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Naturally occurring neuron death during postnatal development of the gerbil ventral cochlear nucleus begins at the onset of hearing

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THE JOURNAL OF COMPARATIVE NEUROLOGY 387:421–429 (1997)
Naturally Occurring Neuron Death During
Postnatal Development of the Gerbil
Ventral Cochlear Nucleus Begins
at the Onset of Hearing
TRAVIS S. TIERNEY AND DAVID R. MOORE*
University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
ABSTRACT
Postnatal development of the gerbil ventral cochlear nucleus (VCN) was studied
quantitatively under the light microscope in Nissl-stained serial sections at postnatal day 0
(P0), P5, P7, P10, P12, P15, and P140. VCN boundaries were unambiguous at all ages, and
nucleus volume was calculated planimetrically for all groups. Measurements of neuron soma
cross-sectional area and number were made in all groups except P0. Both VCN volume and
soma area doubled between P5 and P10. Although somatic growth did not continue beyond
P10, VCN volume increased a further 57% between P15 and P140. Neuron number did not
change significantly between P5 and P10, averaging approximately 36,000 neurons. Between
P10 and P12, neuron number decreased significantly by 22%, with no further change
thereafter. Our data show that, following significant postnatal growth in the gerbil VCN, a
brief period of naturally occurring neuron death begins at the onset of hearing. J. Comp.
Neurol. 387:421–429, 1997. r 1997 Wiley-Liss, Inc.
Indexing terms: auditory brainstem; degeneration; quantitative development; programmed neuron
death; pyknotic cell
Following proliferation and migration, most developing
neural populations undergo a brief period of neuron death,
which is often referred to as naturally occurring neuron
death (NND). Although dying embryonic cells have been
reported in proliferative zones during neurogenesis (see,
e.g., Carr and Simpson, 1981; Homma et al., 1994; Blaschke et al., 1996), the term NND is usually restricted to a
later ontogenic period when a significant fraction of differentiating neurons die while forming functional connections with their target field (for reviews, see Cowan et al.,
1984; Oppenheim, 1991; Clarke, 1994). NND is thought to
assist in the development of functional connectivity by
eliminating erroneously projecting neurons and by matching numbers of neurons between projection and target
nuclei (Cowan et al., 1984). The primary aim of this paper
is to establish whether NND occurs in the mammalian
ventral cochlear nucleus (VCN) during the period when
postmigratory neurons begin to establish functional connections.
Neurogenesis of VCN neurons in the mouse (Taber
Pierce, 1967; Martin and Rickets, 1981) and rat (Altman
and Bayer, 1980) occurs approximately 1 week before
birth. Newly generated neuroblasts then migrate to form
well-defined nuclear aggregations and begin to extend
growth cone-tipped axons just before birth (Kandler and
r 1997 WILEY-LISS, INC.
Friauf, 1993). Although initial projections from differentiating neurons in the VCN to targets within the brainstem
are present at birth in both the rat (Friauf and Kandler,
1990) and the gerbil (Kil et al., 1995), adult-like collateral
refinement and mature synaptic morphology emerge during the second postnatal week (Kil et al., 1995; Russell and
Moore, 1995). Final maturation of anatomical structure is
accompanied by the onset of hearing between postnatal
days 10-12 (P10–P12; Woolf and Ryan 1984, 1985; McFadden et al., 1996). At the onset of function (the earliest age
at which high-intensity stimuli first elicit neural activity),
many developing neural systems proceed through a period
of NND (e.g., rat dorsal root ganglion: Fitzgerald, 1987,
Coggeshall et al., 1994; chick auditory nuclei: Saunders et
al., 1973, Rubel et al., 1976; rat retinal ganglion: Crespo et
al., 1985, Galli and Maffei, 1988; cat neocortex: Huttenlocher, 1966, Ferrer et al., 1989; and rat somatosensory
Grant sponsor: Medical Research Council of the United Kingdom; Grant
number G9320738.
*Correspondence to: Dr. David R. Moore, University Laboratory of
Physiology, Parks Road, Oxford OX1 3PT, United Kingdom. E-mail:
dmoore@physiol.ox.ac.uk
Received 17 March 1997; Revised 2 June 1997; Accepted 4 June 1997
422
T.S. TIERNEY AND D.R. MOORE
cortex: McCandlish et al., 1993; Spreafico et al., 1995).
This observation does not appear to be limited to sensory
systems, because bursting activity in the chick spinal cord
can be recorded at embryonic day 5, the onset of NND in
chick spinal neurons (Provine, 1972; Hamburger, 1975).
If the developing gerbil VCN follows the general pattern
of neural development i.e., 1) proliferation, followed by 2)
migration, followed by 3) differentiation, followed by 4) a
period of NND as functional connectivity is established,
then a reduction in neuron number might be expected
relatively late during postnatal development. However,
the literature on this point is sparse and equivocal. In the
mammalian auditory system, we are aware of only two
studies (Mlonyeni, 1967; Webster, 1988), both in the mouse
VCN, in which an age-graded series of neuron counts has
been conducted. Both studies reported an increase in
numbers of VCN neurons during the first postnatal week.
However, only Mlonyeni observed a reduction (by about
25%) in the number of VCN neurons during a brief period
in the second postnatal week. Webster, sampling at coarser
time intervals, observed no such reduction. To resolve this
issue, we performed stereological counts of neuron number
in the developing gerbil VCN at closely spaced intervals
between P5 and P15.
Previously, we demonstrated that afferent removal during, but not after, the first postnatal week caused the death
of neurons in the gerbil VCN (Tierney et al., 1997). A
secondary objective was to correlate this sensitive period
for deafferentation-induced neuron death to a period of
NND. If NND is involved in matching neuron number
between projection and target nuclei, then it might be
expected to occur during the period when elimination of
projection neurons results in a substantial loss of target
neurons. Our data show that a substantial (22%) loss of
neurons does occur in the gerbil VCN but that all of the
loss occurs between P10 and P12, after the end of the
sensitive period for deafferentation. Preliminary reports of
this work have been published previously (Tierney et al.,
1996; Tierney and Moore, 1997).
MATERIALS AND METHODS
Animals
All gerbils in this study were obtained from our vivarium at the University Laboratory of Physiology. A
colony was derived from stock originally purchased from B
and K Universal Limited (United Kingdom) and was
maintained on a 12-hour light-dark cycle with food and
water ad libitum. Breeding pairs were checked every
morning for newborn pups, and the day of first appearance
was designated as P0. Three gerbils per age group (i.e., P0,
P5, P7, P10, P12, P15, and P140) were given a lethal
overdose of pentobarbitone sodium (Euthatal; 200 mg/kg
i.p.). The care and use of animals in this study were
approved by the Home Office of the United Kingdom and
were in accord with the Declaration of Helsinki.
Histology
All animals were perfused transcardially with 0.1 M
phosphate-buffered saline followed by a modified Heidenhain’s Susa fixative (20% formalin, 4% glacial acetic acid,
and 2% trichloroacetic acid in distilled water) for at least
15 minutes until forelimbs and mandibular musculature
appeared well fixed. Following perfusion, the skull was
placed in Heidenhain’s Susa fixative overnight. The next
morning, the skull was transferred to a 10% formalin
solution to postfix for a further week. After postfixation,
the brain was removed from the skull, dehydrated in a
graded series of alcohols, embedded in paraffin, and sectioned with a microtome (Jung, Nussloch, Germany) at
10-µm intervals. Serial pairs of frontal sections were
collected every eighth section, mounted on gelatin-coated
slides, dewaxed in xylene, stained with a neutral thionin
solution, and coverslipped with DPX mounting medium.
Analysis
VCN Volume. One-in-eight serial sections of the VCN
were examined under low-power brightfield magnification
with a 36.3 objective (Zeiss, Thornwood, NY) on a Dialux
22 microscope (Leitz, Wetzlar, Germany). At all ages, the
medial edge of the VCN was clearly bounded by the spinal
track and principal sensory nucleus of the trigeminal
nerve anteriorly and by the restiform body posteriorly. The
dorsolateral edge of the VCN was separated from the
dorsal cochlear nucleus (DCN) by a layer of granular cells.
Part of the caudal boundary was defined by the eighth
nerve root. Fibers comprising the eighth nerve root were
considered to be within the perimeter of the VCN if they
were clearly within the body of the medulla. Fibers of the
nerve root clearly protruding from the medulla were not
considered to be within the perimeter of the VCN. The
perimeter of the VCN was traced on paper through a
drawing tube attached to the microscope. Cross-sectional
areas within all VCN perimeter drawings were calculated
by scanning the drawn image into a digital analysis
system (Jandel Scientific, San Rafael, CA) calibrated with
a reference stage micrometer (Graticules, Ltd., Tonbridge,
Kent, United Kingdom). The total volume of the VCN was
estimated by multiplying each cross-sectional area measurement by 80 µm (the nominal separation between each
measured section) and summing these products. No correction was made for possible tissue shrinkage as a function
of age.
Neuron size. Neuron identification criteria included
1) a single, darkly stained nucleolus; 2) well-defined
nuclear membranes; 3) well-defined plasma membranes;
and 4) abundant Nissl substance in the cytoplasm. Measurements of neuron soma cross-sectional area were collected in the section closest to 25% of the distance through
the rostral-to-caudal axis of the VCN. All cells in this
section that met the above neuron identification criteria
were traced at a magnification of 3520 (340 Zeiss objective on a Leitz Dialux 22 microscope) through a drawing
tube attached to the microscope. We believe that glia and
granular cells were excluded by our identification criteria.
Cross-sectional areas within all perimeter drawings were
calculated by scanning the drawn image into a digital
analysis system (Jandel Scientific) calibrated with a reference stage micrometer (Graticules, Ltd.)
Neuron number. Neurons in the 25%, 50%, and 75%
VCN sections were counted at a final magnification of
3520 in the brightfield (340 Zeiss objective on a Leitz
Dialux 22 microscope). By using the physical dissector
method in adjacent serial sections (Sterio, 1984), a neuron
was counted if its nucleolus appeared in the reference
section but not in the look-up section. In practice, we found
that split nucleoli occurred less than 5% of the time. The
same neuron identification criteria as in the above analy-
NATURALLY OCCURRING NEURON DEATH IN THE VCN
423
Fig. 1. Low-magnification (310 Lecia objective) photomicrographs
comparing Nissl-stained frontal sections of the gerbil brainstem
through the 50% section of the ventral cochlear nucleus (VCN) at
postnatal day 140 (P140; A) and P0 (B). Arrow in B indicates the
eighth nerve root. Asterisks mark spiral ganglion cell bodies. CP,
cerebellar peduncle; GC, differentiating granular cell zone; F, flocculus
of the cerebellum; S5, spinal tract of the trigeminal nerve; TN,
principal sensory nucleus of the trigeminal nerve. Scale bar 5 250 µm.
sis were used. The number of neurons in the whole VCN
was estimated by using the method of Moore (1990), which
is an estimate based on the calculated density of neurons
in each of the three counted sections (rostral, 25%; middle,
50%; and caudal, 75%) multiplied by the segment volume
for which each section is assumed to be representative.
Neurons counts in the three segments were summed to
give the total number of neurons within the VCN.
Statistics. VCN volume, neuron cross-sectional area,
and numbers of neurons were compared between groups
by using analyses of variance (ANOVAs) with NewmanKeuls post-hoc comparisons (SPSS for Windows, R6.1.1).
Statistical errors for all data presented in this study are
standard deviations (S.D.).
RESULTS
General qualitative observations
In Nissl-stained frontal sections, the cochlear nucleus
can be recognized on the day of birth as a relatively large
aggregate of darkly staining cells on the dorsolateral edge
of the medulla just ventral to the flocculus of the cerebellum (Fig. 1B). Even at birth, the VCN can be distinguished
clearly from the DCN, which, at this age, is filled with
small, densely packed, granular cells that appear to be
contiguous with the cerebellum (Fig. 1B). The medial edge
of the VCN is bounded by the spinal tract and the principal
sensory nucleus of the trigeminal nerve anteriorly and by
the restiform body posteriorly (Figs. 1, 2). The cochlear
Fig. 2. Photomicrographs (310 Leica objective) in the left column
compare Nissl-stained frontal sections of the gerbil brainstem through
the 25% section of the VCN at P5 (A), P10 (B), P15 (C), and P140 (D).
Higher magnification photomicrographs (3100 Leica oil-immersion
objective; n.a. 5 1.40) in the middle column (A8–D8) show VCN
neurons from the same section. In A8, asterisks mark representative
neurons, and arrowheads mark representative glia or granular cells.
Arrowheads in B8 mark pyknotic profiles. Histograms in the right
column (A88–D88) compare the distributions of neuron cross-sectional
areas measured in the 25% section at P5, P10, P15, and P140. From
original photographic negatives, individual TIF images were gener-
ated with a digital scanner (LS-1000; Nikon, Tokyo, Japan). In Adobe
Photoshop 3.0 (Adobe Systems, Mountain View, CA), images were
composed and altered to visually match contrast and brightness
between images. The composite image was annotated in Corel Draw
6.0. The final figure was printed on a high-resolution photographic
printer (NP-1600; Codonics). 4, Lateral recess of the fourth ventricle;
CP, cerebellar peduncle; GC, differentiating granular cell zone; F,
flocculus of the cerebellum; S5, spinal tract of the trigeminal nerve.
Scale bars 5 500 µm in D (also applies to A–C), 20 µm in D8 (also
applies to A8–C8).
NATURALLY OCCURRING NEURON DEATH IN THE VCN
nerve root, with large, well-differentiated spiral ganglion
cell bodies, can be seen exiting the VCN, suggesting that
primary afferents have entered the brain by birth (Fig.
1B). At low magnification, the cochlear nucleus complex
appears to have assumed an adult-like position and general morphology. However, at higher magnification, both
divisions of the cochlear nucleus were found to contain
small, densely packed, and relatively undifferentiated
cells compared with those at older ages. Mitotic profiles
were not observed at P0, suggesting that neurogenesis is
complete. Within the newborn DCN, no clear laminar
organization was present, and few somata with nucleoli
were recognized among the dense field of small granular
cells. Many somata in the newborn VCN contained a
darkly stained nucleolus. However, poorly stained nuclear
and plasma membranes and a lack of Nissl substance
within the cytoplasm hampered neuron identification and
precluded analysis of either neuron size or number at this
age.
By P5–P7, the dense zone of granular cells in the DCN
had become restricted to the dorsolateral margin of the
nucleus and to a thick layer ventromedially separating the
nucleus from the VCN. At these ages, the DCN had become
distinct from the flocculus as the lateral recess of the
fourth ventricle expanded and as the dorsolateral granular
cell region no longer appeared to be contiguous with the
cerebellum. A laminar organization of well-stained pyramidal neurons within the center of the DCN had begun to
emerge. Neurons within the VCN were clearly, although
relatively lightly, stained, with a dark nucleolus and
well-defined nuclear and plasma membranes (Fig. 2A8).
The nucleus was located eccentrically in the cytoplasm.
Relative to older ages, neuron packing density within the
VCN was high, and cytoplasm-to-nucleus ratio was low. No
mitotic or pyknotic neurons were observed in these groups.
From P10 to P15, the VCN was separated from the DCN
by a thin layer of granular cells. Neurons within the VCN
were adult-like in size, and the nucleus was located more
concentrically within the cytoplasm compared with younger neurons (Fig. 2B8,C8). Neuron packing density at these
ages appeared greater than at P140 (Fig. 2B8–D8). Pyknotic profiles were occasionally seen at P10 and P12 (Fig.
2B8), but not at P15. Microcytic lesions, which are known
to occur in the gerbil VCN (Ostapoff and Morest, 1989;
Faddis and McGinn, 1993), were not found at these ages.
By P140, the density of neuron soma profiles had diminished noticeably (Figs. 1A, 2D,D8). No pyknotic profiles
were observed. Microcytic lesions were present, particularly in the posterior VCN. To summarize, the major
qualitative observations include 1) gross adult-like cochlear nucleus morphology and location at birth with
undifferentiated cellular profiles, 2) rapid somatic and
nucleus growth from P5 to P10, 3) decreased neuron soma
density with appearance of pyknotic profiles between P10
and P15, and 4) continued growth of extracellular space
after P15.
Nucleus volume
Boundaries of the VCN were unambiguous at all ages.
Mean nucleus volume (Fig. 3) varied significantly between
groups (ANOVA; F6,14 5 89.5, P , 0.0001). Post-hoc
comparisons suggested that nucleus growth kinetics are
biphasic (Table 1). After an initial period of rapid growth
that more than tripled nucleus volume between P0 and
P10, VCN size remained significantly unchanged through
425
Fig. 3.
Mean volume of the VCN (61 S.D.) for each group.
TABLE 1. Post Hoc Comparisons of Mean Ventral Cochlear Nucleus
Volume Between Groups
Group
(n 5 3)
P0
P5
P7
P10
P12
P15
P140
Mean VCN
volume
(mm3 )
P0
P5
P7
P10
P12
P15
0.13
0.22
0.35
0.45
0.50
0.52
0.81
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Group
P140
*P , 0.50 (Newman-Keuls test). VCN, ventral cochlear nucleus.
P15. A second growth phase occurred sometime between
P15 and P140 as nucleus volume significantly increased a
further 57%.
Neuron cross-sectional area
Before P5, few cellular profiles met neuron identification
criteria (see Materials and Methods), and no earlier measurements of neuron cross-sectional area were made. By
P5, neurons had become sufficiently differentiated to distinguish a single nucleolus and nuclear and plasma membranes (Fig. 2A8). Smaller glia and granular cells, which
contain virtually no Nissl substance, were clearly distinguished from neurons by this age (Fig. 2A8). Growth of
somata appeared to occur rapidly between P5 and P10
(Fig. 2A8,B8). Comparison of neuron cross-sectional area
histograms (Fig. 2A88–D88) shows that VCN neuron size
more than doubled from P5 to P10, with no further growth
thereafter. Unimodal distributions of neuron size suggest
that the same single population was measured at each age
and that different sized neurons grew at similar rates.
Mean neuron cross-sectional area (Fig. 4) varied significantly between groups (ANOVA; F5,12 5 22.6, P , 0.0001),
ranging from a mean of 92.6 µm2 at P5 to 231.3 µm2 at P15.
Post-hoc comparisons revealed that only the P5 and P7
groups differed significantly from all of the other, older
groups, indicating that VCN neuron somata have completed their growth phase by P10.
Neuron number
Counts of neuron number within the developing VCN
were first possible at P5. Neuron number (Fig. 5) varied
significantly between groups (ANOVA; F5,12 5 9.3, P ,
0.001). Post-hoc comparisons showed that the P5, P7, and
426
T.S. TIERNEY AND D.R. MOORE
TABLE 2. Stereological Estimates of Neuron Number in the Ventral
Cochlear Nucleus of Each Animal, Group Mean (6S.D.), and Coefficient
of Variation for Each Group
Group
Fig. 4. Mean neuron soma cross-sectional area in the 25% section
of the VCN (61 S.D.) for each group except P0.
P5
9642
9643
9644
P7
9646
9647
9650
P10
9635
9636
9637
P12
9657
9658
9660
P15
9652
9653
9654
P140
9421
9422
9423
1VCN,
Fig. 5. Mean number of neurons in the VCN (61 S.D.) for each
group except P0.
P10 groups were not significantly different from each
other, collectively averaging 35,932 neuron per VCN.
Groups P12, P15, and P140 were not significantly different
from each other, collectively averaging 25,512 neurons per
VCN. However, all three of the youngest groups differed
significantly from all three of the oldest groups, showing
an age-related and, within the limits of our analysis,
step-wise reduction in neuron number. The step occurred
between P10 and P12, with a reduction in neuron number
of 22%. Table 2 lists individual neuron counts, group mean
(6 S.D.), and coefficient of variation (CV) for each group.
Note that variability was greatest at P12, an age at which
one animal in the group (9657) had many more VCN
neurons than its two litter mates. Presumably, neurons in
the VCN in this animal had not yet completed the cycle of
NND, whereas those in the other animals had completed
the cycle. This observation suggests to us that the period
for NND in individual animals may be even less protracted
(perhaps less than 24 hours) than our acute, crosssectional time series suggests.
DISCUSSION
Methodological considerations
One methodological problem needs to be addressed
before conclusions from the results can be drawn. We have
Number of
VCN neurons1
Group
mean (61 S.D.)
Coefficient of
variation (%)
36,199
40,851
33,065
36,705 (3,918)
10.7
36,089
34,066
40,373
36,843 (3,220)
8.7
36,455
33,878
32,408
34,247 (2,049)
6.0
33,292
23,022
24,004
26,773 (5,667)
21.2
26,113
25,325
23,441
24,960 (1,373)
5.5
24,542
26,118
23,749
24,803 (1,206)
4.9
ventral cochlear nucleus.
not determined possible differences in tissue shrinkage
between groups due to fixation and histological processing.
Differential tissue shrinkage as a function of age might be
expected to affect two quantitative measures in this study:
nucleus volume and neuron cross-sectional area. However,
total number of neurons, an absolute figure, is relatively
independent of tissue shrinkage insofar as shrinkage is
homogenous throughout the nucleus. This problem has
been examined previously in some detail by Rübsamen et
al. (1994) in the developing gerbil brain. That study
achieved absolute estimates of brain size as determined by
microcaliper measurements taken from whole brains prior
to histological processing. Because our measurements of
whole cochlear nucleus volume in both adult and neonatal
(P0, P7, P15) gerbils did not differ significantly from those
obtained in that study (two-tailed Student’s t tests; P .
0.05), we believe that the effect of tissue shrinkage on our
measurements was negligible.
Common developmental time course
of VCN in rodents
An account of the major developmental events in the
VCN makes it possible to 1) place NND chronologically in
relation to other ontogenic events, 2) compare the relative
timing of NND in the VCN to other developing systems,
and 3) speculate on underlying mechanisms regulating
NND. Although the histogenesis of the gerbil brainstem
has not been studied prenatally, comparison of gestation
period (gerbil, 24 days; mouse, 20 days; rat, days 21; Poole,
1987) and postnatal maturation suggests that the gerbil
shares a common neurodevelopmental time course with
other rodents. For example, the onset of hearing occurs
between P10 and P12 in the gerbil (Woolf and Ryan, 1984,
1985; McFadden et al., 1996), at P12 in the mouse (Shnerson and Willot, 1979; Shnerson and Pujol, 1981), and
between P11 and P12 in the rat (Blatchley et al., 1987;
Puel and Uziel, 1987). Eye opening occurs at P16 in the
gerbil, at P14 in the mouse, and at P13 in the rat (Poole,
1987).
NATURALLY OCCURRING NEURON DEATH IN THE VCN
Neurogenesis and migration of nongranular VCN neurons occurs prenatally in the rodent. In the rhombic lip of
the embryonic rat (Altman and Bayer, 1980) and mouse
(Taber Pierce, 1967; Martin and Rickets, 1981), VCN
neurons are born approximately 1 week before birth and
migrate laterally to their characteristic place on the dorsolateral edge of the medulla by birth. Our observations are
consistent with the idea that postnatal VCN neurons in
the newborn gerbil are also postmitotic (no mitotic profiles
were observed at P0) and postmigratory (a full complement of neurons was present by P5 at the latest). Neurons
in the rodent VCN begin to differentiate before birth.
Afferent projections from neurons in the spiral ganglion to
the VCN (mouse: Taber Pierce, 1967; Fritzsch and Nichols,
1993; rat: Angulo et al., 1990) and efferent projections (rat:
Kandler and Friauf, 1993) from neurons in the VCN to
second-order auditory nuclei begin to form before birth.
Differentiation of neurons in the VCN continues during
the postnatal period. Although initial connections appear
to be in place at birth (gerbil: Kil et al., 1995; Russell and
Moore, 1995; rat: Friauf and Kandler, 1990), terminal
maturation of both afferent (end bulbs of Held in the VCN;
rat: Mattox et al., 1982; Neises et al., 1982; hamster:
Schweitzer and Cant, 1984) and efferent (calyces of Held in
medial nucleus of trapezoid body; rat: Kandler and Friauf,
1993; gerbil: Kil et al., 1995; Russell and Moore, 1995)
projections occurs postnatally. Somatic growth of VCN
neurons in the mouse (Webster and Webster, 1980) and
gerbil (Gleich et al. 1997; present study) is complete by the
onset of hearing. Mature synaptic morphology is observed
a few days later (rat: Mattox et al., 1982; Neises et al.,
1982; Kandler and Friauf, 1993; gerbil: Kil et al., 1995;
Russell and Moore, 1995), following the onset of hearing.
Taken together, the above studies indicate that development of the rodent VCN begins approximately 1 week
before birth with neurogenesis in the rhombic lip, followed
by prenatal migration of neuroblasts to the dorsolateral
edge of the medulla as connections from the spiral ganglion and to second-order nuclei are formed. Although
most of the major afferent and efferent projections are in
place at birth, significant growth and differentiation of
VCN neurons continues into the postnatal period. Mature
somatic and synaptic morphology emerge during the second postnatal week, near the onset of hearing. Our data
from the gerbil show that a period of NND also occurs
during this relatively late stage of development.
The existing literature concerning NND in the VCN
appears to be somewhat contradictory. Webster (1988)
reported that neuron number in the developing mouse
VCN at P1, P3, P6, P12, and P24 increased monotonically
to adult-like levels by P12, suggesting that postnatal NND
does not occur. However, the sampling intervals used in
that study could not have detected the 25% decrease in
neuron number between P9 and P12 reported by Mlonyeni
(1967), who sampled at daily intervals from P4. The latter
study agrees quite well in both the magnitude and timing
of NND with our 22% loss of VCN neurons in the gerbil
between P10 and P12. However, our data differ from both
studies, which reported an increase in the of number VCN
neurons during the first postnatal week. Our counts in the
gerbil VCN show that neuron number is relatively constant between P5 and P10. Because neurogenesis and
migration of nongranular neurons in the mouse VCN occur
prenatally (Taber Pierce, 1967; Martin and Rickets, 1981),
no new neurons would be expected to be added to the
427
population after birth. It is possible that the above observations of increasing numbers of VCN neurons during postnatal development are methodological artifacts. Because
significant somatic differentiation occurs during this period, apparent increases in neuron number could occur
merely as a result of identifying more neurons at successively older ages without any actual change in neuron
number.
In most developing neural systems, a period of NND
happens concurrently with the establishment of synaptic
contact in the target field (Oppenheim, 1981). Although
this also appears to be true for developing VCN neurons,
no study has directly addressed putative target control of
NND by manipulating VCN efferent access to the target
population. However, several studies have found that
destruction of the primary afferent population (i.e., the
spiral ganglion) before the period of NND results in
significant VCN neuron loss (Trune, 1982; Nordeen et al.,
1983; Hashisaki and Rubel, 1989), suggesting that afferents play a role in regulating VCN neuron number during
the developmental period. However, it is not known
whether the onset of deafferentation-induced neuron death
following early afferent removal is delayed until the period
of NND, as it is in other developing systems (Okado and
Oppenheim, 1984; Clarke, 1985; Furber et al., 1987),
including the chick auditory nuclei (Levi-Montalcini, 1949;
Rubel et al., 1976).
In a previous study (Tierney et al., 1997), we found that
afferent removal performed during, but not after, the first
postnatal week reduced the number of VCN neurons that
survived to maturity. Remarkably, no difference in VCN
neuron number was found between adult animals deafferented at P9 or P11 and normal adult controls. Because we
now know that a 22% reduction in VCN neuron number
(due to NND) occurs between P10 and P12 in normal
animals, it must be the case that VCN neuron number is
similarly reduced following deafferentation at P9 or P11.
Whether neuron death following deafferentation at these
ages is a result of NND or afferent removal, per se, bears
upon the question of mechanisms controlling NND in
intact systems. If VCN neurons deafferented at P9 or P11
proceed through a normal period of NND, then afferent
control of NND seems unlikely. However, if VCN neuron
death is induced following deafferentation at P9 or P11,
then afferent regulation of neuron survival would extend
through the period of NND, suggesting that afferent
control of NND is possible in intact systems. It may be
possible to resolve this issue if, as in the chick ciliary
ganglion (Pilar and Landmesser, 1976), the ultrastructure
of degenerating neurons following induced neuron death is
markedly different from that occurring during the period
of NND. Although it is not yet clear whether afferent
regulation of NND occurs in the VCN, neuron survival
after the period of NND does not depend upon afferent
input.
Concomitant onset of hearing and NND
The period of NND in the gerbil VCN coincides with the
onset of hearing, between P10 and P12 (Woolf and Ryan
1984, 1985; McFadden et al., 1996). Similarly, in the
chicken nucleus magnocellularis, the avian homologue of
the mammalian VCN, a small reduction in neuron number
occurred over a 2-day period from embryonic day 11 (Rubel
et al., 1976), the earliest day at which acoustically evoked
responses can be detected (Saunders et al., 1973). Concur-
428
T.S. TIERNEY AND D.R. MOORE
rent onset of function and NND also occurs in other
developing sensory systems. For instance, neuron death in
the rat dorsal root ganglion (Coggeshall et al., 1994) and
somatosensory cortex (Spreafico et al., 1995) peaked immediately following the time that evoked responses could first
be elicited (Fitzgerald, 1987; McCandlish et al., 1993,
respectively). Furthermore, this observation does not appear to be limited to sensory systems, because bursting
activity in the chick spinal cord can be recorded at
embryonic day 5, the onset of NND in chick spinal neurons
(Provine, 1972; Hamburger, 1975).
A causal relationship between neural activity and NND
has been inferred from studies that have applied pharmacological agents to block neural activity within a developing population of neurons (Wright, 1981; Meriney et al.,
1987; Maderdrut et al., 1988), within the target field
(Pittman and Oppenheim, 1978, 1979; Harris and McCaig,
1984; Meriney et al., 1987; Maderdrut et al., 1988;
Péquignot and Clarke, 1992), or within the afferent population (Galli-Resta et al., 1993). During the period of NND,
blockade of activity within the developing neural population or within the afferent population led to increased
neuron death, whereas blockade of activity within the
target field led to increased neuron survival. Taken together, the above studies suggest that the presence or level
of spontaneous neural activity derived from afferent inputs may control the onset and magnitude of NND within
a developing population of neurons. Because spontaneous
activity has been reported in the gerbil VCN as early as
P10 (Woolf and Ryan, 1985), it is intriguing to speculate
that such activity plays a role in regulating NND.
ACKNOWLEDGMENTS
This work was sponsored by grant G9320738 from the
Medical Research Council of the United Kingdom to
D.R.M. T.S.T. is a Fulbright scholar.
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