1Department of Surgery/Otolaryngology and 2Department of Physiology, School of Medicine, University of Missouri-Columbia, Columbia, Missouri 65212
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ABSTRACT |
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Jones, Timothy A. and Sherri M. Jones. Spontaneous Activity in the Statoacoustic Ganglion of the Chicken Embryo. J. Neurophysiol. 83: 1452-1468, 2000. Statoacoustic ganglion cells in the mature bird include neurons that are responsive to sound (auditory) and those that are not (nonauditory). Those that are nonauditory have been shown to innervate an otolith organ, the macula lagena, whereas auditory neurons innervate the basilar papilla. In the present study, single-unit recordings of statoacoustic ganglion cells were made in embryonic (E19, mean = 19.2 days of incubation) and hatchling (P6-P14, mean = 8.6 days posthatch) chickens. Spontaneous activity from the two age groups was compared with developmental changes. Activity was evaluated for 47 auditory, 11 nonauditory, and 6 undefined eighth nerve neurons in embryos and 29 auditory, 26 nonauditory, and 1 undefined neurons in hatchlings. For auditory neurons, spontaneous activity displayed an irregular pattern [discharge interval coefficient of variation (CV) was >0.5, mean CV for embryos was 1.46 ± 0.58 and for hatchlings was 1.02 ± 0.25; means ± SD]. Embryonic discharge rates ranged from 0.05 to 97.6 spikes per second (sp/s) for all neurons (mean 18.6 ± 16.9 sp/s). Hatchling spontaneous rates ranged from 1.2 to 185.2 sp/s (mean 66.5 ± 39.6 sp/s). Discharge rates were significantly higher for hatchlings (P < 0.001). Many embryonic auditory neurons displayed long silent periods between irregular bursts of neural activity, a feature not seen posthatch. All regular bursting discharge patterns were correlated with heart rate in both embryos and hatchlings. Preferred intervals were visible in the time interval histograms (TIHs) of only one embryonic neuron in contrast to 55% of the neurons in posthatch animals. Generally, the embryonic auditory TIH displayed a modified quasi-Poisson distribution. Nonauditory units generally displayed regular (CV <0.5) or irregular (CV >0.5) activity and Gaussian and modified-Gaussian TIHs. Long silent periods or bursting patterns were not a characteristic of embryonic nonauditory neurons. CV varied systematically as a function of discharge rate in nonauditory but not auditory primary afferents. Minimum spike intervals (dead time) and interval modes for auditory neurons were longer in embryos (dead time: embryos 2.88 ± 6.85 ms; hatchlings 1.50 ± 1.76 ms; modal intervals: embryo 10.09 ± 22.50 ms, hatchling 3.54 ± 3.29 ms). The results show that significant developmental changes occur in spontaneous activity between E19 and posthatch. It is likely that both presynaptic and postsynaptic changes in the neuroepithelium contribute to maturational refinements during this period of development.
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INTRODUCTION |
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There has been considerable recent interest in the
study of spontaneous neural discharge patterns in peripheral and
central sensory projections during ontogeny. The interest in
spontaneous activity stems in part from the idea that such activity may
play a significant role in the refinement of synaptic connections in developing sensory relays. There is convincing evidence that
modifications of spontaneous afferent discharge patterns can result in
altered synaptic configurations (Shatz 1996). There is
also a long-standing general interest in the role of primary afferent
activity, spontaneous and driven, in the regulation of postsynaptic
cells, including synaptic junctions, trophic influences, cell survival,
and gene expression (Constantine-Paton et al. 1990
;
Harris 1974
; Levi-Montalcini 1949
;
Moore 1992
; Parks 1979
; Peusner
and Morest 1977
; Purves 1994
; Rubel and
Hyson 1994
). Detailed studies of both peripheral and central
spontaneous activity in the immature visual system have been offered
for many species (Constantine-Paton et al. 1990
; Galli and Maffei 1988
; Masland 1997
;
Meister et al. 1991
; Mooney et al. 1996
;
Shatz 1990
; Wong et al. 1993
, 1995
).
Spontaneous discharge patterns also have been characterized in
developing mammalian peripheral vestibular (Curthoys 1978
, 1979
,
1982
, 1983
; Desmadryl et al. 1986
, 1992
;
Romand and Dauzat 1982
), somatosensory (Fitzgerald 1987
; Fitzgerald and Fulton
1992
), and auditory systems (Gummer and Mark
1994
; Kettner et al. 1985
; Romand
1984
; Romand and Dauzat 1981
; Walsh and
McGee 1987
, 1988
).
The focus of the current work is on the functional development of the
avian basilar papilla and macula lagena. The bird is a widely used
developmental model that has provided considerable insight into the
ontogeny of auditory and vestibular systems. Despite thorough
documentation of the morphological details of ontogeny (see
Cohen and Cotanche 1992), there are few descriptions of
spontaneous discharge patterns in developing avian central and
peripheral auditory circuits. To our knowledge, there are no
descriptions of avian embryonic vestibular discharge patterns.
Manley et al. (1991a) described spontaneous discharge
patterns of single auditory primary afferents at two ages of young
maturing chickens (days 2 and 21 posthatch). No remarkable differences in spontaneous activity were observed for the two ages [20.5 and 23 spikes/s (sp/s), respectively]. However, Salvi et al.
(1992)
reported substantially higher spontaneous discharge
rates for adult chickens (86 sp/s), thus suggesting the possibility of
maturational changes between early posthatch and adult animals.
Richter et al. (1996)
described spontaneous discharge
patterns of auditory primary afferents in the neonate pigeon. The
pigeon is an altricial species that is substantially less mature than
the chicken during the early posthatch period. In this case, the
investigators described lower discharge rates for neonates in
comparison with adults. Lippe (1994
, 1995
) has
characterized patterns of multiunit activity in the region of brain
stem relay nuclei including nucleus magnocellularis (NM) and nucleus
laminaris (NL) in chicken embryos at ages between 14 and 19 days of
incubation. In addition to low overall discharge rates, Lippe described
bursting patterns reminiscent of early bursting patterns reported for
other sensory modalities, especially the visual system. Spontaneous
synchronous bursting is thought to be a key feature of patterned
activity important in developmental synaptic refinements. Whether this
is a dominant pattern in embryonic primary afferents is of considerable
interest and remains to be shown. The purpose of the present study was
to characterize spontaneous primary afferent discharge patterns in the
statoacoustic ganglion of the late chicken embryo (E19) and compare
embryonic discharge characteristics with those of hatchlings aged
P6-P14.
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METHODS |
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The methods used in the present study have been described
elsewhere in detail (Jones, S. M. and Jones
1995a,b
). These methods and the details of additional analyses
used here are summarized in the following text.
Embryos [19.2 ± 0.6 days of incubation (DOI), n = 39] and posthatch (P6-P14, 8.6 ± 2.1 days posthatch;
n = 18) domestic chickens (Gallus
domesticus) were used. All embryos except one (18.0 DOI) were
18.9 DOI. Collectively therefore the group was representative of late
embryos and will be designated as E19 in the current report. For
embryos, mean beak and toe lengths were 5.5 ± 0.5 mm
(n = 36) and 19.8 ± 1.2 mm (n = 32), respectively, corresponding to stages 43-45 of Hamburger
and Hamilton (1951)
. The head of each embryo was removed from
the eggshell through a small opening and secured in a holder.
EquiThesin was diluted 1:5 with normal saline, and 0.1 ml was
administered subcutaneously along with 1 mg of gallamine triethiodide.
(EquiThesin is a mixture of 0.972 g Na pentobarbital, 4.251 g chloral
hydrate, 2.125 g magnesium sulfate, 12.5 ml ethanol, and 42.6 ml
propylene glycol in distilled water to a total volume of 100 ml.)
Surface electrodes were used to record the electrocardiogram (ECG).
Eggs were placed on a heated platform in a sound-attenuating booth. Egg
and brain temperatures were monitored, and brain temperature was
maintained on average at 38.8 ± 0.9°C. Posthatch birds were
anesthetized using an intramuscular injection of EquiThesin (0.003 ml/g). A tracheotomy was completed, and abdominal and thoracic air sacs
were cannulated bilaterally (Nazareth and Jones 1998
).
Lungs were perfused with an oxygen enriched, humidified warm
air/CO2 mixture (~50%
O2, 5% CO2, 39% nitrogen,
and 6% water vapor). Spontaneous respiratory rate was adjusted by
varying the percent of CO2 to maintain normal
respiratory rates between 15 and 20/min. Immediately before electrode
descents, gallamine triethiodide (1 mg) was injected intramuscularly. A deep plane of anesthesia was maintained throughout the experiment with
hourly maintenance doses of EquiThesin (0.05 ml im). Brain and cloacal
temperature probes were placed, and brain temperature was maintained at
an average of 38.4 ± 1.6°C, whereas cloacal temperature was not
allowed to go above 41.0°C. The University of Missouri Institutional
Animal Care and Use Committee approved the care and use of the animals
described in this report (Nos. 2386 and 2446).
In both embryos and hatchlings, the beaks were embedded beak-down in
plaster to stabilize the head, and the naso-occipital axis was placed
~30° off vertical to the right and posterior. The scala tympani of
the cochlea was accessed surgically using the posterolateral approach.
Fluid in the middle ear was removed, and the oval window was
visualized. A small opening was made through the bony plate overlying
scala tympani and the membranous labyrinth opened. Glass micropipettes
were filled with 0.5 M KCl and 0.05 M Tris (pH 7.6) alone or the
KCl/Tris solution included 10% horseradish peroxidase (HRP) or 4%
biocytin. Electrode impedance ranged from 20 to 100 M. Micropipettes
were lowered into the perilymph-filled recessus scala tympani.
Chlorided silver wire electrodes were used for the reference (neck) and
ground (thorax for posthatch birds, extraembryonic fluid for embryos).
Microelectrode descent was completed in 0.5-µm steps, and the tip was
directed to the desired position along the papilla. Ganglion cell
activity was amplified and recorded on analogue tape.
Sound stimuli were delivered using an ER-2 earphone sealed at the left external auditory meatus (EAM). At 0-dB attenuation, stimulus intensity measured at the end of the earphone tube was 86 dB SPL (re: 20 µPa) or 100 dB SPL for some studies in embryos. Clicks, tones, and pure-tone or noise bursts were used as stimuli to determine whether individual cells responded to sound. Pure tones or tone bursts (5- ms onset/offset ramp, 50-ms plateau duration, 50-6,000 Hz) were used manually to estimate the frequency eliciting the maximum level of firing (estimate of characteristic frequency), or, when possible, a computerized threshold tracking procedure was used to obtain a frequency tuning curve (FTC, tone bursts: 40 ms rise/fall, 80-ms plateau). Characteristic frequency (CF) was determined from the FTC and was defined as the frequency corresponding to the lowest threshold (i.e., tip) of the FTC. Threshold at CF (i.e., lowest threshold) was documented for each FTC.
Once single primary afferents were isolated, several determinations
were made and generally in the following order. First, test stimuli
were used to determine whether the cell responded to sound (described
in foregoing paragraph). If cells responded to sound, they were
considered auditory; if they did not, then they were classified as
nonauditory. If it was not possible to complete this test, then the
neuron was assigned to the "undetermined" group. Individually
labeled nonauditory neurons in posthatch chickens have been shown to
innervate the macula lagena (Manley et al. 1991b). In
the embryo, this may not be the case because it is possible that
immature papillar neurons will not respond to the sound levels used in
the present study. Therefore embryonic nonauditory neurons may project
to either the papilla or lagena. Second, if a response was obtained,
then the CF was estimated manually or by computerized threshold
tracking. Third,
10 min of spontaneous activity data were recorded on
tape. Spontaneous activity is defined as neural spike activity recorded
from statoacoustic ganglion cells in the absence of stimulus
presentation. Noise levels (in dB SPL) for the sound coupling system
and booth under our standard recording conditions in the absence of
stimuli were: 31.5 Hz = 38; 63 Hz = 33; 125 Hz = 25; 250 Hz = 15; 500 Hz = 6; 1,000 Hz = 9; 2,000 Hz = 6;
4,000 Hz = 6; 8,000 Hz = 7; and 16,000 Hz = 6.
Data were taken from the tape record and from digital FTC files saved to diskette for each study and analyzed off-line. Firing rate (total number of spikes/total time period), interspike intervals, and coefficient of variation (CV = standard deviation of interspike intervals divided by the mean interspike interval) were determined. Spike interval data were collected at a time resolution of either 40 or 50 µs per point. Interval histograms were constructed and used to evaluate the patterns of spike intervals. Linear regression analysis and the Mann-Whitney U Test (MWU) were used to evaluate results statistically.
Autocorrelation and Fourier transforms
To quantify the frequency and magnitude of periodic activity in
the time interval histograms of spontaneously active neurons, we
analyzed spike trains using autocorrelation and Fourier analyses. Probability density functions (autocorrelation functions, ACFs) were
produced off-line using the strategies outlined by Møller (1970). Bin size was 0.1 ms, and a total of 512 or 1,024 bins was used for the correlation array. Autocorrelation functions were
calculated and then subjected to Fourier analysis to identify the
fundamental frequency and magnitude of harmonic activity (sampling frequency 104/s, 256 or 512 points).
Identifying and quantifying bursting patterns
Regular bursting patterns are easily identified and can be characterized readily quantitatively, for example, by measuring the time period between regular bursts and spike rates during bursts. Nonregular bursting patterns, bursts of neural discharge that occur at irregular intervals separated by relatively long periods of silence, are not as amenable to simple description. In the present study, we employed a numerical burst factor (BF) to help identify and rank bursting discharges. It was reasoned that such a burst factor should be proportional to the relative amount of time the cell spent in long periods of silence during the entire sample, and this component, identified as A, was calculated as the sum of the longest four silent periods expressed as a percentage of the entire sample time. The burst factor also should take into account the relative amount of activity occurring during the active periods where the greater the contrast between silent and active periods the larger the burst factor would be. This latter component of the burst factor, identified as B, was calculated as the ratio of the mean spike interval of the four longest silent periods divided by the mean spike interval over the entire sample. This component expresses the relative time spent in an average long silent interval compared with the cell's average period of silence. The product of the two components A*B was defined as the burst factor and used as a numerical indicator of bursting level for spontaneous neural activity.
Quantitatively BF = A*B = [(total time of the 4 longest intervals)/(total sample time)]*[(mean interval length of 4 longest intervals)/(total sample time/total number of intervals)].
Our testing revealed that the BF magnitude performs well in comparison
with other indicators of "burstiness" such as the correlogram described by Sernagor and Grzywacz (1999) that they used
to identify bursting retinal ganglion cells. BF provides, in addition,
an objective means of quantifying the magnitude of irregular bursting.
Hazard functions
To help identify and compare quasi-Poisson discharge processes
in spontaneously active auditory neurons of embryos and hatchlings, the
Hazard function (conditional probability) for the time interval histogram (TIH) was calculated (Gray 1967; Harris
and Flock 1967
; Harris and Milne 1965
; Li
and Young 1993
). The overall relation is given by Eq. 1
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RESULTS |
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A total of 120 primary afferents were isolated for study in the present report. Sixty-four primary afferent neurons were recorded in embryos and 56 were studied in posthatch birds. Of the 64 embryonic neurons, 47 were classified as auditory, 11 nonauditory, and 6 undetermined. In the posthatch animals, there were 29 auditory, 26 nonauditory, and 1 undetermined primary afferents. The range of auditory CFs for embryos was 200-2,000 Hz and for posthatch animals was 60-4,545 Hz. Thresholds were obtained for 40 neurons, and values ranged between 14 and 46 dB SPL (mean = 32.2 ± 10.3 dB SPL; n = 13) in posthatch animals and between 32 and 87 dB SPL (mean = 54.8 ± 16.2 dB SPL; n = 27) in embryos. Thresholds at CF were significantly lower for posthatch animals in this sample (P < 0.001). The general features of spontaneous activity can be expressed in terms of simple mean firing rate, variation in discharge interval (e.g., CV), spike interval distribution and overall firing patterns (e.g., preferred intervals, bursting, and other periodicities) as described in the following text.
Discharge rates, modal interspike intervals, dead times
A unimodal distribution of afferent discharge rates was found across all neurons of each age group (Fig. 1). However, the distributions were considerably different for the two ages such that embryonic neurons were restricted to lower discharge rates. The discharge rates for all embryonic neurons (19 ± 17 sp/s, n = 64) were significantly lower than those of posthatch birds (67 ± 40 sp/s, n = 56, P < 0.001). Rates ranged from 0.05 to 97.6 sp/s in the embryo and 1.2 to 185.2 sp/s in the posthatch animals. Discharge rates were also significantly lower in embryos when the subgroups of auditory (P < 0.001) and nonauditory (P = 0.001) neurons were considered for each age (Fig. 2, Table 1). Table 1 summarizes numerical data for neural subgroups including mean rates, CV, dead times, and modal intervals.
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The relationships between mean discharge rate and CF for hatchlings and embryos are illustrated in Fig. 3. In posthatch animals, there was a significant relationship such that spike rates were lower for the highest CFs (P = 0.009, R2 = 0.3). No systematic relationship between discharge rate and CF was seen for embryos. A restricted range of high CFs in embryos is also apparent from Fig. 3.
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In posthatch birds, spontaneous discharge rates varied as a function of threshold at CF such that neurons with the lowest thresholds had the highest rates (P = 0.02, R2 = 0.4, Fig. 4). This was true even when one considered only neurons having CFs in the range of those found in embryos (i.e., <2,000 Hz). Generally, a relationship between spontaneous discharge rate and CF did not hold in the embryo (Fig. 4). There was one highly unusual embryonic neuron that had very mature features including a spike rate comparable with the higher rates observed in posthatch animals. Indeed the spike rate of this outlier neuron was 5.7 SD above the mean rate for the group of neurons shown. For the remaining neurons, rates were quite low, and there was no significant relationship between spike discharge rate and threshold at CF. In cases where there were sufficient numbers of spikes to characterize discharge patterns, means were determined for both modal and minimum interspike intervals. It was not possible to obtain well-formed TIHs for neurons having very long modes or minimum intervals >80 ms because spike rates were too low. In those cases where there were too few intervals, the data were not included in the calculation of means for modes and dead time.
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Modal spike intervals ranged from 1.5 to 134 ms in embryos and from 1.0 to 33.5 ms in posthatch animals. The modal spike interval across all cell types was significantly longer in embryos (12.3 ± 21.3 ms, n = 45) compared with hatchlings (6.8 ± 6.5 ms, n = 51, P < 0.04). For all auditory units and for nonauditory irregular neurons (CV >0.5), modal spike intervals were independent of mean spontaneous rate (Fig. 5). In contrast, modal intervals of nonauditory regular neurons (CV <0.5) varied systematically as a function of discharge rate (Fig. 5).
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The minimum spike interval, often referred to as the dead time, ranged from 1.0 to 89 ms in embryos and from 0.6 to 13.7 ms in hatchlings. The dead time across all cell types was not significantly different between embryos (7.0 ± 15.2 ms, n = 46) and posthatch animals (3.4 ± 3.7 ms, n = 51). However, when auditory and nonauditory neurons were considered separately, embryonic modes were significantly longer than those of hatchlings (auditory: P = 0.001; nonauditory: P = 0.013; Table 1).
Variability of discharge rate: interspike interval coefficient of variation (CV)
Figure 6 presents recordings from three embryos and one posthatch bird. The neurons represented illustrate the variation in action-potential discharge patterns seen in the present study. These can be described as regular (A), irregular (B), and bursting (C and D). CV varies as a function of the regularity of discharge interval. The neuron in Fig. 6A fires in a relatively regular pattern and has a CV of 0.12, whereas B fires in a more irregular pattern and has a CV of 0.92. These are typical values for CVs of regular and irregular neurons in the present study. Nonetheless, CV varied widely and ranged from 0.04 to 3.1. The highest CV was observed for an embryonic auditory neuron, whereas the lowest CV was found in a posthatch nonauditory neuron. Different distributions of CV were obtained for auditory and nonauditory neurons. The distribution of CV for auditory neurons is shown in Fig. 7. CVs ranged from 0.6 to 3.1 in auditory neurons, whereas the CVs of nonauditory cells varied from 0.04 to 1.8. Figure 8 illustrates the distribution of CVs found in nonauditory neurons where discharge patterns appearing regular, those with CV <0.5, and irregular, those with CV >0.5, were observed.
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All auditory cells appeared to fire irregularly and had CVs > 0.5 regardless of age. Both embryos and posthatch birds evidenced regular and irregular firing in nonauditory neurons. Although the CV was >0.5 in all auditory cells, most had values near 1.0 in posthatch animals, a fact that is consistent with discharge patterns approximating a Poisson process (because for a homogeneous Poisson process the ratio of the standard deviation to the mean is exactly 1.0). The CV of one posthatch neuron was ~2.1 and is identified easily in Fig. 7. The exceptionally high CV in this neuron is associated with a bursting pattern synchronized to the heart beat. Figure 7 also reflects the substantially higher range and mean of CVs found in embryos (P < 0.001, Table 1). Embryos evidenced greater variability in discharge patterns implying that some neurons show considerable deviation from Poisson discharge distributions. Higher embryonic CVs were not simply due to the fact that the mean rates were lower for embryos because CV was independent of discharge rate for auditory neurons in hatchlings and embryos (Figs. 9 and 10). Bursting neurons having BFs between 1.8 and 7.6 produced the highest embryonic CVs (see Bursting patterns). Conspicuous in Fig. 10 are the exceptionally high CVs found for bursting embryonic neurons (BFs >2.0).
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For nonauditory neurons, CV decreased systematically with increasing rate. In hatchlings, nonauditory CVs were related to the mean spike rate (MSR, sp/s) by a power function. This is illustrated in Fig. 9 where CV is plotted as a function of the mean spike interval (MSI, ms) for hatchlings. There was no similar tendency for CV to change as a function of spike rate in auditory units. Figure 10 summarizes the comparable CV data for embryos and illustrates that although rates were considerably lower, relationships similar to the posthatch bird also held in the embryo for auditory and nonauditory neurons.
Discharge interval histograms and patterns of activity
AUDITORY NEURONS. Auditory neurons in embryos evidenced discharge intervals having a quasi-Poisson distribution as illustrated for representative examples of TIHs in Fig. 11. This pattern also was found in posthatch birds, a finding that has been widely reported for posthatch birds and mammals. However, in embryos, the shortest intervals occurred with reduced probability compared with the mature TIH. The insets in Fig. 11 illustrate the rounded appearance in the region of shortest intervals in the embryonic TIH. This contrasts with the sharp appearance of the posthatch TIH. As interval lengths increase beyond the dead time in the hatchling, there is an abrupt rise and then exponential fall in the number of spikes per bin. In the embryo there is a more gradual rise and fall in spikes per bin over comparable interval lengths. The minimum and modal intervals are about twice as long for embryos compared with posthatch animals. Hazard functions can be used to illustrate the altered probabilities of intervals <10 ms. Figure 11 shows Hazard functions for the TIHs of an embryo and hatchling. In both cases, the function tends to level off and varies around a relatively stable mean value indicative of a Poisson process. However, note the significantly longer interval times required to achieve the Poisson plateau in the embryo. This is a typical observation and in this example, there was significant bias against spike intervals less than ~2 ms, whereas action potentials occurred at much shorter intervals in hatchlings. These results are consistent with and provide graphic illustration of the substantially longer modal intervals and dead times found in embryos.
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NONAUDITORY NEURONS.
Figure 15 illustrates representative
time interval histograms of nonauditory neurons. Nonauditory neurons,
especially those discharging in a regular pattern (Fig. 15,
top), displayed a Gaussian or modified Gaussian
distribution. Such distributions are typical of neurons innervating the
lagena in posthatch animals (Manley et al. 1991b), and
this is our hypothesis for the embryo as well. In a few cases,
nonauditory cells in the embryo did not show a typical Gaussian
distribution but rather had a more irregular discharge pattern (CV
>0.5) and demonstrated a long trailing skew toward longer intervals
(Fig. 15, bottom). Variations in TIH discharge patterns of
nonauditory neurons in hatchlings are illustrated in Fig.
16, some of which were similar to those
of auditory neurons. Generally, the dead times and modal intervals of
nonauditory cells were long compared with auditory neurons (Table 1).
Rather than quasi-Poisson, the time interval distribution tended to be
a modified Gaussian, multimodal or in a few cases even mixed
Poisson-Gaussian in appearance (Figs. 15 and 16).
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Bursting patterns
Figure 6 illustrates two bursting patterns displayed by primary afferents. Figure 6C is the record of a cell that has relatively long silent periods broken intermittently with spontaneous bursts of activity. Silent periods and bursts of spike activity occurred at irregular intervals. This type of pattern was found only in embryos and was present in ~30% of the neurons. In contrast, the pattern shown as Fig. 6D is a regular bursting pattern. This pattern was correlated with the heart rate of the animal. This is the only bursting pattern found in posthatch birds (5% cells studied) and was present in 3% of the embryonic cells studied. To provide a quantitative measure of bursting, the burst factor (BF, as defined in METHODS) was calculated for each neuron. Figure 6 shows the BF for each spike train represented. In general, the magnitude of BF was well correlated with pronounced irregular bursting. Figure 18 contrasts BF for auditory and nonauditory neurons in embryos and hatchlings and best illustrates that significant amounts of irregular bursting were found only in embryonic auditory neurons. Pronounced bursting was not a feature of discharge patterns in nonauditory neurons at any age. BF was near or well below 1.0 in nonauditory neurons (embryo: 0.3 ± 0.36, n = 10; hatchling: 0.2 ± 0.29, n = 24; range across all animals: 0.004-1.02). The BF for auditory cells varied considerably for embryos (range: 0.17-7.64). In contrast, the BF in posthatch neurons remained <1.0 in all cases except one (range: 0.09-1.29). In the one exception (BF = 1.29), bursting was regular and synchronized to the heartbeat.
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DISCUSSION |
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The findings of the present study show that statoacoustic ganglion
cells of the late embryo (E19) are capable of generating most of the
basic types of spontaneous primary afferent activity found in mature
posthatch animals. This includes both auditory and nonauditory neural
activity in concert with interval histograms displaying both
quasi-Poisson and Gaussian distributions, respectively. These
relatively mature patterns of spontaneous activity are consistent with
the mature tuning characteristics and tonotopic map that has been
described for embryonic neurons (E19) innervating the apical 60% of
the papilla (CFs ~100-2,000 Hz) (Jones, S. M. and Jones
1995a,b
). However, many findings of the present study reveal that these statoacoustic systems are not fully mature at E19. In
comparison with posthatch animals, primary afferent neurons of the E19
embryo have significantly lower spontaneous discharge rates, greater
variability in discharge rate as reflected by a larger range of CVs,
significantly longer modal interspike intervals and dead times (minimum
intervals), and irregular bursting discharge patterns in 30% of the
neurons (not correlated with heart rate). Moreover, preferred intervals
were rare in embryos and when they were present they were not obvious
from the TIH alone despite being widely reported in posthatch birds.
There were no CFs higher than 2,000 Hz in embryos of the present study,
a finding consistent with previous observations (i.e., 2,000 Hz)
(Jones, S. M. and Jones 1995a
,b
). We attribute the
differences between posthatch animals and embryos to immature
statoacoustic assemblies at late embryonic stages as discussed in
subsequent sections.
There have been many reports describing the physiology of
vestibulocochlear primary afferent neurons in neonatal mammals
(Carlier et al. 1975; Curthoys 1978
, 1979
, 1982
,
1983
; Desmadryl et al. 1986
; Gummer and
Mark 1994
; Kettner et al. 1985
; Romand
1984
; Romand and Dauzat 1982
; Walsh and
McGee 1987
) and in embryonic or early posthatch birds
(Jones, S. M. and Jones 1995a
,b
; Manley et
al. 1987
, 1991a
; Richter et al. 1996
;
Sheppard et al. 1992
; Valverde et al.
1992
; Yamaguchi and Ohmori 1990
, 1993
). The
present study provides the first report on in situ recordings of
spontaneous activity in embryonic statoacoustic primary afferents of
the bird.
Spontaneous rates
Low spontaneous discharge rates have been reported widely for
auditory and vestibular primary afferents in developing mammals. The
maturational patterns for mean spontaneous rates in chickens are less
clear. Manley et al. (1991a) reported no difference in spontaneous rates for chicken hatchlings at ages P2 (20 sp/s) and P21
(23 sp/s). Similar rates were found in emu chicks aged P1-P14 (26 sp/s) (Manley and Koppl 1997
). These values contrast markedly with rates in the adult chicken of 86 sp/s (Salvi et al. 1992
). Higher rates also are reported for adult animals of other avian species including the pigeon (65 sp/s, Gummer
1991
; 34 sp/s, Hill et al. 1989
; 67.4 sp/s,
Klinke et al. 1994
; 90 sp/s, Sachs et al.
1974
; 78 sp/s, Temchin 1988
), starling (48 sp/s) (Manley et al. 1985
), and barn owl (72 sp/s)
(Koppl 1997
). The reported rate differences between
hatchling chicks and adult animals could reflect maturational
refinements in peripheral sensory elements. However, some investigators
have argued that the rate differences in these studies can be
attributed to temperature or factors other than age (Smolders et
al. 1995
). Similarly, Manley et al. (1991a)
have
suggested anesthesia may play a role in producing the variability in
rates for posthatch animals. This latter suggestion is supported by the
findings of Anastasio et al. (1985)
showing that, on
average, spontaneous rates of vestibular primary afferents were reduced by ~55% in anesthetized (93 sp/s) compared with unanesthetized (168 sp/s) adult pigeons. In anesthetized hatchlings of the present study,
the mean spontaneous discharge rate was ~74 sp/s. This rate is close
to that reported for adult chickens (Salvi et al. 1992
)
as well as other species as described in the preceding text. Embryos of
the present study produced mean spontaneous rates that were
significantly lower than these values (19.5 sp/s). In the present
study, temperatures were comparable across all ages thus ruling out the
argument that slower spontaneous rates in embryos could have been due
to significant temperature differences between groups. Moreover,
because all animals were anesthetized (using the same agents) the
differences are not likely to be explained on the basis of this factor.
We conclude that the differences in spontaneous discharge rate found
here between embryos and posthatch animals reflect maturational
changes. Richter et al. (1996)
also have reported
increases in spontaneous discharge rates for neonate pigeons during the
first 4 wk posthatch.
Despite being widely reported across many species, it is not clear why spontaneous discharge rates increase during maturation. Ambient acoustic noise may be a factor in the generation of spontaneous activity as we have defined it. In the present study, ambient noise levels were the same for both age groups. As a result, decreases in discharge rate from this source could arise only as a consequence of reduced sensitivities to ambient external or internal physiological noise in embryos.
Although the mean threshold at CF reported here for embryos (middle
ears cleared of fluid) was similar to those described for hatchlings by
Manley et al. (1991a), they were significantly higher
than hatchlings of the present study. Our thresholds in hatchlings were
comparable with the mean thresholds in adult chickens reported by
Salvi et al. (1992)
for CFs between 500 and 1,500 Hz.
Moreover, it is reported here and elsewhere that spontaneous rates
tended to increase with decreasing threshold at CF in posthatch animals
(Manley et al. 1991a
; Salvi et al. 1992
).
These finding, on the whole, tend to support the hypothesis that rates
were lower in embryos due to higher thresholds. Despite this
possibility, other observations suggest that ambient noise levels and
the relative sensitivity of sensory elements may not be the only, or
the most important determinants, of discharge rate. First, spontaneous rates were significantly lower in embryonic versus hatchling
nonauditory neurons, which presumably do not depend on the middle ear
or ambient acoustic noise for activation. Second, spontaneous discharge
rates did not increase generally with decreasing thresholds at CF in embryos, an observation that is not consistent with the hypothesis that
rates in embryos are related to ambient sound levels. Third, there is
evidence that a substantial portion of spontaneous activity in
vertebrate hair cell sensory systems is unrelated to ambient noise and
that it arises autogenously within the neuroepithelium.
Support for the autogenous nature of spontaneous activity is based on
several lines of inquiry. Isolating afferent neurons from
(Santos-Sacchi 1993) or destroying hair cells of the
auditory and vestibular system (Kiang et al. 1976
;
Li and Correia 1998
; Muller et al. 1997
;
Salvi et al. 1994
, 1998
) reduces or eliminates spontaneous afferent activity in birds and mammals, suggesting that
spontaneous activity relies critically on the peripheral sensory
epithelium. Uncoupling ambient stimuli (e.g., removing the cupula)
(Harris and Flock 1967
; Harris and Milne
1965
) fails to block spontaneous discharge, showing that the
neuroepithelium alone is sufficient to generate such activity and that
ambient noise is not required. The possible independence of spontaneous discharge from ambient noise has been emphasized by many investigators (Kiang 1965
; Kiang and Sachs 1965
;
Sachs et al. 1980
; Walsh et al. 1972
) and
this likely holds for vertebrates in general (Hudspeth 1986
). In the absence of ambient noise, a stochastic excitatory process is thought to result from the steady, spontaneous,
calcium-dependent release of chemical transmitter from the presynaptic
hair cell (Annoni et al. 1984
; Flock and Russel
1976
; Flock et al. 1973
; Furukawa and
Ishii 1967
; Furukawa et al. 1972
; Harris
and Flock 1967
; Hudspeth 1986
; Ishii et
al. 1971
; Katz 1969
; Rossi et al. 1977
; Schessel et al. 1991
; Siegel
1992
; Siegel and Dallos 1986
). In practice,
spontaneous discharge may include both a component linked to external
ambient noise and an independent endogenous "resting" process.
Maturational refinements affecting spontaneous rates could include
changes in both this endogenous discharge process and elements
affecting responses to external stimuli. The extent to which each
contributes to rate differences observed throughout maturation remains
to be determined.
A unimodal distribution of spontaneous discharge rates has been
reported widely for auditory neurons of birds (Hill et al. 1989; Manley and Koppl 1997
; Manley et
al. 1985
, 1991a
; Sachs et al. 1974
, 1980
;
Salvi et al. 1992
), and this was true for both age
groups in the present study. Mean discharge rate did not vary systematically as a function of CF in embryos although discharge rates
decreased at higher CFs in hatchlings. Such a relationship has been
reported occasionally for the bird (Manley et al. 1985
).
Discharge regularity
A number of investigators have evaluated the developmental
appearance of spontaneous discharge regularity in vestibulocochlear ganglia. Regular firing patterns in nonauditory fibers are uniformly reported to be either absent or to represent a small proportion of
neurons in mammals at or near the time of birth. The number of regular
units increases markedly with age (Curthoys 1983;
Desmadryl et al. 1986
, 1992
; Romand and Dauzat
1982
). Mean discharge rates also increased with age in mammals
and irregular activity was found in progressively fewer cases as
animals matured. These results indicate that regular firing patterns
reflect a relatively mature status for nonauditory hair cell-primary
afferent assemblies. No information is available regarding the
appearance of regular discharge patterns during development of the
statoacoustic ganglion of the bird. The present study shows at E19,
however, that regular patterns are present. Although our embryonic
sample of nonauditory neurons was small, the proportion of regularly
discharging nonauditory neurons at E19 was actually larger compared
with those reported for mature animals. Furthermore there was no
evidence of substantial change in mean CV between embryonic and
posthatch ages for nonauditory cells. These results suggest that, in
terms of spontaneous discharge regularity, nonauditory neurons are
relatively mature at E19. It is worth noting that the minimum CV in
posthatch animals (0.04) was less than that of embryos (0.11). This
could be interpreted as a tendency toward more regular firing patterns
in the hatchling. The lower CVs in hatchlings could be related to the
higher rates in these neurons, to maturational refinements in
neuroepithelial assemblies, or both. Given the limited sampling of
nonauditory cells, this issue deserves more detailed study.
Very little is known in any species about changes in CV during development of auditory neurons. Although all auditory cells exhibited an irregular discharge pattern, there was a trend for CV to decrease toward 1.0 as a function of age in the present study. The higher CVs in embryos suggest a deviation from a Poisson distribution in the form of a larger than expected variance in spike discharge. This may be associated with increased bursting patterns and the overall variability of spontaneous discharge in embryos, a finding commonly reported for developing primary afferents in general. The CVs of neurons with burst factors >2.0 are identified in relation to all others in Fig. 10. The relative importance of the contribution bursting activity makes to higher CVs is suggested in Fig. 10 and by the finding that 31% (14) of embryonic neurons had CVs >1.5 and 93% (13) of these were bursters with BF substantially larger than 1.5. Embryonic CVs also are affected by the apparent bias against the occurrence of the shortest spike discharge intervals during spontaneous activity.
Preferred intervals
Preferred intervals are a common feature of spontaneous discharge patterns in primary afferents of the posthatch chicken. Preferred intervals occurred rarely in the embryos of the present study (3/47) and could be detected unambiguously in the TIH of only one neuron. For the other two neurons that exhibited weak preferred intervals, autocorrelation was required for detection, suggesting that this may be an emerging pattern in the late embryo.
Thoughts on the origin and importance of preferred intervals in birds
fall primarily into two contrasting categories. Klinke et al.
(1994) have argued that preferred intervals are driven by
ambient noise. In this view, a sharply tuned cochlear filter of any
type will produce PIs in afferent discharge patterns provided noise is
presented at levels near threshold. The absence of preferred intervals
in this case could be simply a result of reduced amounts of effective
ambient noise or the presence of generally higher thresholds. We cannot
definitively rule out this hypothesis. Nonetheless a strict
relationship between threshold at CF and the occurrence of preferred
intervals is not supported by our data. Preferred intervals were
present in neurons having the lowest thresholds; however, many neurons
(34% posthatch, 50% embryo) having CF thresholds at these same levels
did not have PIs at all.
A second view holds that preferred intervals represent the outward
expression of a specific endogenous electromechanical tuning mechanism,
which is linked to the tuned electrical resonance found in some hair
cells (Fuchs and Evans 1990; Fuchs and Mann
1986
; Fuchs and Sokolowski 1990
; Fuchs et
al. 1988
; Manley 1979
, 1990
; Manley et
al. 1985
). No requirement for ambient noise has been invoked by these investigators. Fuchs and coworkers have argued that
hair cell electrical resonance may be critical for the development of
sharply tuned auditory selectivity in the bird and that the resonance
requires functional calcium-activated K channels [K(Ca)]. These
investigators have shown further that the K(Ca) channels do not appear
in great number before E18 in the chicken. However, these channels did
appear in apical regions of the cochlea at E19 (Fuchs and
Sokolowski 1990
). If this is the case and if such channels are
the principal significant prerequisite for preferred intervals as
suggested, then why were preferred intervals not well manifested in the
embryos of the present study? One possible explanation may be that the
presence of K(Ca) channels alone is not sufficient to ensure the
occurrence of robust preferred interval patterns. This hypothesis
remains to be critically tested. Manley (1990)
also has
noted that preferred intervals do not occur in the caiman and yet the
appropriate K(Ca) channels are present.
The number of spikes recorded per neuron in embryos tended to be fewer
than older animals primarily due to the significantly lower spike rates
found. It is more difficult to resolve preferred intervals in cases
where spike numbers are low. Therefore an important alternative
explanation for the absence of preferred intervals may be that there
were too few intervals available in records from embryos to resolve
preferred intervals. This is not a completely satisfactory explanation
since the number of spikes recorded for embryos ranged 919, yet clear
preferred intervals were seen in TIHs of posthatch animals with as few
as 300 spikes. Perhaps more compelling is the fact that PIs were barely
discernable in only three embryos even when autocorrelation functions
were used for their detection. The absence of robust preferred
intervals in embryos thus remains an interesting finding that deserves
further evaluation.
Membrane channels and immature discharge patterns
It has been recognized for some time that the distribution of the
shortest intervals in mature auditory neurons is related to
refractoriness of the dendrite (Gray 1967; Kiang
1965
; Li and Young 1993
). Longer absolute and
relative refractory periods limit the occurrence of the shortest spike
intervals and highest discharge rates. Generally in hatchlings there is
a sharp rise to the modal interval as shown in Fig. 11. This pattern is
typical for mature auditory primary afferents in both birds and mammals
(mammals: Kiang 1965
; Walsh et al. 1972
;
bird: Manley and Koppl 1997
; Manley et al. 1985
,
1991a
,b
; Sachs et al. 1974
; Temchin
1988
). The period of exponential decay in interval counts often
is described as a Poisson process where spike intervals are independent
of previous activity. In embryonic TIHs, there is general agreement
with this pattern for intervals longer than the modal intervals. This
suggests indirectly that a stochastic excitation process is likely
present in most neurons in the late embryo. However, the reduced
probability of short intervals and longer interval modes and dead times
also suggest some limitation on the process that is not present in mature animals.
In mammals, there is very little quantitative information available
regarding overall dead times and modal intervals for spontaneous activity in neonates. Romand (1984) noted that shorter
intervals became more prevalent with age. Gummer and Mark
(1994)
found a significant decrease in the modal interspike
interval with increasing pouch age. Most investigators reported reduced
maximum discharge rates and prolonged onset latencies for evoked
primary afferent activity in neonates. To explain how mature patterns
were achieved gradually, investigators commonly emphasized end organ
structural changes, changes in hair cell stereocilia or membrane
properties and changes at the synapse (Desmadryl et al.
1992
; Romand 1984
; Walsh and McGee
1987
). These explanations remain plausible for some aspects of
maturing function; however, they are somewhat nonspecific and remain
difficult to test.
Prolonged dead times and modal spike discharge intervals are functional signs of immaturity that may be understood in simpler and more direct terms. These particular features are determined, at least in part, by the functional status of ion channels in the membrane trigger zone of primary afferents. The presence of longer dead times and modal intervals at E19 could be indicative of altered ion channel kinetics. Alternatively, biases in discharge probability could arise presynaptically, for example, as a result of an immature presynaptic mechanism operating to modify the stochastic excitation process. The transformation from embryonic patterns to those of posthatch animals may involve changes at many levels including refinements in the presynaptically derived excitation, postsynaptic activation and action potential cycle. There is no direct evidence for a "presynaptic excitation bias" in the embryo. On the other hand, there is direct evidence to support a role for membrane channels and the refractory state of the postsynaptic membrane.
Yamaguchi and Ohmori (1990) described the kinetics of
Na, Ca, and K channels found in isolated primary afferents taken from chicken embryos aged E16-E19. Compared with mature mammalian afferents (Santos-Sacchi 1993
), channel kinetic rates were
substantially lower in the embryos. These observations support the
working hypothesis that the long dead times and delayed modal intervals
found in embryonic neurons are, at least in part, a result of lower
kinetic rates for ion channels. However, a direct comparison of channel kinetics in avian embryos and posthatch animals has not been made.
Patterns in nonauditory neurons
TIH patterns distinctly different from those of auditory neurons
were found for most nonauditory neurons. Both regular (CV <0.5) and
irregular (CV >0.5) discharge types were present. Manley and
coworkers (1991b) have shown in the posthatch bird that these ganglion cell types project to the lagena, an otolith organ occupying a
position at the distal end of the cochlear duct. For the cells of
mammalian and avian eighth nerve ganglia, regular spontaneous discharge
patterns are produced only by afferents innervating vestibular organs.
The fact that regular firing nonauditory afferents were most abundant
in the present study contrasts markedly with results from neonate
mammals as noted in the preceding text and suggests that these
afferents are somewhat mature at E19 in the chicken. This is consistent
with work using vestibular evoked potentials in the chicken. In the
latter case, vestibular compound action potentials were present only in
animals older than E18 (Jones and Jones 1996
). On the
other hand, other features of nonauditory primary afferent activity may
point to immature channel kinetics comparable with those of auditory
neurons discussed above. For example, E19 nonauditory cells have
prolonged spike interval modes and dead times much like embryonic
auditory neurons. Significantly slower channel kinetic rates could
explain these findings as argued for auditory cells. Moreover,
progressive increases in channel kinetic rates also would account for
the observations that vestibular compound action potentials steadily
mature after E19 (latencies decrease and amplitudes increase to mature
values) over a period of several weeks posthatch (Jones, T. A. and Jones 1995
). These late maturational changes occur in
both auditory and vestibular systems and are likely to accompany
changes in membrane channel kinetics, myelination and synaptic
refinements. The extent to which each of these mechanisms contributes
to functional maturation remains to be explored.
Many of the TIH variations of irregular nonauditory neurons shown here
have been described for avian semicircular canal neurons (Correia and Landolt 1973, 1977
; Landolt and
Correia 1978
; Lifschitz 1973
). In the posthatch
chicken, Manley and coworkers (Manley et al. 1985
,
1991b
) described the distinctive TIH patterns as well as long
dead times and modes for irregular nonauditory neurons of the chicken
macula lagena. In the mature mammal, Walsh et al. (1972)
found that all auditory fibers had modal intervals <10 ms, whereas
nonauditory neurons had longer modes. In the bird, one cannot rely on
minimum and modal intervals alone to distinguish nonauditory TIHs from
auditory since some auditory neurons do have longer modes and dead
times especially those with lower frequency CFs (Fig. 14)
(Manley et al. 1985
, 1991b
; Salvi et al.
1992
).
Another difference between auditory and nonauditory neurons was the
relationship between CV and discharge rate. The CV of nonauditory
neurons is a function of discharge rate whereas it is not for auditory
neurons. We have described the relationship between CV in nonauditory
neurons and spontaneous discharge rate using a power function. Other
investigators have reported similar relationships for semicircular
canal afferents in pigeons (Anastasio et al. 1985;
Dickman and Correia 1989
; Lifschitz
1973
). Although qualitatively similar, the quantitative
features across these studies vary markedly. CV has been shown to be a
function of discharge rate in mammalian vestibular primary afferents
also (Fernandez and Goldberg 1976
; Fernandez et
al. 1972
; Goldberg and Fernandez 1971
). Indeed,
a particular neuron may have wide ranging values of CV depending on its
rate of discharge.
It is important to note an exception to the rule that the CV of
nonauditory neurons decreases with increasing rate of discharge. Figure
9 illustrates two outliers from the general trend of nonauditory cells
(). These nonauditory neurons had relatively high rates (114 and 127 sp/s) coupled with CVs approaching 1.0 (0.85 and 0.89, respectively).
Schermuly and Klinke (1990a
,b
) have identified high
discharge rates and lack of substantial rate change with traditional
auditory stimulation as key discharge features of infrasound neurons.
It is clear that the outlier neurons of Fig. 9 have a combination of CV
and discharge rate that is inconsistent with other nonauditory units.
It is possible that these two neurons are infrasound neurons of the
papilla, and we have indicated this hypothesis in Fig. 9.
Bursting patterns and synaptic refinements
The basis for bursting activity in embryonic auditory ganglion cells is unknown. The occurrence of bursting patterns implies a priori that the normal quasi-Poisson process is altered, and this appears to be born out here by the generally higher CVs of bursting neurons. Whether it represents an altered presynaptic excitatory process and/or a modified postsynaptic response compared with mature systems are reasonable first questions to address in the future.
Numerous observations have led to the idea that spontaneous activity
may play a significant role in the refinement of synaptic connections
during development and maturation (see Shatz 1996). Most
notably, synchronous bursting patterns have been identified in ganglion
cells in the visual system prior to the onset of sight (Galli
and Maffei 1988
). The collective firing patterns are thought to
provide a key signal that identifies cells originating in the same eye
as they project to primary receiving areas of the cortex. Modification
of these early signals has been shown to alter synaptic refinements in
the visual cortex and lateral geniculate nucleus (see Mooney et
al. 1996
; Shatz 1990
). In the chicken,
Lippe (1994
, 1995
) has described patterns of spontaneous
discharge for small populations of neurons in the region of central
auditory relays including the cochlear nucleus and nucleus laminaris.
Using multiple-unit recordings he reported rhythmic synchronized
discharge patterns which disappeared by E19. Gummer and Mark
(1994)
recording from the region of the cochlear nucleus also
reported action potential bursting patterns with rhythmic interspike
intervals in pouch young of the wallaby. In the present study, bursting
patterns for individual neurons were observed clearly in embryos but
not in hatchlings. We regard this pattern as a sign of immature sensory function. However, the bursting did not appear rhythmic nor was there
any evidence of a collective bursting of a population of neurons. The
only rhythmic bursting patterns found were synchronized to the cardiac
cycle and were found in both embryos and hatchlings. Therefore although
the irregular bursting reported here in primary afferents may be a
remnant of some putative primordial bursting pattern, we do not have
reason to believe that it could serve as the basis for the central
bursting patterns reported by Lippe. Indeed, Lippe reported that
synchronous rhythmic bursting disappeared by E19.
Summary
The results of the present study suggest that the essential features of adult auditory and nonauditory TIH patterns are present in the late embryonic chicken. Furthermore embryonic auditory and nonauditory neurons evidenced fundamentally different spontaneous discharge characteristics as is the case in mature animals. Embryonic auditory neurons displayed irregular activity producing a quasi-Poisson TIH distribution, and the majority of nonauditory cells displayed regular activity producing Gaussian TIH distributions. In addition, CV varied systematically as a function of discharge rate in nonauditory but not in auditory primary afferents. The regular activity of embryonic nonauditory neurons is remarkable because adult-like regular discharge patterns are the last feature to develop in mammals. This points to the precocious nature of statoacoustic end organs in the chicken at E19.
The present results also reveal key features of immature spontaneous activity in auditory and nonauditory primary afferents at E19 including lower overall spontaneous discharge rates, longer minimum discharge intervals (dead time), a reduced occurrence of the shortest spike interval accompanying longer modes, and notable absence of preferred intervals. In addition, irregular bursting patterns were commonly observed in embryonic auditory neurons. Regular bursting patterns were found only where discharges were synchronized to the heartbeat.
The basis for reduced spontaneous discharge rates in embryos is unknown
but may include immaturities in both an endogenous resting
neuroepithelial excitation process (e.g., resting rate of transmitter
release) and/or elements affecting the cells' responses to external
stimuli (e.g., transfer of sound through the middle ear). The lower
kinetic rates of sodium and potassium membrane channels reported
elsewhere for embryonic primary afferents (Yamaguchi and Ohmori
1990) provide one simple and direct hypothetical explanation for the longer dead time and modal spike intervals observed here in
embryos. Other presynaptic and postsynaptic maturational refinements also may contribute significantly.
Auditory and nonauditory spontaneous discharge patterns are fundamentally different with regard to relationship between discharge rate and interval regularity. The basis for this difference remains to be shown. The contrasting discharge behavior may be helpful in distinguishing primary afferent groups physiologically.
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ACKNOWLEDGMENTS |
---|
This research was supported by National Institute on Deafness and Other Communication Disorders Grants R03 DC-02573 and R01 DC-02753.
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FOOTNOTES |
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Address for reprint requests: T. A. Jones, Dept. of Surgery/ENT, 207 Allton Bldg., DC375.00, University of Missouri School of Medicine, Columbia, MO 65212.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 June 1999; accepted in final form 28 October 1999.
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REFERENCES |
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