Hearing Research, 55 (1991) 81-91 Q 1991 Elsevier Science Publishers B.V. All rights reserved 0378-5955/91/$03.50

81

HEARES 01603

Postnatal saturation

of spiral ganglion neurons: A horseradish peroxidase study

D . D 1 Simmons 1,2,L. Manson-Gieseke

‘, T.W. Hendrix *, K. Morris

’ Natara~ Science Di&ion, Peppe~dine U~i~e~s~ty,~a~ibtl, Ca~iforrl~a, ’ Department California, U.S.A.

ofBiologyand

’

and S.J. Williams

’

the Brain Resea~cb ~~siitute, UCLA, Los Angeles,

(Received 15 November 1990; accepted 27 February 1991)

Using an in vitro cochlear preparation from postnatal hamsters, spiral ganglion cells WXsl were labeled retrogradely following extracellular injections of HRP into the cochlear nerve. In 24 cochleae from hamsters between postnatal days iP) 0 and IO, the neuronal morphology of 201 SGCs and their peripheral axons were analyzed. From P 0 to 3, labeled SGCs had few distinguishable features. Although SGCs could be traced separately to inner hair cells (IHCs) and outer hair cells (OHCs), they all had roughly bipolar-shaped cell bodies. Approximately half of the labeled SGCs had peripheral axons that spiraled some distance before entering radial fiber bundles. From P 3 to 7, SGCs increased in size by nearly 30% and the number of SGCs with spiraling peripheral axons decreased to near zero. At P 10. the central axon diameter to peripheral axon diameter ratios distinguished two ~pulations of SGCs. The hair-cell inne~ation patterns of SGCs also changed morphologically as a function of postnatal age. At P 0, radial fiber (RF) terminals of peripheral axons contacted as many as 8 1HCs; by P 3, RFs contacted typically one or two IHC’s. The terminal portions of peripheral axons contacting OHCs did not show any appreciable spiral until P 2. By P 5, individual outer spiral fibers (OSFs) had greater spiral lengths underneath row-3 OHCs and the number of OHC contacts was also greatest for row-3 OSFs. These data suggest that SGCs undergo a systematic maturational process. Furthermore, the morphological differentiation of SGCs occurs after they have established separate inner and outer hair cell innervations. Development; Cochlea; Organ of Corti; Neuronal morphology; Branching patterns

introduction

Recent studies of cochlear innervation in adult animals suggest there are significant differences between the neuroanatomy of the neonate and adult. In adult mammals, two fundamentally different types of spiral ganglion neurons contact separately the two types of hair cells. Type-I neurons send peripheral axons to terminate on inner hair cells (IHCs) via large radial fibers (RFs) and type-II neurons send their peripheral axons to terminate on outer hair cells (OHCs) via thin outer spiral fibers (OSFs). The type-1 neurons constitute between 90-95% of the afferent neuron population, whereas type-II neurons comprise the remainder of the afferent neuron population (Spoendlin, 1972; Spoendlin, 1974). The cochlear connections and response properties of type-1 neurons are well described Wang et al., 1965; Liberman and Oliver, 1984): most type-1 neurons contact only a single IHC. On the other hand, there is only a single report that gives a possible type of a response property for type-II neurons (Robertson, 1984). Recent anatomical studies show type-II neurons with very complicated cochlear connections:

Correspondence

to: Dwayne D. Simmons, Department of Biology, 405 Hilgard Avenue, UCLA Los Angeles, CA 90024-1606, U.S.A.

(i.e., OSFs) the vast majority of OSFs terminate on OHCs in a single row; the third row of OHCs receives the majority of afferent innervation; and single OSFs contact anywhere from 6 to 98 OHCs depending on cochlear location (Simmons and Liberman, 1988a). How adult innervation patterns are established is not explained by previous anatomical studies in neonatal and young animals which have relied mostly on the Golgi stain (Ginzberg and Morest, 1983; Lorente de No, 1937; Perkins and Morest, 1975; Smith and Haglan, 1973). Although a great deal has been learned from the Golgi work, few of these studies attempted to provide systematic data on the maturation of branching patterns and spiral ganghon cell bodies. Additionally, these Golgi studies suggest a different view of cochlear innervation patterns. For example, RFs branch and terminate on multiple IHCs and OSFs typically contact more than one row of OH&. The present study applied a horseradish peroxidase (HRP) technique to neonatal cochleae which has been used successfully in adult animals to achieve a Golgi-like filling of spiral ganglion neurons (Liberman and Simmons, 1985; Kiang et al., 19821. The present report describes the morphology and peripheral innervation patterns of spiral ganglion neurons from neonatal hamster cochleae. An in vitro preparation mitigated significantly the difficulty associ-

x2

ated with surgery on newborn animals (Simmons et al., 1990). To minimize the confusion associated with different rates of maturation and morphological gradients along the cochlear spiral, this study includes only data from spiral ganglion neurons innervating the basal 40% of the cochlear spiral. These data allow a systematic analysis of the maturation of spiral ganglion neurons as a function of postnatal age.

Materials

The in vitro procedure has been described elsewhere (Simmons et al., 1990). Briefly, hamsters were perfused intracardially with an oxygenated media (typically Eagle’s Minimum Essential Media) that had been sterilefiltered. Immediately, the bullae were located and the cochleae dissected free and placed in an organ chamber (at room temperature1 with oxygenated media continuously supplied. A micropipette (broken to the desired O.D. between 30 and 40 pm) filled with a 40% solution of horseradish peroxidase (Sigma type VI in 0.05 M Tris buffer, pH 8.6) was inserted into the auditory nerve stump and small (nanoliter) quantities of HRP solution were pressure ejected (20-50 psi at I-50 ms pressure pulses). One to 4 h after the last injection, the cochleae were immersed in a fixative solution (2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3) with 0.008% CaCI, at 10°C for a minimum of 4 h. Cochleae from hamsters older than six days were decalcified in a

and Methods

All retrograde labeling data came from newborn hamsters (Mesocricetus aurutus) between postnatal days (P) 0 and 10. The day of birth was defined as P 0. Hamsters were either bred in house or obtained already pregnant. In all experiments, healthy postnatal hamsters were anesthetized with a near-lethal injection of sodium pentobarbital, IP (15 mg/kg body weight).

B

CELL BODY c--

CENTRAL

PERIPHERAL

AXON

AXON

!-

RADIAL

FIBER TERMINATION

J

;,

0 Fig. I. A. Photomicrograph P 3 hamster

cochlea.

reconstructed

of an HRP

labeled spiral ganglion neuron. The neuron was labeled extracellularly

The scale bar represents

IO Km.

in a light microscope using a 100~

B. A digitized

oil-immersion

tracing

of the same neuron

objective. The terminal

20 pm

000,o IHC NUCLEI at the 30-20%

distance region of a

as in A. The spiral ganglion

neuron

radial fiber contacts two inner hair cells (IHCsI.

was

83

phosphate buffered after fixation.

solution of 0.1 M EDTA at 10°C

Histological processing

Cochlear tissue was sectioned at 100 pm on a vibratome after being embedded in a gelatin-albumin mixture cured with glutaraldehyde. Tissue sections were incubated in a cold (4°C) solution containing a 1% solution of cobalt chloride and a 1% solution of nickel ammonium sulfate added to a 0.05% solution of diaminobenzidine (DAB) in phosphate buffer with 1% dimethylsulfoxide, 0.5% imidazole and 3% hydrogen peroxide (Adams, 1981). After a total incubation time of 30 min, the sections were rinsed several times, mounted on slides, dehydrated, and coverslipped with Permount. Light microscopic analysis

Neurons that were judged to be completely filled and unambiguously interpreted formed the primary database. Unacceptably labeled neurons were those that had either their cell body or its peripheral axon weakly labeled; generally, such neurons had their peripheral axon fade within 20 pm of the cell body. All completely reconstructed neurons had to be labeled darkly throughout their peripheral axon and had to have either a distinct swelling or an abrupt ending marking its termination. It should be noted that cell morphometry data also included measurements from labeled neurons that may not have been traced completely to their peripheral terminations, whereas all hair cell innervation data were obtained from completely reconstructed neurons. Labeled afferent neurons were reconstructed in the light microscope using a 100 X oil-immersion objective and traced either with a drawing tube or with a stylus on a monitor (Fig. 1). Tracings were always of the maximum silhouette area of a labeled profile. All tracings were digitized, and morphometric data generated by computerized planimetry. Cell body size was estimated as the maximum silhouette image obtained from a cell that had both central and peripheral axons within the tissue section. The angle that the peripheral axon (over a 30 pm segment from the cell body) projected was determined against the radial-most direction from the cell body to the organ of Corti. The average diameter of axon segments was estimated by tracing the maximum diameter (over a minimum of 20 pm), computing the silhouette area of the segment, and dividing the silhouette area by the path length.

Results General observations

To minimize the amount of morphological variation due to differences in cochlear location at any given

age, spiral ganglion cell (SGC) data were limited to the basal 40-10% distance region along the cochlear spiral. However, it should be noted that the cochlear spiral is lengthening during this period. Over the course of the first 10 postnatal days, the cochlear spiral nearly doubled in length (about 4 mm at P 0 to 7 mm at P 13). Thus for each postnatal day, the entire cochlear spiral was reconstructed, and spiral lengths normalized. When extracellular HRP injections were successful (typically 1 out of 5 experiments), it resulted in massive numbers of SGCs being labeled, thus obscuring their reconstruction. Occasionally as seen in Fig. lA, regions were found with isolated, labeled SGCs whose peripheral axons were clearly traceable to their hair-cell terminations. In addition to spiral ganglion neurons being labeled, frequently (roughly one out of two cases>, efferent axons to the cochlea were labeled (Simmons et al., 1990). A total of 201 cell bodies were labeled retrogradely and traced to the organ of Corti in a total of 20 hamsters ranging in age from P 0 through P 10. Roughly twenty-five percent (N = 50) of these labeled cells had abrupt terminations in the organ of Corti. At birth (P O), there were few differences that distinguished SGCs (N = 41). Qualitatively, they were all bipolar and had uniform, oval-shaped cell bodies. Mean cell body size was 51.1 pm* (SD + 10.1). SGCs gave rise to peripheral axons with an average diameter less than 0.2 pm (SD + 0.1) immediately adjacent to the cell body. Although the peripheral axon was larger near the cell body, there was only gradual diminution as the axon reached the organ of Corti. Additionally, labeled cells innervating IHCs (N = 10) were indistinguishable from cells innervating OHCs (N = 5). Maturation of cell bodies

Consistent with developmental literature (e.g., Panesse, 1974; Uray and Gona, 1978), there were several characteristics that changed as a function of postnatal age. Digitized tracings of representative ganglion cells from four different postnatal ages are shown in Fig. 2. Although SGCs remained roughly bipolar in shape from P 0 to P 10, cell body size increased by as much as 50% from P 0 to P 3 and 150% over the P 0 to P 10 period. The increase in cell size is plotted in Fig. 3. The growth in cell size was roughly linear (r2 = 0.84). Another feature that changed dramatically was the orientation of the peripheral axon leaving the cell soma. At birth, about 50% of the SGCs had cell body orientations that were roughly parallel to the cochlear spiral. These SGCs gave rise to peripheral axons that deviated by more than 30 degrees from the radial direction (toward the organ of Corti), thus resulting in peripheral axons that spiraled before joining one of the radial bundles (Fig. 4A). In some cases, the peripheral axon that spiraled in a direction parallel to the organ

Day 0

Day 3

Day 7

Day 10

4

ORGAN OFCORTI

Fig. 2. Digitized

tracings of labeled spiral ganglion cell bodies traced to the organ of Corti. The peripheral

axon toward the right in all cases. These are representative

of Corti (i.e., at 90” to the radial direction). Several of these peripheral axons were traced to radial fiber terminations on IHCs. As shown in Fig. 4B, the percentage of spiraling axons decreased from P 0 to P 10. At each postnatal age measured there was greater than a 50% decrease in the number of cell bodies found with spiraling axons. On P 10 and thereafter, spiraling axons could not be found.

1

0

2

3

4

5

POSTNATAL

Fig. 3. Mean as a function individual

silhouette fN=

age. The

from a minimum

8

9

10

are

calculated

line is calculated

(see Methods). the standard

Sample sizes

deviation

from the equation

+ 59 with rz = 0.84.

from

the sample of all spiral

of 14 cells (at P 10) to a maximum

cells fat P 3). The error bars represent mean. The regression

means

10) and represent

ganglion cells that met labeled criteria varied

7

AGE

areas of labeled spiral ganglion cells plotted

of postnatal

animals

6

axon is toward the left and the central

of cells found in cochleae of hamsters at four different

of 100 of the

Y = 6.6X

ages (as indicated).

Axonal dimensions near the cell soma also changed as a function of postnatal age. In the newborn cochlea, the mean diameter of the peripheral axon was roughly equal to the central axon diameter. As the hamster aged, more cells had larger central axons compared to peripheral axons within the immediate vicinity of the cell soma. This phenomenon is graphically represented in Fig. 5. The average ratio of the central axon diameter (DC) to peripheral axon diameter (DP) was statistically unchanged from P 0 (mean: 1.6 f 0.8) to P 7 ( mean: 1.8 f 1.4). The maximum DC to DP ratio increased from 3.7 to 5.1 P 0 to P 7 (Fig. 5). However, an abrupt change in the DC to DP ratio occurred at P 10: the mean DC to DP ratio was about 4.1 (SD k 2.7) with a large degree of scatter (variance: 7.5). This increase in the DC to DP ratio was due largely to an increase in the DC, whereas the DP remained almost unchanged within the immediate vicinity of the soma postnatal ages. The DC to DP ratio also appeared to differentiate a subpopulation of SGCs in P 10 hamsters. This separation was independent of cell body size as shown in Fig. 5D. Only three SGCs had a DC to DP ratio less than one and all three gave rise to OSFs underneath OHCs. In the remainder of the sample (N = ll), all SGCs had a DC to DP ratio greater than one and five of these could be traced definitively to IHCs. Furthermore as shown in Fig. 6, diameter measurements along the course of the peripheral axon revealed another characteristic that separated neurons but only at P 10 (Berglund and Ryugo, 1987): 1) those whose peripheral

85

A SPIRALGANGLION

CELLS

!

ORGAN

OF CORTI

+ lOllm 1OOJ

case after an HRP extracellular injection. A total of 118 RFs were traced to cell bodies. In the newborn cochlea (P 0 and P l), observations were made from 15 labeled RFs. At P 0, it was admittedly difficult to distinguish labeled fibers that contacted IHCs from those that contacted OHCs since the separation between IHCs was not always clear especially in cases where the cochlea was sectioned parallel to the basilar membrane. With this in mind, no RF was observed contacting both IHCs and OHCs. However, the vast majority (roughly 80%) of RFs to the IHCs were branched, contacting two or three IHCs (Fig. 7A). Many RFs spiraled short distances (three or four IHC lengths) underneath IHCs before a final termination (Fig. 7A). The maximum number of IHCs contacted by a single RF was eight. Frequently, RFs had both terminal swellings and en passant swellings that abutted against IHCs. Thus, assuming swellings in contact with IHCs to be of significance (Simmons and Liberman, 1988a; 1988b), the maximum number of hair cell contacts (swellings) made by a single RF was 12. By P 2 branching and multiple hair cell contacts decreased abruptly: greater than 60% of the RFs had only a single IHC termination. Although branching and multiple terminations were still present at P 3 (Fig. 7A), they were even less common. By P 8, branching and multiple terminations were not observed (Fig. 7B). In

A IO

0 DAY

9

3 DAY

8 7

POSTNATAL

AGE

Fig. 4. A. Digitized tracings of two spiral ganglion neurons from a P 3 hamster and located in the same region of the ganglion. Both cells terminated on IHCs. The cell of the left had its cell body oriented orthogonal to the direction of the organ of Corti and gave rise to a spiraling peripheral axon. B. The percentage of spiral ganglion cells that gave rise to peripheral axons which spiraled at least 30 pm is plotted versus postnatal age.

;j 0

,&, , , 50

loo

150

200

CELL BODY SIZE

7 DAY

D

IO 9

axon was initially thin and subsequently increased in size, and 2) those whose peripheral axon continued to taper once leaving the cell body. The three SGCs that innervated OHCs also had gradually tapering peripheral axons whereas the five SGCs that innervated IHCs had peripheral axons with increasing diameter before reaching the organ of Corti. Maturation of hair cell innervation

The terminal portions of 151 SGCs were reconstructed in the organ of Corti from P 0 to 10. As in adult animals (Simmons and Liberman, 1988a), labeled RFs greatly outnumbered labeled OSFs in any given

8 7 &

6

1

10 DAY

A A

A

es x4 3 2 1

1

0

0, 0

50

loo

150

CELL BODY SIZE

200

A 0

I 50

, loo

A

A , 150

, 200

CELL BODY SIZE

Fig. 5. Ratio of the central axon diameter (DC) to peripheral axon diameter (DP) plotted versus the silhouette areas of the cell bodies. Data are taken from a single cochlea at the ages specified. At P 0, 3, 7, and 10, the sample sizes are 41, 64, 17, and 14, respectively.

86

A

7 DAY 6 5

4

1

E i

l

3 2i l

1-d

0

0

%

0 0

2

4

6

8

10

&dLL.~AL IHC NUCLEI

DC/DP 10 DAY

0

l

B

l P7

0

2

4

6

8

RFs

c

10

DC/BP Fig. 6. Axon diameter ratios for two animals at P 7 and P 10. The data are from a subset of the same cells as in Fig. 5C,D. The ratio of the diameter of the peripheral axon at two locations (DPl and DP2) is plotted versus the ratio of the DC to DP. DPI is the average diameter of the peripheral axon at 20 pm from the cell body and DP2 is the average diameter at greater than 100 pm. Open symbols represent cells traced to OHCs.

general, RFs at this postnatal age exhibited adult characteristics as described in other studies (Liberman and Oliver, 1984). Another feature of RFs that changed within a very short postnatal period was the fiber caliber. In the newborn cochlea, the average diameter of RFs was less than 0.2 pm in the region (20 pm> immediately adjacent to their IHC termination. Their small caliber made it very difficult to reconstruct individual RFs. As branching pared, the average diameter increased substantially from P 0 to P 2. As shown in Fig. 8, mean RF diameter was 0.6, 0.8 and 1.0 pm at P 2, 5, and 8, respectively. Additionally, both the maximum diameter and the range of diameters also increased during this postnatal period. From P 2 to P 8, the maximum diameter increased from 1 to 2 pm and the range of diameters increased from 0.6 to 1.0, respectively. Underneath OHCs, a total of 33 labeled fibers that had cell bodies in the spiral ganglion were reconstructed at various postnatal ages. Since the tunnel of Corti is not yet formed in the newborn cochlea, these reconstructions are probably skewed to fibers innervating OHC rows 2 and 3 especially in the P 0 and P 1

25 pll

Fig. 7. Drawing tube reconstructions of the terminal portions of RFs. A. Representative RFs taken from hamsters at P 0 and P 3. Shown semi-schematically are the nuclei of the IHCs. Most RFs at P 0 contacted at least two IHCs with a combination of terminal and en passant swellings. The scale bar is given in B.B. Representative RFs taken from hamsters at P 7 and P 10. The IHC is shown semi-schematically in cross section as a RF ascends and makes a single termination.

hamster. Nonetheless, seven fibers were reconstructed in the P 0 cochlea and of these, three were seen with branches that contacted a single IHC. These fibers 2d ‘s

1.5.

2

5

8

POSTNATAL AGE Fig. 8. Mean RF average diameter for spiral ganglion neurons in hamsters at P 2. P 5 and P 8. The error bars represent the standard deviation of the mean.

87

4

-1

a

TUNNEL OF CORTI

BASE

TUNNEL OF CORll

\

t

8OHCs

C TUNNEL OF CORTI

OHC CONTACTS

b

OSF

+

26 OHCs

Fig. 9. Drawing tube reconstructions of the terminal portions of OSFs. A. Representative fibers contacting OWCs taken from hamsters at P 0. The leftmost fiber sends a thin branch which contacts the base of an IHC. All of these fibers had en passant swellings. The cochlear base is toward the left as indicated. Outer hair cell nuclei are represented as circular profiles. B. An OSF from a P 2 hamster. This fiber spirals underneath a total of eight OHCs. Outer hair cell nuclei are represented as circular profiles. C. An OSF from a P 6 hamster. The fiber contacts a total of 13 OHCs and spirals underneath 26 OHCs before termination. Outer hair cell nuclei are represented as circular profiles.

spiraled in a basal direction for a maximum distance of 60 pm (underneath OHCs), with as many as five hair cell contacts. Several labeled fibers did not have any appreciable spiral and just terminated on OHCs as soon as they traversed underneath IHCs. Although the exact OHC row was often difficult to define in the newborn cochlea, in all cases labeled fibers contacted OHCs in more than one row. Additionally, all labeled fibers had at least one en passant swelling in contact with an OHC.

Fig. 9 presents drawings of the terminal distributions for representative OSFs, typical of postnatal ages 0, 2 and 6. From P 0 to P 2, the most obvious change was that all fibers spiraled a minimum distance of 50 pm and the mean spiral length more than doubled (Fig. IO). Even in P 2 hamsters, it was still difficult to discern the exact OHC row innervated; however, such was not the case in the hamsters of more advanced postnatal ages. Not only did OSFs show a large increase in their spiral underneath OHCs but it was

88 800

11

Discussion

Postnatal morphology of SGCs

0

5

6

POSTNATAL AGE Fig. 10. Mean bars represent

spiral length for OSFs in P O-8 hamsters. The error the standard deviation of the mean. The data are not separated by individual OHC row.

possible to discern the row in which they contacted OHCs. For example in a P 5 hamster, five OSFs were reconstructed: two contacting OHCs in row 1, one contacting OHCs in row 2, and two contacting OHCs in row 3. In P 5 and older hamsters, OSFs spiraled in a basal direction for at least 170 pm, contacted a single row of OHCs, and did not have branches terminating on IHCs (Figs. 9 and 10). From the data presented in Fig. 10 it might appear that certain characteristics of OSFs do not increase very much after P 5. However as in adult animals (Simmons and Liberman, 1988a), OSFs have characteristics which are highly correlated to the OHC row in which they contact (Table I>. For example, a comparison of individual fibers indicates that spiral length is yet increasing from P 5 to P 8: OSFs (N = 7) contacting row-l OHCs have a mean spiral length of 291.7 km (S.D. * 83), while OSFs (N = 7) contacting row-3 OHCs have a mean spiral length of 495.8 pm (SD i 118). Similarly, the number of contacts which abut OHCs is dependent on OHC row. Outer spiral fibers contacting row-l OHCs have a mean of 7 contacts (per OSF) while OSFs contacting row-3 OHCs have a mean of 18 contacts.

TABLE OHC

I

ROW

INNERVATION

POSTNATAL

DAYS 5-10 Sample size

(wm)

Mean OHC contacts per OSF

293+ 83 391 f 159 495*117

7.3 k 2.9 8.3 f 2.4 17.8+8.3

7 4 7

Mean spiral length OCH row I OHC row 2 OHC row 3

FOR

The present study suggests that although at least two types of SGCs exist at birth in the hamster based on their separate hair cell innervations, their cell bodies are not morphologically distinguishable until after the first postnatal week. Previous light-microscopic studies of neonatal cochleae have also shown bipolar neurons that innervate IHCs to be indistinguishable from those that innervate OHCs (Lorente de No, 1937; Perkins and Morest. 1975). In a Golgi study of rat and kitten, Perkins and Morest (1975) reported that there were no consistent differences in size or shape between the cell bodies of spiral ganglion neurons giving rise to RFs or OSFs. Similarly, in a study of protargol-stained SGCs in the neonatal cat, Kiang et al. (1984) could not differentiate two discrete classes using various criteria that had been used successfully in adult animals. Furthermore, electron-microscopic observations in the neonatal cochlea also suggest that most, if not all, the SGCs in the neonatal animal are initially bipolar in their shape and unmyelinated (Romand and Romand, 1984; Romand and Romand, 1985; Schwartz et al., 1983). The lack of morphologically distinguishable neurons in the early stages of cochlear development is consistent with the view that immature sensory neurons may all be bipolar in shape and will only assume distinctive morphologies later in their development Wang, et al., 1984; Pannesse, 1974). Similar to descriptions of bipolar SGCs found in older studies of cochlear innervation (e.g., Lorente de No 1937), we observed that SGCs in newborn cochleae could have a variety of neuronal geometries: both radial and nonradial orientations. Cells with nonradial orientations typically sent their peripheral axons spiraling some distance before joining radial bundles. This is in contrast to the SGC orientations found in adult animals where cell bodies are almost always oriented radially within the spiral ganglion and send their peripheral axons more or less in an orthogonal direction towards the cochlear spiral. To date, HRP studies show radially oriented SGCs in the adult mouse (Berglund and Ryugo, 1987), guinea pig (Brown, 1987), and cat (Liberman and Oliver, 1984). The radial orientation of cell bodies probably aids in maintaining cochleotopic organization; this assures that neighboring SGCs are receiving input from the same or adjacent regions of the organ of Corti (Liberman and Oliver, 1984; Simmons and Liberman, 1988aI. Thus, the spiral ganglion should have the same spatial representation of frequency found along the basilar membrane, and this has been recently demonstrated (Keithley and Schrieber, 1987). Are SGCs in the newborn similar in their cochleotopic organization as adult animals‘? At least the possibility is raised by the data

89

presented that at birth some SGCs may not project to the same cochlear regions as their nearest neighbors. Since the present study also suggests that by the end of the first postnatal week, only radial orientations and nonspiraling axons are found, this raises several interesting questions: Are neurons with nonradial geometries still present and if so, how does either the change in cell geometry or the direction of the peripheral axon occur (assuming it does)? The presence of neurons with multiple geometries at birth appears to be a common developmental observation. Throughout the vertebrate nervous system, studies of neonatal neurons have shown that many of them experience significant changes in cell geometry and shape as they mature (Hume and Purves, 1981; Pannesse, 1974; Uray and Gona, 1978). Additionally, our findings suggest that the majority of SGCs go through a significant morphological change between P 7 and P 10. Although the sample in the present study was small, by P 10 we could differentiate two types of SGCs based on morphological criteria that correlated with separate inner and outer hair cell innervations; this was not possible in hamsters at P 7 or before. Consistent with this, Romand and Romand (1984; 1985) in an electron-microscopic study reported that at birth they could observe only a single, morphological type of SGC; however, by the end of the first postnatal week, they were able to distinguish a second cell type. To date in adult animals, the most successful morphologic criterion used to define SGCs which project to IHCs versus OHCs appears to be the morphology of the central and peripheral axons as they leave the cell body (Berglund and Ryugo, 1987; Brown, 1987; Kiang et al., 1982). In cat, guinea pig and mouse, type-1 neurons traced to IHCs have a very fine caliber in the initial segment (from the cell body) of the peripheral axon compared to the central axon, whereas type-II neurons traced to OHCs have similar axon diameters. At greater distances from the cell body, the peripheral axon of type-1 neurons enlarges in caliber. The analysis of the axon caliber of type-l neurons in the adult cat has led Liberman and Oliver (1984) to hypothesize that the thin region of the peripheral axon nearest the cell body is a node of Ranvier. Whether or not this is true can be confirmed only by electron microscopy. Since adult-like innervation patterns are well established prior to the ability to distinguish separate populations of SGCs, this raises the interesting possibility that type-1 and type-II neurons only become morphologically distinct when SGCs become myelinated. According to Pujol and Abonnec (1977), myelination of SGCs begins to occur in hamster about P 8. Thus, the process of myelination by the Schwann cells may serve as the developmental marker that gives rise to two morphologically distinct cell types in the postnatal cochlea. This hypothesis would not only be consistent

with the present results but several other light and electron microscopic studies (Kiang, et al., 1984; Romand and Romand, 1985; Schwartz, et al., 1983). However, one can not exclude the possibility that there may be other influences such as signals from hair cells or even efferent synaptogenesis that also play a role in SGC differentiation (Simmons et al., 1990). Postnatal hair-ceil innervation

It has not been known whether the morphological discrepancies of hair cell innervation patterns between recent adult studies and previous neonatal studies are due to technical differences, age differences or possibly sampling differences. The present study suggests very strongly that anatomical discrepancies between the adult and neonate are due to maturational processes as described elsewhere in the nervous system. In the cochleae of adult cats, there are clear morphological gradients in the afferent innervation both longitudinally and radially that are nonmonotonic. For example, the number of RFs contacting IHCs is maximal near midbasal regions and decreases toward the apex and basal hook. Similarly, the number of OHC contacts by single OSFs is maximal in the middle regions of cochlear spiral. Additionally, both types of fibers are more highly branched in the apex than in other regions. Thus depending on the region sampled, there could be significant differences in the innervation data obtained. An added complexity also exists in the neonatal cochlea where there are apparently different rates of maturation, that is, the upper basal regions mature before either the extreme base or apex. For these reasons, the sampled data was restricted to the basal portion of the cochlear spiral (excluding the extreme base). However, previous studies in the neonate did not have knowledge of such morphological variations and may have sampled somewhat indiscriminately across the cochlea. This would lead to a misinterpretation of morphological maturation. Other HRP studies have concluded that there are few, if any, differences due to technique or species (Berglund and Ryugo, 1987; Brown, 1987; Ryugo and Fekete, 1982). Neonatal neurons, in general, tend to be more highly branched and have more expansive innervation fields than neurons found in adult animals (Ivy and Killackey, 1981; Stanfield et al., 1982; Whitehead and Morest, 1985). As an animal ages, the immature neurons are either lost or retract branches where functional connections are not made. The present study confirms that there are transient developmental features of RFs and OSFs in the newborn animal as suggested by previous studies (Berglund and Ryugo, 1987; Ginzberg and Morest, 1983; Ginzberg and Morest, 1984; Simmons and Liberman, 1988a; Simmons et al., 1990). For example in a study of progressively older postnatal ages in the cat, Ginzberg and

YO

Morest (1983) noted that many anatomical features of afferent fibers in their youngest animals such as growth cones, filopodial branches and varicosities are seen less frequently and are even lost by the end of the second postnatal week. In the one-month postnatal cochlea, most RFs were unbranched and contacted the bases of IHCs by means of simple swellings, and OSFs with IHC branches were no longer observed. In regard to OSFs, the maturational situation may be different. In agreement with other studies (Ginzberg and Mores& 1983), branches to IHCs as well as multiple row innervations seem to disappear as a function of postnatal age; however we found that both the length and number of contacts with OHCs increased as a function of postnatal age. By limiting our data to neurons reconstructed within the basal 40-100/o distance regions it is unlikely that this developmental phenomenon is due to OSF variations along the cochlear spiral as found in other studies (Ginzberg and Morest, 1983; Simmons and Liberman, 1988a). Additionally, we found OSFs contacting OHCs in row 3 had longer spirals and more OHC contacts than OSFs contacting row 1 OHCs. Extensive studies in adult animals have shown that OSFs contacting primarily row 3 OHCs spiral for greater distances and that afferent terminals are distributed preferentially to row3 OHCs (Brown, 1987; Liberman and Simmons, 1985; Simmons, 1986; Simmons and Liberman, 1988a). This suggests that an adult-like pattern as well as distribution of afferent OHC terminals are established as early as P 2 in the neonatal hamster. In a similar study on cochlear efferent axons, we found that the efferent OHC innervation was preferentially distributed to OHCs in row 1 by P 6 which was the earliest day they could be detected underneath OHCs (Simmons et al., 1990). In combination with the present results that OSFs have a greater number of contacts to row 3 OHCs, we hypothesize that the afferent terminals on OHCs in rows 2 and 3 may be responsible, in part, for limiting most of the efferent terminations to row-l OHCs. This assumes that the number of OSFs to all three rows is equal. A simple prediction from this hypothesis is that the number of afferent terminals per OHC should be greatest on row 3 OHCs and least on row 1 OHCs during this period. However, the prediction of a lower number of afferent terminals on row 1 OHCs is exactly opposite to what most investigators have observed. Electron microscopic studies report numerous afferent terminations on row-l OHCs just prior to and during efferent synaptogenesis (Ginzberg and Morest, 1984; Pujol and Abonnec, 1977; Pujol and Carlier, 1982; Pujol et al., 1978). It is also reported that the afferent terminals decrease significantly after efferent synaptogenesis. Such observations led Pujol et al. (1978) to hypothesize that efferent terminals compete with afferent terminals

contacting OHCs. The numerous afferent terminals seen on row-l OHCs in the neonate could be the result of OSF sprouting similar to observations from denervation studies in the adult animal (Iurato et al., 1978). In an efferent denervation study performed on kitten, Pujol and Carlier (1982) could find no evidence for such afferent sprouting. Where do these additional afferent terminals originate? It is possible that the present study missed a class of OSFs that exhibit such sprouting or that there may be more OSFs contacting row-l OHCs in the neonate than in the adult. An intriguing suggestion is that RFs may be responsible for some, if not most, of the afferent contacts on row-l OHCs (Pujol et al., 1985; Simmons, 1991).

Acknowledgements

This research was supported by NSF grants Rll 8709611 and DIR 8900930, a grant from the Ralph M. Parsons’ Foundation, and a fellowship award from the Alfred P. Sloan Foundation. A preliminary report of this research has appeared in abstract form (Simmons, et al., 1988~).

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