.9 (n= 10).

Deoelopment of the magnocellular synapses in NL, NM axons project to NL before the time of hatching, and at E28, the NL neuropil was made up of closely packed neurons surrounded by growth cones. Growth cone synapses were found on the short dendrites and the cell body. The large pale growth cones (Fig. 8A) were assumed to originate from NM because of their uniform appearance and because of the congruence between their size and shape and the size and shape of Golgi impregnated NM axons and terminals. The growth cone synapses were asymmetric, with a prominent dense undercoating on the cytoplasmic face of the NL neuron (Figs. 8B and 9A). The synaptic cleft was about 40 nm wide, and the presynaptic side was marked by small clusters of 50 nm clear vesicles. The growth cone terminals contained small numbers of the same size vesicles, distributed throughout the cytoplasm. The terminals also contained a few dense core vesicles, and lacked the many puncta adhaerens that connect the terminal and the postsynaptic cell in adult birds.

By P12, the NL neurons were much increased in size (Table 1). The numerous NM terminals no longer resembled growth cones, but formed large terminals with many synapses on dendrites and cell body (Fig. 8). They differed from the adult in that the axons and terminals were largely unmyelinated. Myelination of the NM axons in NL had begun, but had not spread as far as the terminals (Cheng and Carr, 1992). Synaptic form did not change with age, and at P12, the synapses were asymmetric, like those described for E28, with a prominent dense undercoating on

Fig. 6. Bar diagram of changes in number of dendrites with age and position in the NM. E30 data were not plotted because adult dendrites were not present a t this age. The number of dendrites in all Gola impregnated NM neurons were counted from E30 ln=24), P6 (n=36i, P12 (n=56i, PI5 (n=35), P21 (n=20i, toadult ln=207). A: Summaryof all cells showing growth and retraction of dendrites during development. Black, no dendrites; narrow stripe, one dendrite per cell; wide stripes, two dendrites; and blank, threeor more dendrites. B Break- down of data in A with NM divided by best frequency into three rostrocaudal divisions. Note the gradual emergence of the adult differences between the number of dendrites in rostral and caudal cells. Also note that the adult profile had yet not been acquired by P21.

** P6

P12

$if t P21

I

Fig. 7. Drawings of selected Golgi impregnated NL neurons at E30, P6, P12, and P21. Some NM growth cones were also shown (arrows). The nucleus was divided into four zones from caudal to rostral, with unique cells from the caudolateral low best frequency region on the far left (stars), and with cells from caudal, central and rostral regons shown from left to right. NL neurons produced many short dendrites by

E30. The number of dendrites decreased in the first week posthatch, and the dendrites grew thicker and more spinous. Note that a t all ages, the cells of the caudal low best frequency regon tended to have the longest dendrites. Short axon segments (a ) shown in many cases. Scale bar = 20 pm.


Fig. 8. Development of presumptive NM synapses within NL. A: Large pale growth cone (*) forms synapses (arrowheads) on both dendrite and soma of a P2 nucleus laminaris (NL) neuron. B: E28 synapses on NL neurons are morphologically very similar to mature

synapses, with clusters of vesicles opposed to a postsynaptic density (arrow). Small dendrites (stars) interpenetrate the growth cone terminals. Scale bars = lpm.

the cytoplasmic face of the NL neuron. At P12, however, the symmetric membrane densities or puncta adhaerens that connect the terminal and the postsynaptic cell, or two NM terminals, had begun to be more numerous. In the adult, these membrane densities are very much more common than actual synapses; in the P12 chick they occurred at about the same density as synapses (mean number of synapses per thin section = 7.6?3.5; puncta adhaerens = 6.621.7, n= 15 neurons examined).

By 3 weeks after hatching, or P20, the NL neuropil closely resembled that of the adult. NL dendrites received numerous NM synapses which formed large terminals on both the dendrites and cell body (Fig. 9). The synapses were asymmetric, with a prominent dense undercoating on the cytoplasmic face of the NL neuron. In the adult, putative GABAergic terminals surround NL neurons (Carr et al., 1989; Carr and Boudreau, 1993). Similar electron dense terminals with pleomorphic vesicles were also observed in young owls (Fig. 9B). One month after hatching (P301, the NM endings on NL cells were morphologically indistinguishable from the adult.

NL neurons have an unusual axon. In the adult, these axons lack a conventional

Development of NL neurons.

spike initiation zone and have large diameter axon that becomes myelinated when it leaves the cell body (Fig. 1OC). This unusual morphology was observed in E28 NL cells. These cells had a very irregular outline with an asymmetric nucleus located close to where the axon leaves the cell body (Figs. 7 and 10A). NL axons project out of the nucleus towards the auditory midbrain. A few glial wrappings surround the axon, which does not taper into a cone shape to form an initial segment (Fig. 10B). By P8, the axons have increased in diameter and some myelination had occurred. By P12, the axon had taken on its adult appearance, with myelination of the axon at the soma, although the number of myelin lamellae were fewer than found in the adult, and by P20 the axon was indistinguishable from that of the adult (Fig. 1OC).

NL neurons showed a steady increase in size over the first month posthatch (Table 1). At E28, NL neurons were small (270 pm2) and closely packed in the nucleus. By P12, NL neurons were much increased in size and the distances between had begun to increase as the neuropil in the nucleus expanded. NL neurons continued to increase in size past P21 (Table 1).


DISCUSSION The young owl’s skull more than doubles in width during

the first month after hatching. Since the connections between NM and NL are formed before hatching, the developing time coding circuit must be subject to constantly increasing interaural time difference cues as the distance between the two ears increases during the first month. We have analyzed the normal development of the cells of the time coding circuit with reference to the growth of the head. Since discrimination of small time differences requires accurate transduction and processing of the stimulus, we have concentrated on the development of a number of features which may be related to coding timing information in the auditory system. We have found that the auditory brainstem circuits grow and mature after hatching, and the end of growth in the auditory brainstem circuits is generally correlated with the attainment of stable interaural time difference cues.

Owls are altricial, and when they hatch are unable to produce a motor response to sounds (Haresign and Moiseff, 1988; Knudsen, 1984). The eyes open at about P9, and the head and the rest of bird grows rapidly between P8 and P24. The feathers of the facial ruff, which serves a purpose similar to the mammalian pinna, start to appear by P21 (Haresign and Moiseff, 1988; Knudsen 1984). The skull, ruff, and ear canals grow rapidly and linearly for the next three weeks, reaching adult dimensions at about 6 weeks. At this stage the skull is 40-50 mm wide, the ruff is 82-92 mm wide, and the diameter of the ear canals is 9-10 mm (Haresign and Moiseff, 1988). Thus the owl appears to receive stable, adult interaural time difference cues by about 1 month after hatching and stable adult interaural intensity cues by two months post hatch (Knudsen et al., 1984).

Knudsen and his colleagues have shown that the owl’s auditory system is plastic during the 2 months posthatch. They have analyzed developmental changes in sound localization ability under various conditions of monaural occlusion or ear plugging, and have shown that these first 2 months constitute a sensitive period. The end of the sensitive period coincides with attainment of an adult sized head and facial ruff (Knudsen, 1984; Knudsen and Knud- sen, 1986). Ear plugs cause the owl to experience changes in both interaural intensity and interaural phase. Interaural time and intensity cues stabilize at different times, and the sites and timing of plasticity in the time and intensity pathways appear to be different (Mogdans and Knudsen, 1994). Interaural time cues stabilize at around P24 days (Fig. 11, while interaural level difference cues depend on ruff development and stabilize after P45 (Haresign and

Fig. 9. Maturation of synapses within NL. All terminals adjoin NL neurons. A P12 terminal forms a synapse (arrow) on a thin NL dendrite. Note that, unlike the E28 case, puncta adherens are now also common. B: P20 putative GABAergic terminal forms a small electron dense bouton packed with pleomorphic vesicles. These terminals make small symmetric synapses with small pre- and post-synaptic densities (arrow) on NL neurons. The development of the GABAergic input will not be further described in this paper. C: P30 club-shaped NM terminal forms numerous synapses (large arrows) on a NL dendrite. Note the many coated vesicles (arrowhead) in the presynaptic terminal and the puncta adherens joining both adjacent terminals (small arrow) and terminals and their postsynaptic targets. Scale bars = 1 pm.


Moiseff, 1988). The first site of binaural interactions in the adaptive changes in interaural level difference tuning ob.- intensity pathway is the posterior portion of the ventral served at higher levels of the auditor pathway (Mogdans nucleus of the lateral lemniscus (VLVp); Manley, 1988). and Knudsen, 1994). It is not yet known where the neural Plastic changes in VLVp are partly responsible for the sites of remodeling may be in the time pathway. Remodel-

ing could occur a t both the initial stages of auditory processing, in the magnocellular-laminaris circuit, or in the lemniscal nuclei and inferior colliculus. It is possible that the plasticity in the processing of interaural time difference cues during the sensitive period may be reflected in the changes in the organization of NM and NL. The long period of normal development of the brainstem time coding nuclei described in this paper suggests that plastic changes could take place at the level of the auditory brainstem.

We have found that time coding circuits are formed before auditory cues stabilize because the connections between the auditory nerve, NM and NL are made before the time of hatching. The elements of the circuit are immature, however, and the appearance of an adult morphology occurs near or after the time that stable interaural time difference cues are acquired. Thus, connections are made early and the circuit matures over the first months posthatch.

Development of the auditory nerve endbulbs The development of the auditory nerve projection to NM

in the barn owl is very similar to that described for the chicken (Jhaveri and Morest, 1982b; Parks and Jackson, 1984), although the time courses are different, in part because of differences in incubation times. In the chicken, the egg is incubated for 21 days and in the barn owl for 32 days. In both the owl and the chicken, however, endbulb terminals are formed a few days before the embryo pips and is exposed to airborne sound.

In the E l 0 chicken, auditory nerve axons in NM form growth cones. These endings ramify between El l -13, after a period of growth of immature NM dendrites. On E14, the terminal auditory nerve axon branches form a compact highly branched plexus which coalesces into a endbulb-like structure between E16-17. A recognizable endbulb is formed by embryonic days 19-20 (Jhaveri and Morest, 1982b). The transformation of the plexus to the endbulb occurs around the same time as the cell loses its embryonic dendrites. Although auditory nerve terminals were not examined in barn owl embryos of a comparable age to the Ell-13 chickens, owl E28 auditory nerve terminals in the caudal portion of NM formed fine branching terminal arborizations similar to those described by Jhaveri and Morest (1982a,b) for chicken E14-15. In both the chick and the barn owl, immature synapses were formed by cochlear nerve fibers on immature dendrites, while synapses on the

Fig. 10. Embryonic and mature NL neurons and axons in the barn owl. A E28 neuron with an asymmetric nucleus and many short fine dendrites (arrow), surrounded by growth cones and fine unmyelinated NM axons (arrowhead). B: E28 N L axon with no initial segment, and as yet, no myelin lamellae, although presumptive oligodendrocyte processes wrap the axon (arrowhead). C: P20 NL neuron with large myelinated axon which leaves the cell body a t the axon hillock (arrow). The cytoplasm near the axon had characteristically reduced levels of endoplasmic reticulum, while the axon itself has many fascicles of microtubules. The neuron is surrounded by dendrites (arrowheads), oligodendrocytes ( ’ ), and the myelinated axons of the inputs from NM. Scale bars = 2 pm in A, 1 pm in B. 5pm in C .


magnocellular somata were rare. Similarly, the older E30 owl embryo’s examined with the Golgi technique in this study showed large spoon shaped swellings with lamellate expansions (type I endings) which were similar to the chicken E16-17 endbulbs. Thus it appears that the owl E28 auditory nerve development is comparable to that of the E15-16 chicken.

In both the barn owl and the chicken, the rapid transition between the immature terminal arbors and the acquisition of the endbulb morphology coincided with the disappearance of the embryonic NM dendrites (Jhaveri and Morest, 198213). In the chicken, this change was also accompanied by a reduction in the number of synaptically evoked auditory nerve inputs onto NM neurons (Jackson and Parks, 1982). Since the loss of the embryonic dendrites coincides with the formation of the endbulb, Jhaveri and Morest (1982a) had previously suggested that the many embryonic dendrites and branching auditory nerve terminals would provide an opportunity for interactions between the incom- ing auditory nerve and its magnocellular targets. Once the endbulb was formed, there would no further need for the embryonic dendrites. This hypothesis is supported by parallel observations on the development of GABAergic terminals in NM of the chick (Code, 1989). In the chick, GABAergic fibers with varicosities appear between E12-14. These then form somatic puncta around E16-17. Thus the movement of both auditory nerve and GABAergic synapses from neuropil to soma occurs at about the same time in development. The movement of both terminal types may be driven by removal of their dendritic targets, or by signals from either presynaptic input. The dramatic change in the postsynaptic cell’s geometry suggests that these synaptic rearrangement’s are more likely to be driven by the postsynaptic cell (Hume and Purves, 1981; Jackson and Parks, 1982). Experimental evidence supporting this hypothesis comes from Parks and Jackson (1984) who showed that the auditory nerve afferents do not cause dendritic retraction in the chicken. One otocyst was surgically ablated early in development, preventing the formation of the auditory nerve. They found that NM neurons go through the normal sequence of dendritic loss without auditory nerve input. Thus either NM neurons control endbulb formation, or the endbulbs and NM neurons develop independently (Parks and Jackson, 1984). Regardless of the underlying mechanism, the change in synapse location to the soma is functionally significant, since it allows the formation of the specialized endbulb and its precise transmittal of phase- locked spikes between auditory nerve and cochlear nucleus. In the barn owl, the formation and maturation of the endbulb is also accompanied by an increase in the number of synapses per cell, which could further serve to increase the physiological security of the synapse.

The barn owl endbulbs matured after hatching, and their development followed a similar pattern to that described for cat auditory nerve (Ryugo and Fekete, 1982). We adhered to the Ryugo and Fekete nomenclature in our descriptions of endbulb maturation (see Results). In the cat, the three stages of endbulb development follow head development. At birth, cats’ eyes and ears are closed, and the auditory nerve has formed type I endings in the anteroventral cochlear nucleus. By P10, the eyes and ear canals have just begun to

open, and a mixture of type I and I1 endings are present. Twenty-day-old cats had a mixture of type I1 and I11 and a fairly competent although immature auditory system (Brugge, 1988; Ryugo and Fekete, 1982). The time course of the endbulb development in the barn owl is similar to that of the cat, although the development in the owl followed a more rapid time course. The barn owl had type I endbulbs in the nucleus at the time of hatching when the eyes are closed. By P6, before the eyes open, there is a predominance of type I1 endings. By P12, when the owl’s eyes have begun to open, most endings were either type I1 or 111, and by P21, all endings observed were type 111. Auditory nerve record- ings should therefore show competent responses by P12, and be mature before the interaural time cues stabilize.

Development of the nucleus magnocellularis Adult NM neurons in both barn owls and chickens have

large round or oval cell bodies with a few or no medium length dendrites (Smith and Rubel, 1979; Jhaveri and Morest, 1982b; Conlee and Parks, 1983; Carr and Bou- dreau, 1993). In barn owls these mature NM dendrites appear after hatching, beginning around P6. Over the first month, the dendrites change in length and number, with the changes dependent upon their rostrocaudal position or best frequency. This period of dendritic development in the owl is lengthy, since the adult morphology had not been achieved by P21, when these NM neurons still have more dendrites than in the adult owl. Adult NM neurons also show a decrease in the number of dendrites from low to high best frequency regions of the nucleus (Carr and Boudreau, 1993). This adult pattern begins to form when cells in the rostra1 high best frequency regions show a decrease in both dendritic number and length, beginning about 2 weeks after hatching.

Despite the considerable differences between the owl and chicken’s patterns of development, the time course and developmental changes observed in the growth of NM dendrites in the barn owl are very similar to those seen in the chicken (Conlee and Parks, 1983). In the chicken, the first mature dendrites are first seen at E l 7 (mean length 14 pm). They grow from a mean length of 48pm at P4 to a maximum of 100 pm at P10. These mean lengths are unchanged at P60 (Conlee and Parks, 1983). In order to compare our results with those of Conlee and Parks, we calculated the mean dendritic length from Table 1, by dividing mean total length of all dendrites by the mean number of dendritedcell. In the barn owl, the first mature dendrites were seen around P6 (mean length 15 pm). The dendrites grew to a mean length of about 170 pm at P20. These dendrites then decreased in length, with mean adult lengths of about 80 pm. Thus, in both the owl and the chicken, mature dendrites appear late in development and undergo their most rapid growth after hatching, although development took longer in the owl (less than 10 days in the chick compared to more than 20 days in owl). In general, adult NM neurons end up with a few medium length dendrites (Smith and Rubel, 1979; Jhaveri and Morest, 1982b; Conlee and Parks, 1983).

The major difference between the barn owl and the chicken is that the owl shows a differential loss of dendrites in the high best frequency region of the nucleus, and thus

DEVELOPMENT OF AUDITORY BRAINSTEM 48 1

the adult owl had a much lower mean number of dendrites per cell in the rostromedial region of the nucleus. This loss of dendrites in the high best frequency region of the cell had begun by P21, the last age we examined in this study. By adulthood ( > 8 months age), the majority of cells in the high best frequency (6-10 kHz) rostral region had no dendrites (see Fig. 4, Carr and Boudreau, 1993). Thus, in the barn owl, slow changes in dendritic number and length continued past the first month posthatch. Since these dendrites continued to grow and change weeks after the NM neurons had received mature endbulb inputs, it is unlikely that the development of the synapses per se is directly responsible for the morphological changes. There is evidence for an afferent role in dendritic sculpting. Conlee and Parks (1983) demonstrated that a conductive hearing loss produced substantial deprivation of dendritic growth in chicken NM neurons during the first 2 months posthatch, although it did not produce a change in dendritic number. Second, in the owl, the best frequency of the afferent inputs is a good predictor of dendritic number. The barn owl has exceptional high frequency hearing (Konishi, 1973) and the loss of dendrites in the rostral regions of the nucleus may indicate specializations for phase locking to high frequencies (Sullivan and Konishi, 1984). The reduction in dendritic area might decrease the time constant of the cell and improve the speed and accuracy of the phase locked response to synaptic inputs. Similarly, the increase in cell size should improve the robustness and accuracy of the responses to stimulation. Physiological developments in frequency tuning and phase locking may follow similar time courses to the morphological development.

Development of the nucleus laminaris In the barn owl, sensitivity to interaural time differences

arises in NL. NL neurons receive bilateral input from NM such that the ipsilateral axons enter from the dorsal edge of the nucleus, while the contralateral axons enter ventrally. The dorsoventral arrays created by the interdigitating afferent delay lines form multiple maps of interaural time difference within the nucleus (Carr and Konishi, 1990). The organization of the barn owl NL is very different from the pleisiomorphic pattern seen in the chicken, where the nucleus is composed of a monolayer of bipolar neurons which receive input from ipsi- and contra-lateral cochlear nuclei onto their dorsal and ventral dendrites, respectively (Smith and Rubel, 1979; Jhaveri and Morest, 1982a,b). In the chicken, there appears to be a single map of interaural time difference in the mediolateral direction (Parks and Rubel, 1975; Young and Rubel, 1983, 1986; Overholt, 1992), as opposed to the many dorsoventrally directed maps in the barn owl.

We have described the normal development of NL neurons and will compare their development with that of the chicken. Unlike the NM, the neurons of the owl NL did not show changes in dendritic number past the first week posthatch, but produced large numbers of short dendrites before hatching. The numbers of dendrites decreased in the week after hatching, and then did not change significantly. Thus, dendritic number did not appear to be affected by the maturation of the magnocellular-laminaris circuit.

The development of NL neurons in the owl differs from that in the chicken. Chicken NL development has been

described a t both the ultrastructural level and with the Golgi technique (Cajal, 1908; Smith and Rubel, 1979. Smith, 1981; Deitch and Rubel, 1984; Parks e t al., 1987). In the chicken, a steep anteromedial to posterolateral gradient of increasing dendritic length develops in NL about 10 days before hatching (Smith, 1981). This gradient parallels the tonotopic gradient, with low best frequency neurons having long dendrites and high best frequency neurons having short dendrites. Otocyst removal experiments have shown that this gradient develops in the absence of auditory cues and suggest that the gradient depends upon intrinsic developmental cues (Parks and Jackson, 1987). In the ages we examined (E30-adulthood), we did not find such a gradient. Barn owl NL neurons from the main body of the nucleus resembled the most anteromedial high best frequency chicken NL neurons, which are characterized by numerous short dendrites from early in embryogenesis (E10; Smith, 1981). The owl may either not recapitulate the gradient, or may do so earlier in development.

The two species also differed in that chicken NL neurons show a distinct dorsoventral polarity from the earliest times in embryogenesis, while barn owl neurons from the main body of NL showed no polarity, a t least after E30. Instead, dendrites were distributed evenly over the cell body. Furthermore, the chicken dendrites were longer and more highly branched, and they continued to decrease in number and extent of branching until P25 (Smith, 1981). The number of owl NL dendrites decreased until about P6 (Table 1) but did not show further changes in number. Since NL neurons are susceptible to afferent regulation in the chicken, this lack of dendritic variation is curious. For example, in the chicken, removal of afferent input from either side results large losses in the affected dendritic arbor (Deitch and Rubel, 1984; Parks and Jackson, 1987). It is possible that the owl NL neurons have no mechanism for differentiating between inputs from either side, and thus cannot employ conventional mechanisms for afferent regulation. Unless some signal permitted differentiation of ipsilateral from contralateral NM inputs, owl NL neurons should not be able to determine the ipsi- versus contralateral nature of their inputs, since the dendrites show no dorsoventral polarity, the NM inputs from each side are evenly distributed over the soma and dendrites, and all inputs phase-lock to the auditory stimulus (Carr and Konishi, 1990).

Comparisons between altricial and precocial species

The rearing of young birds follows two distinct patterns. One type, the precocial bird, hatches out covered with down, legs well developed, eyes open and able to feed itself. The other altricial type is born nearly naked, blind, and unable to support itself on its legs (Welty, 1979). One of the greatest differences between altricial and precocial birds is in brain development; in altricial birds, the brain a t hatching is small in proportion to those parts of the body involved in metabolism. The growth of myelination is also delayed with respect to precocial birds. Once hatched, altricial birds grow more rapidly than precocial birds, and tend to have larger brains for their body weight (Welty, 1979).

The effects of these two strategies may be compared in the development of the auditory system of the precocial


chicken and the altricial barn owl. Despite dramatic differences in their life histories, the two species showed very similar patterns of development, except that in the barn owl, the duration of development and developmental plasticity was longer in the barn owl. The major differences between the barn owl and the chicken appear in this later period of posthatch development. The late development of NM dendrites in the owl parallels that described in the chicken (Conlee and Parks, 1983), but owl neurons grow larger, have longer dendrites, and show a longer period of dendritic growth and regression than that described for the chicken. The gradient of decreasing dendrite number with increasing best frequency which is not seen in the chicken appears fairly late in development, and may be related to the barn owl's extended frequency range (Carr and Bou- dreau, 1993). In keeping with the longer time that the barn owl chick spends in the nest, both NM and NL continue to show small changes past P21, the time when interaural time difference cues stabilize, while chickens show few changes in NM and NL past P10 (Conlee and Parks, 1983; Smith, 1981).

ACKNOWLEDGMENTS We would like to thank M. F. Kubke and 2 referees for

providing helpful comments on the manuscript. The work was supported by NIH DCD 00436 to CEC.

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Development of the time coding pathways in the auditory brainstem of the barn owl

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