Spontaneous Activity in the Statoacoustic Ganglion of the Chicken Embryo

Timothy A. Jones1,2 and Sherri M. Jones1

 1Department of Surgery/Otolaryngology and  2Department of Physiology, School of Medicine, University of Missouri-Columbia, Columbia, Missouri 65212


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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
<IT>H</IT>(<IT>k</IT>)<IT>=</IT><FR><NU><IT>y<SUB>k</SUB></IT></NU><DE><LIM><OP><IT>&Sgr;</IT></OP><LL><IT>n</IT><IT>=</IT><IT>k</IT></LL><UL><IT>∞</IT></UL></LIM> <IT>y<SUB>n</SUB></IT></DE></FR> (1)
where H(k) is the Hazard function, k is the bin number, and yk is the number of times interval k occurred in the TIH. H(k) is a number representing the probability that a spike will occur in bin k given that k units of time have elapsed since the last spike. The bin size or time unit used in the present study for calculating H(k) was 0.5 ms. For a random (homogeneous Poisson) process, the magnitude of H(k) is a function of the average spike rate and is a constant for all spike intervals. Factors such as neural refractory periods tend to bias discharge patterns. Neural refractory periods reduce the probability of spike discharge to as low as zero at very short intervals. The Hazard function of a neural TIH therefore rises from zero to a relatively stable range of probabilities as interval length increases. By comparing H(k) across ages, one can estimate maturational biases imposed on spontaneous discharge patterns as a function of age.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Distribution of afferent discharge rates for embryos (top) and posthatch animals (bottom). All neurons are represented (n = 120). Numbers of neurons as a function of rate [spikes/s (sp/s)] are represented. Bin size is 4 sp/s.



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Fig. 2. Auditory and nonauditory primary afferent discharge rates for embryos and posthatch animals. Means ± SD are represented. Auditory and nonauditory discharge rates were significantly higher for posthatch animals.


                              
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Table 1. Summary for embryos and posthatch animals

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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Fig. 3. Discharge rate (sp/s) of auditory neurons plotted against CF (Hz) for embryos and hatchlings.

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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Fig. 4. Auditory primary afferent discharge rate (sp/s) plotted as a function of threshold (dB SPL) at characteristic frequency (CF). Generally low rates and high-thresholds of embryos can be seen.

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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Fig. 5. Arithmetic mode of the spike interval (ms) for each neuron is plotted as a function of mean discharge rate. - - -, reciprocal of the corresponding discharge rate (Theory). Only nonauditory regular neurons fall on this line. Emb, embryonic; PH, posthatch.

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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Fig. 6. Spontaneous discharge recordings from 1 regular (A), irregular (B), and 2 bursting neurons (C and D). Age of each animal is shown (in parentheses). Spike-interval coefficient of variation (CV) and burst factor (BF) are represented for each neuron. Neural bursting pattern in D was synchronized to the heartbeat. A time scale of 250 ms is represented by a line above each trace.



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Fig. 7. Distribution of spike-interval CV for auditory neurons in embryos and hatchlings is represented here. CVs were >0.5 for all auditory neurons. A binwidth of 0.1 is used.



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Fig. 8. Distribution of spike-interval CV for nonauditory neurons in embryos and hatchlings is depicted. A binwidth of 0.1 is used.

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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Fig. 9. Spike-interval CV is plotted as a function of mean spike interval (MSI, ms) for posthatch chicks. CV was independent of mean interval for auditory neurons (). For nonauditory neurons, CV was a power function of mean interval as shown by the regression line and equation depicted (). Linear regression equation is given by CV = 5.48 × 10-4(MSI)2.092, where MSI is the mean spike interval. In terms of the mean spike rate (MSR), the function is given by CV = 1,036(MSR)-2.092 (R2 = 0.66, P < 0.01). Two nonauditory neurons have discharge features matching those for infrasound neurons as described by Schermuly and Klinke (1990a,b), and these are indicated (down-triangle).



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Fig. 10. Spike-interval CV plotted as a function of MSI (ms) for embryonic primary afferents. CV of auditory neurons was independent of mean interval (filled circles and triangles). Triangles identify bursting neurons (BF >2.0). In nonauditory neurons, CV was a power function of mean interval as shown by the regression line and equation depicted (open squares). Linear regression over nonauditory embryonic cell data for discharge intervals produced CV = 2.52 × 10-3(MSI)1.26 and for MSR produced CV = 14.7(MSR)-1.26 (R2 = 0.87, P < 0.01). Note the wider scale for intervals required here and compare with Fig. 9.

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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Fig. 11. Time interval histogram (TIH, left, binwidth = 0.5 ms) and Hazard functions [H(k); right] for representative primary afferents in embryos and posthatch animals. Both posthatch and embryonic neurons produce a quasi-Poisson distribution of spike intervals. This is suggested by Hazard functions that rise sharply to a relatively constant average level at spike intervals 3 ms. However, a notable difference in the time required to achieve Poisson probabilities can be seen, where embryos required 2-3 times longer spike intervals than hatchlings to achieve a random spike interval distribution. This also is reflected in the longer dead times and rounded appearance of the region of shortest intervals in embryonic TIHs. Insets: each inset represents the same data as the parent TIH except the x axis is expanded to better resolve distributions at the shortest intervals. x axis: range 0-15 ms, 5-ms major tick marks. y axis: same as the parent TIH.

Auditory neurons in posthatch birds also displayed a pattern of preferred intervals (PIs) (Fig. 12). Preferred interval patterns are widely reported for posthatch birds; however, we found only one example of unambiguous PIs in the standard time interval histograms of E19 embryos. Autocorrelation analysis revealed only two additional examples of weakly periodic discharge patterns in embryos. One of the best examples of PIs from these embryos is shown in Fig. 12 (left). Figure 12 contrasts the traditional TIH, ACF, and fast Fourier transform (FFT) for one embryonic neuron with those from a hatchling. There are no apparent PIs in the TIH of the embryo. Nonetheless, a low-amplitude periodicity was clearly present in the ACF of the same TIH. The amplitude of the FFT indicates a fundamental near 1,000 Hz for the embryonic ACF (bottom). Unlike the case of embryos, preferred intervals were found in a large fraction of posthatch neurons (16 of 29 or 55%) using the autocorrelation technique. The frequency corresponding to PIs (FFT fundamental, PIf) is plotted against CF in Fig. 13, illustrating that PI frequency was a function of CF for hatchlings of the present study. PI frequencies tend to distribute in regions near and parallel to the lines corresponding to PIf = CF and PIf = CF/2. The patterns of ACFs were quite varied as has been reported by Temchin (1988) for the posthatch bird.



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Fig. 12. TIH (top, 0.1 ms/bin), autocorrelation functions (ACFs, middle, 0.5 ms/bin), and fast Fourier transforms (FFTs) of ACFs (bottom, sampling rate 104/s) are shown for 1 embryonic neuron (left) and 1 hatchling neuron (right). Axis scales are identical for the 2 animals. Twenty-three seconds of spontaneous activity are represented for the embryo and 3.2 s for the hatchling. Total number of spikes were 693 spikes (30.1 sp/s) for the embryonic neuron and 396 spikes (120.8 sp/s) for the posthatch neuron. Preferred intervals (PIs) are present in the hatching TIH but are not obvious in the TIH of the embryo. ACF and FFT reveal PIs and the corresponding PI frequency (PIf) in the discharge pattern of the embryo.



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Fig. 13. ACFs were calculated for all neurons (see Fig. 12). ACFs of hatchling auditory neurons were subjected to FFT to identify the fundamental frequency (PIf in Hz) for the PIs detected. Here the FFT fundamental for PIs is plotted against the CF of each neuron. - - -, position where the frequency of the PI (FFT fundamental, PIf) equals CF (PIf = CF) and PI frequency equals CF/2 (PIf = CF/2).

Although most auditory cells (~60%) showed a classical quasi-Poisson distribution or, in hatchlings, exponentially decreasing PIs, other distributions in the TIH were observed. Figure 14 illustrates the most notable variations in TIH patterns for both ages. An early plateau (i.e., a stepped or gradual increase in number of spikes for successive intervals at the left edge of the TIH) and late mode were occasionally present in both age groups (Fig. 14, A-C; compare with Fig. 11). Late modes and long dead times occurred in two posthatch neurons having low CFs near 100 Hz (Fig. 14D). In hatchlings, normal dead times accompanying unusually long modal intervals occasionally produced multimodal distributions with significant periods of spike suppression (2-5 ms) immediately preceding the modal region (Fig. 14E). Unusual TIH shapes also were present for some low-frequency neurons (Fig. 14F).



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Fig. 14. Examples of variations in TIH distributions for auditory neurons: early shoulders and late modes (A-C); long dead times and late modes (D) and multiple modes and spike suppression (E and F). Bin width = 0.5 ms.

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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Fig. 15. TIH discharge patterns in nonauditory neurons. Examples of embryos (left) and hatchlings (right) are shown. Regular neurons are shown at the top and irregular in the bottom row. Bin width is 0.5 ms. Inset: same data viewed with time scale expanded to show interval lengths from 6.0 to 14.0 ms with axis ticks at 2-ms intervals.



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Fig. 16. Examples of variation in the TIHs of nonauditory neurons. Bin width = 0.5 ms.

Autocorrelation analysis of the TIH of nonauditory neurons revealed robust periodicities that varied in magnitude as a function of CV. The more regular the neural discharges, the more striking were the ACF oscillations. FFTs were used to calculate the frequency of the periods. Figure 17 shows that the period correlated well with the rate of spike discharge (P < 0.001, R2 = 0.8). Hence this type of periodicity is related to discharge rate and regularity. Although not shown in Fig. 17, we note that those cells falling some distance from the rate line had the highest CVs and thus had discharge patterns that were more irregular. Those nonauditory neurons that had CVs approaching 1.0 had little or no periodic activity in the ACF. This is in contrast to PIs of auditory neurons where CV and spike rate were unrelated to ACF-FFT frequencies.



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Fig. 17. Fundamental frequencies of periodic waveforms found in the ACFs of nonauditory neurons are plotted against discharge rate. Plot illustrates that the FFT fundamental reflected the basic firing rate of regular firing neurons. Linear regression: R2 = 0.8, P < 0.001.

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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Fig. 18. Scatter plots of burst factors (BFs) for individual auditory (left) and nonauditory neurons (right). All nonauditory and posthatch auditory neurons had BFs near or below 1.0. BFs substantially >1.0 are all embryonic bursting neurons.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (down-triangle). 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.


    ACKNOWLEDGMENTS

This research was supported by National Institute on Deafness and Other Communication Disorders Grants R03 DC-02573 and R01 DC-02753.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society