Departments of 1Biomedical Engineering and 2Otolaryngology, Hearing Research Center, Boston University, Boston, Massachusetts 02215-2407
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ding, Jiang, Thane E. Benson, and Herbert F. Voigt. Acoustic and Current-Pulse Responses of Identified Neurons in the Dorsal Cochlear Nucleus of Unanesthetized, Decerebrate Gerbils. J. Neurophysiol. 82: 3434-3457, 1999. In an effort to establish relationships between cell physiology and morphology in the dorsal cochlear nucleus (DCN), intracellular single-unit recording and marking experiments were conducted on decerebrate gerbils using horseradish peroxidase (HRP)- or neurobiotin-filled micropipettes. Intracellular responses to acoustic (tone and broadband noise bursts) and electric current-pulse stimuli were recorded and associated with cell morphology. Units were classified according to the response map scheme (type I to type V). Results from 19 identified neurons, including 13 fusiform cells, 2 giant cells, and 4 cartwheel cells, reveal correlations between cell morphology of these neurons and their acoustic responses. Most fusiform cells (8/13) are associated with type III unit response properties. A subset of fusiform cells was type I/III units (2), type III-i units (2), and a type IV-T unit. The giant cells were associated with type IV-i unit response properties. Cartwheel cells all had weak acoustic responses that were difficult to classify. Some measures of membrane properties also were correlated with cell morphology but to a lesser degree. Giant cells and all but one fusiform cell fired only simple action potentials (APs), whereas all cartwheel cells discharged complex APs. Giant and fusiform cells all had monotonic rate versus current level curves, whereas cartwheel cells had nonmonotonic curves. This implies that inhibitory acoustic responses, resulting in nonmonotonic rate versus sound level curves, are due to local inhibitory interactions rather than strictly to membrane properties. A complex-spiking fusiform cell with type III unit properties suggests that cartwheel cells are not the only complex-spiking cells in DCN. The diverse response properties of the DCN's fusiform cells suggests that they are very sensitive to the specific complement of excitatory and inhibitory inputs they receive.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cochlear nucleus (CN) provides the first stage
for processing acoustic information in the central auditory pathway. It consists of three tonotopically organized subnuclei: the anteroventral, posteroventral, and the dorsal cochlear nuclei (Lorente de
Nó 1981; Osen 1969
; Ramon y Cajal
1909
; Rose et al. 1960
). Of these, the laminated
dorsal CN (DCN) is the most complex. The DCN contains a diverse set of
morphologically distinct neuron types, including fusiform/pyramidal
cells, giant cells, cartwheel cells, granule cells, Golgi cells,
unipolar brush cells, stellate cells, chestnut cells, and
vertical/corn/tuberculoventral cells (Brawer et al. 1974
; Floris et al. 1994
; Kane
1974
; Lorente de Nó 1981
; Mugnaini et al. 1980a
,b
; Osen 1969
; Osen and
Mugnaini 1981
; Weedman et al. 1996
;
Wickesberg and Oertel 1988
; Wouterlood and
Mugnaini 1984
; Wouterlood et al. 1984
;
Zhang and Oertel 1993a
-c
). These neurons participate in
complex neural circuitry thought capable of performing sophisticated
signal processing tasks (Davis et al. 1996
;
Nelkin and Young 1994
; Young et al.
1992
). Extensive extracellular studies have revealed a variety
of response patterns from DCN units responding to acoustic stimulation.
These responses have been classified according to response maps
(Davis et al. 1996
; Evans and Nelson
1973
; Young and Brownell 1976
; Young and Voigt 1982
) and/or temporal discharge patterns (Gdowski
and Voigt 1997
; Pfeiffer 1966
; Rhode and
Smith 1986a
,b
; Shofner and Young 1985
;
Young et al. 1988
). Little is known, however, about
which neurons give rise to these responses.
To understand DCN neural circuitry and function, it is important to
associate acoustic response properties with morphologically defined
neuron types. Several in vivo intracellular studies on cat have been
successful in this regard for a few cell types (Rhode et al.
1983a,b
; Smith and Rhode 1985
, 1989
). For
example, Rhode et al. (1983a
,b
) showed that
fusiform/pyramidal cells possessed pauser-buildup and chopper
peristimulus time histogram response patterns depending on
stimulus parameters. These studies were performed in
barbiturate-anesthetized preparations, and units were classified
according to their temporal discharge patterns. Response map properties
for identified DCN neurons still remain largely unknown.
The effects of barbiturates on the response properties of DCN neurons
is a confounding factor in these past studies. Since 1973, the profound
effects of barbiturates on the response maps of presumed
fusiform/pyramidal cells have been well known (Evans and Nelson
1973). Young and Brownell (1976)
showed how
injections of pentobarbital sodium (Nembutal) reduced much of the
inhibition for moderate level best frequency (BF) tones transforming
type IV units into type III units. In gerbil, the effects of Brevital, an ultrashort-acting barbiturate, and Nembutal reduce the spontaneous activity of all spontaneously active DCN cells in decerebrate preparations, and transform units with one response map into another (Fan and Voigt 1997
). Gdowski and Voigt
(1997)
found that nearly 90% of all units recorded in the
barbiturate-anesthetized gerbil DCN have no spontaneous activity and
that some measures used to classify DCN units in unanesthetized,
decerebrate animals (e.g., relative noise response and normalized tone
slope) are not useful for this purpose for anesthetized preparations.
Thus is it hardly surprising that the response maps of DCN units
recorded in the presence of barbiturates are not well known.
This study is an attempt to achieve a better understanding of the
neural circuitry and function of the DCN by investigating acoustic
response properties of morphologically defined DCN neurons and
establishing relationships between unit physiology and cell morphology.
Intracellular single-unit recording and horseradish peroxidase (HRP)-
and neurobiotin-marking techniques in vivo were used to achieve this
goal. An unanesthetized, decerebrate preparation was selected to
eliminate the influence of barbiturates on neural response properties
(Evans and Nelson 1973; Fan and Voigt
1997
; Young and Brownell 1986
). Portions of this
work have been reported at scientific meetings (Ding et al.
1994a
,b
; Voigt et al. 1998
).
Portions of this work were submitted by J. Ding in partial fulfillment of the requirements for a doctoral degree in Biomedical Engineering at Boston University.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal choice
Mongolian gerbils (Meriones unguiculatus Tumblebrook
Farms) were chosen as subjects because they have well-developed
auditory systems capable of low-frequency hearing (Ryan
1976), have neurons and a basic organization similar to cats
(Benson and Voigt 1995
; Schwartz et al.
1987
), have relatively large bullae providing easy access to
the DCN (Frisina et al. 1982
; Lay 1972
;
Plassmann et al. 1987
), and allow a stable preparation
for in vivo intracellular studies (Ding and Voigt 1997
).
In addition, an extensive database of DCN unit response properties from
both extracellular (Davis et al. 1996
) and intracellular
(Ding and Voigt 1997
) studies in the decerebrate
preparation and extracellular studies in the barbiturate anesthetized
gerbil (Gdowski and Voigt 1997
) is available.
Surgical procedures
All experiments were performed in a sound-attenuating chamber
(IAC, model 1202A) using institutionally approved protocols. Detailed
surgical procedures can be found in Davis et al. (1996). Female gerbils (~3 mo old) first were administered atropine (0.04 mg/kg im) to reduce respiratory secretions. The animals then were anesthetized by an intraperitoneal injection of Brevital sodium (methohexital sodium, 65 mg/kg), an ultrashort-acting barbiturate. Supplemental doses of Brevital sodium (32 mg/kg) were administered during surgery as needed. Body temperature was maintained at ~38°C using a Harvard Apparatus heating blanket and controller. After an
incision in the throat, a small slit was made on the ventral surface of
the trachea, and the common carotid arteries were ligated with silk.
After the pinnae were removed, the animal was placed in a stereotaxic
device (KOPF, model 1730). A hole was made in the skull, and all brain
tissue rostral to the superior colliculus was aspirated. The empty
portion of the skull was packed gently with gelfoam to promote blood
clotting and to provide mechanical support for the remaining brain
tissue. The DCN was accessed using Frisina et al.'s transbulla
approach (Frisina et al. 1982
). The animal was allowed
to stabilize for ~30 min after the anesthetic was discontinued.
Acoustic system and calibration procedures
Acoustic stimuli were delivered to the left ear by an earphone
(Beyer Dynamic DT48A, 200 ) coupled to a hollow earbar. A probe-tube
microphone (0.5-in Bruel and Kjaer, model 4134) was placed near the
tympanic membrane to measure sound pressure levels during acoustic
calibration procedures. Pure tone stimuli were generated by a
programmable (Wavetek, model 5100) or manual (Wavetek, model 188)
oscillator with low harmonic distortion (less than
60 dB). Broadband
noise stimuli were generated digitally on a personal computer (Gateway
2000, 486DX) and output through a D/A converter (Tucker/Davis) at a
sampling frequency of 100 kHz (Davis et al. 1996
).
Acoustic stimuli were gated on and off with 5-ms rise/fall times using
an electronic cosine switch (Wilsonics, model BSIT). The signals were
attenuated by a programmable attenuator (Wilsonics model PATT) to
achieve the desired stimulation levels. Stimulus presentation and data
acquisition were under the control of a DEC PDP 11/73 computer. The
acoustic system was calibrated in each experiment by delivering a click
(5 V, 20 µs) to the earphone and measuring the resulting sound
pressure near the tympanic membrane. The system's frequency response
was computed by performing a fast Fourier transform (FFT) of the click
response and dividing through by the FFT of the click. A 10-band
equalizer (BSR, model EQ-3000) was used to achieve a maximally flat
frequency response by modifying the frequency spectrum of the click
before delivery to the ear. To compensate for the nonflat frequency
response, the digital noise stimuli were created with a spectrum the
magnitude of which was the inverse of the magnitude of the system's
frequency response (
25 kHz). Davis et al. (1996)
provide more details and a representative frequency response curve.
Electrodes and recording system
Glass microelectrodes were pulled from capillary tubes (WPI)
with 1-mm ID using a Flaming-Brown micropipette puller (Sutter Instrument, model P80/PC), and filled with 0.5 M potassium chloride, 0.05 M Tris buffer, and either 4% HRP or 2% neurobiotin using standard techniques. The tip diameters were usually <0.5 µm, and the
initial impedances were >100 M, measured at 1 kHz using a microelectrode impedance meter (Winston Electronics, model BL-1000). The electrodes were beveled in a saline-silicon carbide (Buehler) slurry to reduce the impedance to between 40 and 80 M
before use.
Electrodes were advanced into the DCN through the paraflocculus by a stepper-motor microdrive (Kopf Instruments, model 660). A silver/silver chloride (Ag/AgCl) wire was inserted into the electrode and connected to the headstage of an Axoclamp 2A (x1) amplifier (Axon Instruments). Another such wire was placed in the neck musculature as a reference electrode. The electrical activity recorded by the Axoclamp was further amplified by a DC amplifier (Tektronix, model AM 502), digitized, and stored on a PDP 11/73 computer system.
Experiment protocol
MEASUREMENT OF BRAIN STEM AUDITORY-EVOKED RESPONSES.
Click-induced brain stem auditory-evoked responses (BAERs) were
measured in each animal as described in Ding and Voigt
(1997) to gauge the integrity of the auditory brain stem
pathways. The experiment would continue only if BAER threshold was
within normal range (Burkard and Voigt 1989
).
STIMULUS AND DATA COLLECTION PROCEDURES.
As the electrode advanced through paraflocculus, broadband noise bursts
were used to evoke background driving. Once background driving was
observed, signaling DCN penetration, the noise stimuli were replaced
with tone bursts that clearly elicited background responses. After
penetrating a neuron, its BF and threshold () were estimated
audiovisually. The transmembrane voltage then was recorded for various
stimuli. First, three trials of 50- or
100-ms1 tone
bursts (10-ms delay, 250-ms interstimulus interval) were presented at
three frequencies (BF, BF
0.7 octave, and BF +0.7 octave) and 17 sound pressure levels each (0-80 dB SPL, 5-dB steps). This was
followed by three trials of 50- or 100-ms broadband noise bursts (10-ms
delay, 250-ms interstimulus interval) at each of 17 levels (0-80 dB
SPL, 5-dB steps). To collect rate versus level curves, longer-duration
tone and noise bursts (200-ms duration, 1,000-ms interstimulus
interval) were used, and the unit's spike times were recorded.
Stimulus level typically started at 0 dB SPL and increased in 2-dB steps.
CELL MARKING.
After an inital set of data was collected, the cell was
iontophoretically injected with HRP or neurobiotin using 4.0-nA, 4-Hz depolarizing current pulses (150-ms on, 100-ms off) for 2 min. The
membrane potential was closely monitored and recorded to determine whether the electrode remained inside the cell throughout this procedure. A near constant membrane potential was interpreted as strong
evidence that the cell marked was the same cell from which the
physiological data were recorded. To avoid out-of-cell current leakage,
the injection was stopped immediately after observing a sudden change
in the measured voltage.
Data processing
CRITERIA FOR ACCEPTABLE INTRACELLULAR RECORDINGS.
A recording was considered to be intracellular and acceptable if the
cell's resting potential was less than or equal to 50 mV and if the
cell's maximum action potential (AP) was
40 mV in amplitude. One
exception was made, however, for a giant cell, since these cells are
only infrequently encountered. In this case, the resting potential was
less than
45 mV and its maximum AP amplitude was ~30 mV.
DRIVEN AND SPONTANEOUS RATE ESTIMATES. Discharge rates were obtained by counting the number of APs over a specific time window. For estimating driven rates, the analysis window was either the same as the stimulus duration (for electric stimulation) or the last 80% of the stimulus duration (for acoustic stimulation). The time window used for estimating spontaneous activity rate (SpAc) was the same as that for the driven rate but positioned at the end of each trial. For example, when the tonal stimulus duration was 100 ms, the driven rate was estimated during a 80-ms period, starting 20 ms after the stimulus onset, whereas the SpAc was computed during the last 80 ms of the 250-ms trial. The only exception was when the duration of the acoustic stimuli was 50 ms, then the SpAc was estimated at 0 dB SPL during a 100-ms interval.
Rate versus sound pressure level (RSL) curves and rate versus current level (RCL) curves were constructed from the experimental data. To reduce rate variations due to the short time intervals used to estimate acoustically driven rates, the RSL curves were smoothed using a three-point filter that has the following equation:
![]() |
ANALYSIS OF ACOUSTIC RESPONSES: UNIT CLASSIFICATION.
In classifying a unit physiologically, deviations from SpAc were used
to determine excitation and inhibition (Davis et al. 1995,
1996
). A driven rate that was larger (smaller) than SpAc by >2
SD of the SpAc was taken to be excitation (inhibition). In situations
where SpAc was estimated at only one level (0 dB SPL), a criterion of
20% above/below SpAc was used to determine excitation/inhibition. When
SpAc was zero, a driven rate >2.5 spikes/s was considered excitation.
ANALYSIS OF RESPONSES TO CURRENT-PULSE STIMULATION. In addition to obtaining RCL curves, responses to 50-ms current pulses also were investigated for poststimulus hyperpolarization, anode break excitation, and membrane voltage sags. Estimates of current-voltage relationship (I-V curves) were made for hyperpolarizing current pulses by measuring the steady-state voltage responses near the end of the current pulse. In cases where the amplifier bridge was not balanced (normal case), estimates of the voltage drop across the recording electrode were made by fitting the sum of three decaying exponentials to the entire voltage response. The coefficient of the fastest decaying exponential was taken to represent the electrode contribution, and this component was subtracted from the voltage response to yield the cell's responses to the current pulses. Estimates of the cell's input resistance were made from the slopes of these I-V curves.
TISSUE PROCESSING AND CELL RECONSTRUCTION.
At least 2 h after labeling a cell, the animal was killed by
administering a dose of pentobarbital sodium (60.0 mg/kg),
exsanguinated by perfusion with normal saline solution and 1% sodium
nitrite, and fixed with buffered mixed aldehydes. After overnight
fixation at 4°C, the brain tissue was removed from the skull,
embedded in gelatin/albumin, and sectioned serially with a Vibratome
(series 1000) into 50-µm-thick slices. For neurobiotin, the tissues
were processed overnight in Vector ABC reagent solution. The tissue slices then were processed with diaminobenzadine histochemistry (Graham and Karnovsky 1966), intensified with cobalt
chloride (Adams 1981
). The tissue slices were mounted on
glass slides and counterstained with cresyl violet or further processed
for flat-mount epoxy embedment.
CONFIDENCE OF ASSOCIATION BETWEEN PHYSIOLOGY AND MORPHOLOGY. `Confidence of association' is a qualitative measure of the certainty that the recovered cell was the same cell from which data were recorded. The confidence of association would be high if one or both of the following conditions were satisfied: the resting potential was stable throughout the injection of HRP or neurobiotin and response properties recorded before and after iontophoresis were similar. The confidence of association would be low if the data failed to meet either criteria or if no data were available to assess these criteria. In this report, only cells with high confidence of association are discussed. Six other cells that failed to meet either of these criteria were discarded.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unit incidence
In 114 experiments, 133 spiking cells were successfully impaled.
Of these, 120 (90%) cells fired only simple APs (called simple-spiking cells), and 13 cells (10%) fired both simple and complex APs (called complex-spiking cells). A complex AP consists of a burst of simple spikes superimposed on a slow and transient depolarization (see Fig.
10). Intracellularly recorded nonspiking cells frequently were
encountered. These nonspiking cells had much lower resting potentials
(less than 80 mV) than spiking cells, and did not respond to either
acoustic or electric stimulation. They were not studied further.
All simple-spiking cells were acoustically responsive, and 68 of these were classified according to the response map scheme. Table 1 shows the distribution of unit response types for these cells. Notice that type III (including type III-i) units are the most common response type encountered and type IV (including type IV-i) and IV-T units are the least common. Also included in Table 1 are 11 complex-spiking cells. Only two of these could be classified and both were type III units. The other complex-spiking cells were unclassifiable due to poor acoustic responses (high-thresholds and low driven rates). Another 52 simple-spiking cells and 2 complex-spiking cells could not be classified because of a lack of data (cells were either lost or injured during the recording).
|
Recovery of labeled cells and association of morphology with physiology
After recording responses to acoustic and electric stimuli,
attempts were made to mark 61 successfully impaled cells, 40 with HRP
and 21 with neurobiotin. Seven HRP- and 12 neurobiotin-labeled DCN
cells were identified and associated to physiology. These cells will be
referred to as `identified cells,' and some of these will be
described in detail later. For many other recovered cells, the
association of physiology to morphology was unsuccessful because of one
of the following: 1) the confidence of association was low.
In these cases, the membrane voltage was not monitored during the
marking process and the cells were either lost or injured. 2) The number of recovered cells was not equal to the number
of deposits. These mismatches occurred most frequently in experiments using neurobiotin because this small molecule can easily leak out of
the recording electrode and lightly label cells. And 3) recovered cells have questionable identity because of poor labeling or
their associated responses were ambiguous because of injury-discharging APs. An earlier paper describes in detail the responses properties of
all intracellularly recorded units that were not marked or recovered
(Ding and Voigt 1997).
Acoustic response properties of identified DCN neurons
The 19 identified DCN cells, for which the confidence in the association of physiology to morphology is high, include 13 fusiform/pyramidal cells, 2 giant cells, and 4 cartwheel cells. Listed in Tables 2-4 are the response types and several physiological properties for each morphologically defined cell class. Most of the fusiform cells (10/13) have type III unit response properties, although two of these were type III-i units. A small number were associated with either type I/III (2/13) or type IV-T (1/13) unit response properties (Table 2). Both giant cells had type IV-i unit responses (Table 3). One of the fusiform cells with type III unit response properties (J22795-5-1) was a complex-spiking cell. All other fusiform/pyramidal cells and both giant cells were simple-spiking cells. All cartwheel cells were complex-spiking cells and had weak acoustic responses that were difficult to classify using the response map scheme (Table 4).
|
|
|
Detailed results from seven fusiform cells with different response types (2 type III units, 2 type III-i units, 2 type I/III units, and 1 type IV-T unit), two giant cells with type IV-i response properties, and two cartwheel cells with weak acoustic responses are presented. These cells were selected because of the completeness of the data record and the quality of the labeling. To emphasize the link between morphology and physiology, aspects of both are shown in each figure. Also included is a plot of the membrane potential during the marking process and/or selected responses before and after cell marking.
Fusiform/pyramidal cells with type III and III-i unit response properties
Figure 1A is a camera lucida reconstruction of a fusiform/pyramidal cell (Jd0993-3-1) with type III response properties. Its cell body (* in inset) is located in the DCN's fusiform cell layer. This neuron has a well-developed apical dendritic tree that projects into the DCN's molecular layer, reaching its surface. The apical dendrites have dendritic spines (not shown in the drawings). Basal dendrites are less elaborate or complex than the apical dendrites and project into deep DCN. Spines are not apparent on the basal dendrites. The axon does not have local branches and could be traced into the DAS.
|
The cell's resting potential was 61 mV, its largest APs were 61 mV,
and its initial average spontaneous activity (SpAc) was 92 spikes/s. BF
was 1.7 kHz and BF-threshold (
S) was ~25 dB
SPL. Figure 1B shows examples of intracellular responses to
50-ms tonal and broadband noise bursts at various levels. The cell was
excited by BF tones at all levels. The cell's firing pattern and
membrane behavior, however, were level dependent. At levels between 25 and 35 dB SPL, the cell showed a pauser discharge pattern, and the
membrane was hyperpolarized during the pause (e.g., 1.7 kHz, 35 dB
SPL). At higher levels, the firing pattern became more regular. Although the membrane was not notably depolarized during the stimulus, there was a ~10 mV after-stimulus hyperpolarization that lasted for
>20 ms (e.g., 1.7 kHz, 70 dB SPL). Side-band inhibition was seen at
frequencies both above and below BF. When strongly inhibited, the cell
fired an initial spike or two, followed by a silent period, during
which the membrane was hyperpolarized (e.g., 2.76 kHz, 50 dB SPL).
Responses to broadband noise were excitatory and the membrane behavior
was similar to that at BF except that there were no pauser-like
patterns (e.g., noise, 45 dB SPL). After-stimulus hyperpolarization
also was present.
Shown in Fig. 1C are the cell's RSL curves. These were derived from intracellular responses to tones of three frequencies [BF and BF ± 0.7 octave] and broadband noise, with levels ranging from 0 to 80 dB SPL in 5-dB steps. The curves were smoothed by a three-point filter. The BF-driven rate increased monotonically with increasing level until saturating at ~55 dB SPL with a maximum discharge rate of ~160 spikes/s. Side-band inhibition was clearly seen between 30 and 60 dB SPL at the lower frequency and at levels >30 dB SPL, at the higher frequency. Responses to noise were nonmonotonic with a maximum discharge rate ~180 spikes/s.
The cell was labeled by injecting HRP for ~2 min. Figure
1D shows a stable resting potential measured between
current pulses during the injection, indicating the electrode remained
within the cell. After the cell was marked, its response properties did not change drastically. For example, Fig. 1E shows the
cell's RSL curves derived from responses to 200-ms noise bursts before and after HRP injection. The two curves are similar in that they have
similar thresholds (15 dB SPL), reach maximum at similar sound
pressure levels (
45 dB SPL), and became slightly nonmonotonic afterward with similar slopes. Spontaneous rates were also comparable before and after the injection. In fact, this cell was held long enough
after the HRP injection that extensive intracellular responses were
collected. Figure 2A shows
the responses from which a three-dimensional response map was derived
(Fig. 2B). The V-shaped excitatory area at BF and
side-band inhibition above and below BF clearly indicate a type III
unit response map. The similarity of response properties before and
after HRP injection provides high confidence in the association between
morphology and physiology.
|
Figure 3A shows a camera lucida reconstruction of a fusiform/pyramidal cell (J30294-2-1) with type III-i unit response properties. The cell body (* in the inset), located in the DCN's fusiform cell layer, has a darkly stained nucleus, but the processes were lightly stained, probably due to intranuclear labeling. The main apical dendrites project into the molecular layer, whereas it appears that a second dendritic process was thin and unbranched, possibly indicating that the cell was undergoing some degeneration. The basal dendrites project into the deep DCN. The axon was traced out of the DCN through the DAS.
|
Shown in Fig. 3B are some intracellular responses of
this cell to 50-ms tones and noise bursts. The cell had large, over- and undershooting APs (~73 mV in size), and the resting potential was
61 mV. BF-tone (2.68 kHz) responses were excitatory and monotonic. Compared with the previous cell, the firing patterns were more regular
(e.g., 2.68 kHz, 25 and 60 dB SPL). Side-band inhibition, though not
strong, was seen at both higher and lower frequencies. Unlike the
previous cell, there were no sustained hyperpolarizations seen at
off-BF frequencies (e.g., 4.35 kHz, 30 dB SPL). Noise responses were
excitatory and similar to BF-tone responses except that the
after-stimulus hyperpolarization was stronger for noise (e.g., Noise,
70 dB SPL). Such hyperpolarization would totally suppress SpAc.
At suprathreshold levels (>20 dB SPL), the discharge rate for BF tones quickly rose to ~300 spikes/s before starting to saturate (Fig. 3C). After collecting BF-tone responses, the SpAc increased significantly. These changes in SpAc were reflected in shifted RSL curves for off-BF stimuli. The responses to off-BF stimuli and noise were inhibitory for a range of levels and excitatory at higher levels. Although such responses at side-band frequencies are characteristic of type III units, the inhibitory responses for low levels of noise are specific for the type III-i unit subtype. This feature is more clearly seen in the RSL curve derived from responses to 200-ms broadband noise bursts, shown in Fig. 3E, where the stimulus levels were changed in 2-dB steps and SpAc was more accurately estimated at each sound pressure level. The cell was inhibited by broadband noise at low levels (25-40 dB SPL) but excited at higher levels; therefore it was classified as a type III-i unit.
The RSL curve, derived from responses to 200-ms broadband noise after HRP injection, was similar to the curve obtained immediately before the labeling (Fig. 3E), although shifted to the left. The preservation of such unique noise responses, together with the stable membrane potential during the labeling process (Fig. 3D), strongly indicate that the cell marked was the one recorded.
Shown in Fig. 4A is a digitized image of another fusiform/pyramidal cell (J92595-12-1) with type III-i unit response properties. The cell is located in the fusiform cell layer in the caudal DCN. In this plane of section, the soma is more rounded, something that is not typical. Thick branches of apical and basal dendrites extend into the molecular and deep layers, respectively. Some spines are seen at the distal parts of the apical dendrites. The axon arises from a branch of basal dendrite and does not have collaterals. Part of the axon is seen in the DAS, although it cannot be traced farther.
|
Shown in Fig. 4B are examples of intracellular responses
to 100-ms tones and noise bursts at various frequencies and levels. The
cell had a resting potential of 71 mV, and its largest APs measured
67 mV. Responses to BF tones (2.81 kHz) were excitatory, and the
discharges were regular (e.g., 20 and 60 dB SPL). A mild membrane
depolarization was seen at levels >20 dB SPL, followed by a stronger
after-stimulus hyperpolarization. This long-lasting (>200 ms)
hyperpolarization would suppress significantly the interstimulus activity. When inhibited at off-BF frequencies, however, there was no
sustained hyperpolarization (e.g., 4.55 kHz, 65 dB SPL). The noise
responses, although excitatory, were not as strong as the BF responses,
and the firing patterns were less regular (e.g., noise, 60 dB SPL).
The cell responded monotonically to BF tone bursts (Fig.
4C). Starting with an initial average SpAc rate of 40 spikes/s and a BF threshold of 15 dB SPL, the discharge rate increased
with level to ~280 spikes/s at 60 dB SPL, saturating with increased levels. Like other type III units, weak side-band inhibition was seen
within a narrow region (15-30 dB) 0.7 octaves below BF but much
stronger and wider region 0.7 octaves above BF. Unlike the primarily
excitatory responses at the lower frequency, the responses at the
higher frequency were never excitatory. The RSL curve for short-duration (100 ms) noise bursts was linear at suprathreshold levels, and the noise-driven rate did not saturate 80 dB SPL. When
longer duration (200 ms) noise bursts were applied in smaller steps (2 dB), the responses were inhibitory between 5 and 20 dB SPL (Fig.
4E). Such responses indicate that the cell had type III-i unit response properties.
The cell was labeled with neurobiotin. Although the membrane potential
decreased from 50 to about
90 mV during the iontophoresis (Fig.
4D), there was no indication that the electrode left the cell. Thus we
conclude that the recording and labeling were confined to the same cell.
The cells of Figs. 3 and 4 are both type III-i units and are included because they show slightly different morphology and responses to BF tones and noise stimuli. These are rare units and have not been reported in other species to date. Showing two such cells does not seem to be excessive.
Shown in Fig. 5A is a camera lucida reconstruction of a complex-spiking fusiform/pyramidal cell (J22795-5-1) associated with type III unit response properties. The oval-shaped cell body was located in the fusiform cell layer. Thick trunks of apical and basal dendrites extended toward the surface of the DCN and into the deep layer, respectively. Spines (not shown) were seen on the dendrites reaching the surface. No axon was found in the vicinity of the cell body. Sections of an axon, however, were seen in the DAS.
|
This was the only fusiform/pyramidal cell that fired complex APs. These complex spikes were seen in addition to simple APs. Shown in Fig. 5B are some intracellular responses to 100-ms tone and noise bursts. Complex APs from this cell consisted of doublet and triplet bursts of simple spikes of decreasing amplitude superimposed on a small (~5 mV) and transient depolarization (e.g., 2.68 kHz, 25 dB SPL). Notice that many of the other single spikes also rode on small, transient depolarizations, and these spikes had undershoots. When excited by BF tones at high levels (e.g., 2.68 kHz, 65 dB SPL), the cell responded mainly with simple spikes. There was sustained depolarization during the stimulus, followed by a much stronger after-stimulus hyperpolarization as much as 30 mV below the resting potential and as long as 200 ms, during which SpAc was suppressed. When inhibited by off-BF frequencies, the membrane was heavily hyperpolarized (~20 mV; e.g., 4.35 kHz, 80 dB SPL). Responses to broadband noise were excitatory and similar to responses at BF (e.g., noise, 65 dB SPL).
The RSL curves (Fig. 5C) indicate that BF threshold was ~40 dB SPL, the highest of all the fusiform/pyramidal cells, and the maximum BF-driven rate was ~140 spikes/s, which was among the lowest for this cell type. The RSL curve for noise is similar to that for BF tones, both became slightly nonmonotonic at the highest levels. Like all fusiform/pyramidal cells with type III unit response properties, side-band inhibition for this cell was more obvious for frequencies above BF (e.g., RSL curve at BF +0.7 octave).
The membrane potential was quite stable as neurobiotin was injected into the cell (Fig. 5D), decreasing slightly with time. SpAc, however, decreased after the injection, as can be seen in the RSL curves derived from responses to 200-ms noise bursts (0-80 dB SPL in 2-dB steps) presented immediately before and after neurobiotin injection (Fig. 5E). The RSL curve after neurobiotin injection was shifted downward probably because of the decrease in SpAc. The fluctuations in the two curves were due to the bursting APs. The confidence of association between cell morphology and physiology is considered to be high because the membrane potential was flat during cell marking and because the responses before and after labeling were similar.
Fusiform/pyramidal cells with type I/III unit response properties
Figure 6A is a camera lucida of a fusiform/pyramidal cell (J32695-13-1) with type I/III unit response properties. The cell body was located in the fusiform cell layer. Heavy branches of spinous apical dendrites were seen extending into the molecular layer, some reached the ependyma. The basal dendrites had fewer branches and extended into the deep layer. A single axon emerged from a branch of the basal dendrites without forming collaterals. The axon was seen only in the vicinity of the cell body.
|
The cell had nearly no SpAc, and thus side-band inhibition of APs could not be detected. Because responses to noise were as strong as those to BF tones (7.15 kHz), the cell was classified as a type I/III unit. Samples of excitatory intracellular responses at and near BF and for noise are shown in Fig. 6B. These responses were similar in two ways. First, spike discharges occurred mainly within the first half of the stimuli. Second, a slow membrane depolarization, sometimes >10 mV, preceded most spikes. The ensuing repolarization process, however, quickly drove the membrane back toward resting potential. Finally, unlike fusiform/pyramidal cells with type III unit responses, there was no obvious after-stimulus hyperpolarization in this cell, although the cell was strongly excited by both tones and noise bursts. Hyperpolarization was also not evident during the subthreshold stimuli.
The RSL curves for this cell are shown in Fig. 6C. For BF
tones, the responses began at 10 dB SPL (threshold) and were slightly nonmonotonic. The discharge rate decreased slightly after exceeding 100 spikes/s at 25 dB SPL and then rose again to a new peak of 170 spikes/s
at 80 dB SPL. Threshold for tones at BF 0.7 octaves was much higher
(40 dB SPL), and the responses were also nonmonotonic. In this case the
driven rate simply decreased after reaching 95 spikes/s at 60 dB SPL.
Responses to noise, however, were monotonic with a threshold of 25 dB
SPL and a maximum rate of 105 spikes/s. The relative noise response,
, defined as the ratio of maximum noise-driven rate minus SpAc
divided by maximum BF-driven rate minus SpAc (Davis et al.
1996
; Young and Voigt 1982
) was 0.96 for this cell.
Also shown in Fig. 6C are the RSL curves derived from intracellular responses to 100-ms BF tone and noise bursts applied immediately after neurobiotin injection. The labeling of the cell had almost no effect on these response properties because the RSL curves before and after the labeling were almost identical. During the labeling process, however, the membrane potential initially fluctuated and then decreased before finally showing a gradual increase (Fig. 6D). Nevertheless, these changes did not show a sudden upward shift of >50% from the resting potential. On the basis of this fact and the unchanged response properties, we conclude that there is a high confidence of the association between the morphology and the physiology of this cell.
Figure 7A is a digitized image of the other fusiform/pyramidal cell (Jo1895-9-1) with type I/III unit response properties. This cell had a typical fusiform-like cell body located in the fusiform cell layer. More apical dendrites were seen in this plane of section than basal dendrites. Unfortunately, no axon was found either near the cell body or in the DAS. Nevertheless, the cell's identity is clear because of the soma's location and shape and the structure of the dendrites.
|
Examples of intracellular responses to 100-ms tones and broadband noise
bursts are shown in Fig. 7B. The resting potential was 65
mV, the AP amplitude reached 70 mV, SpAc was nil, and threshold for BF
tones (9.74 kHz) was 5 dB SPL. Acoustic responses were excitatory for
frequencies near BF and for noise. This cell was similar to the
previous one with respect to AP features and membrane behavior. For
example, there was often a slow depolarization immediately prior to the
rising phase of each spike (e.g., 9.74 kHz, 20 dB SPL), and there were
no after-stimulus hyperpolarizations, even after strong depolarizations
and high discharge rates (e.g., 9.74 kHz, 40 dB SPL). The major
difference between the two cells was that this cell's responses were
consistently stronger (spike discharge occurred throughout the
stimulus). Accordingly, the cell's monotonic RSL curves (Fig.
7C) achieved a higher maximum BF-driven rate (270 spikes/s).
Noise responses were comparable with BF-tone responses but had a higher
threshold and a lower maximum rate. Responses to BF
0.7 octave tones
had a much higher threshold (50 dB SPL), although the maximum rate was
comparable with that at BF. Responses to BF +0.7 octave tones were
weak; the discharge rate never exceeded 35 spikes/s.
Responses to 200-ms BF tones and noise bursts were collected before and after neurobiotin injection. The resulting RSL curves are shown in Fig. 7D. These curves are very similar with only slight changes in the slopes and steady-state rate, providing strong evidence that the recordings were from the recovered cell.
Fusiform/pyramidal cell with type IV-T unit response properties
Figure 8A is a camera lucida of a fusiform/pyramidal cell (J61793-10-1) with type IV-T unit response properties. The cell body is located in the fusiform cell layer. A single dendritic trunk gave rise to both apical and basal dendrites (indicated by arrow head). Its axon was traced into the DAS without local branches.
|
Type IV-T units are distinguished from type III units by their highly nonmonotonic BF responses. Intracellular responses to 50-ms BF tones (5.66 kHz) at 10, 30, and 75 dB SPL in Fig. 8B show such nonmonotonicity. Although excited at all levels, the responses decline at midlevels (e.g., 30 dB SPL) from those at lower levels (e.g., 10 dB SPL) before increasing again at higher levels (e.g., 75 dB SPL). Membrane depolarizations were usually followed by after-stimulus hyperpolarizations, which would suppress the interstimulus activity (e.g., 5.66 kHz, 75 dB SPL and noise, 65 dB SPL). Side-band inhibition was seen at both lower and higher frequencies (e.g., 11.32 kHz, 75 dB SPL and 2.83 kHz, 25 dB SPL), and responses to noise were strictly excitatory (e.g., 65 dB SPL). Note the absence of sustained hyperpolarization when the cell was inhibited.
The RSL curve at BF is nonmonotonic with one local minimum and two
local maxima (Fig. 8C). The first maximum of 170 spikes/s occurred at 10 dB SPL. The rate dropped to 69 spikes/s at 30 dB SPL
before increasing again at higher levels. Note that the rate exceeded
SpAc (~54 spikes/s) at all levels. This cell is a type IV-T unit
because the minimum occurred within 35 dB of the first peak and was
less than half of the average of the first maximum and SpAc. At one
octave below BF (BF 1 octave), the discharge rate drops below SpAc
between 20 and 30 dB SPL and then increases with increasing levels to a
maximum of 170 spikes/s. One octave above BF (BF +1 octave), however,
the cell was not responsive until 45 dB SPL, when the driven rate
decreased below SpAc. Responses to noise increased monotonically and
exhibited sloping saturation beginning at 20 dB SPL.
The cell's membrane potential was not monitored during HRP injection.
After the injection, the SpAc decreased nearly to zero. The cell's
characteristic type IV-T RSL curve, however, remained nearly unchanged
(Fig. 8C), although it was shifted downward due to the
reduced SpAc. Type IV-T units were among the least common response
types encountered in the decerebrate gerbil (Ding and Voigt
1997). The preservation of type IV-T unit response properties after HRP injection leads us to conclude that the cell labeled was the
same cell from which data were recorded.
Giant cells with type IV-i unit response properties
Figure 9A shows a camera lucida of a giant cell (J22394-11-1) with type IV-i unit response properties. The cell's large soma (~30 µm diam) was located in the deep DCN. Its multipolar dendrites extend isotropically in the deep DCN, and some were found well into the molecular layer. The axon was traced out of the DCN via the DAS toward the midline as far dosal as the floor of the fourth ventricle without visible branches. This is the only cell in this study with APs <40 mV in size. Although it did not meet our criterion for an acceptable unit, the cell still is included because giant cells are among the least recorded and labeled cells in the study.
|
The giant cell was strongly inhibited by acoustic stimuli over a wide
range of frequencies and levels, which is characteristic of type IV
units. Figure 9B shows selected intracellular responses at
various stimulus conditions. At BF (7.47 kHz), the responses were
excitatory only for a small range of low levels (e.g., 7.47 kHz, 15 dB
SPL). No depolarization was evident when the cell was excited.
Inhibitory responses occurred at higher levels (e.g., 7.47 kHz, 40 dB
SPL; 12.14 kHz, 35 dB SPL; and 4.35 kHz, 25 dB SPL). These responses
consisted of onset spikes followed by membrane hyperpolarization, as
strong as 15 mV (e.g., 12.14 kHz, 35 dB SPL). Responses to noise,
however, were mostly excitatory and much stronger than the excitatory
responses at BF (e.g., noise, 25 dB SPL). At higher levels, the noise
responses became onset-like and inhibitory (e.g., noise, 70 dB SPL).
Type IV units with this kind of noise rate versus level curve are
subclassified as type IV-i units (Davis et al. 1996).
Note there were no sustained hyperpolarizations during the inhibition
by noise.
The RSL curves shown in Fig. 9C clearly show the type IV
unit response properties. At BF, the excitatory response region was between 5 to 10 dB SPL, and the maximum driven rate was 110 spikes/s, indicating a weak excitation (average SpAc = 80 spikes/s; SpAc estimated at BF, 0 dB SPL). At higher levels, the driven rate dropped
to zero, indicating strong inhibition. Responses to BF 0.7 octaves
were similar to those at BF except the cell was excited again at very
high levels. At BF +0.7 octaves, there was no excitatory region at all.
The cell became responsive >30 dB SPL by quickly reducing the number
of spikes to zero. Responses to noise were mainly excitatory and highly
nonmonotonic. At 25 dB SPL, the noise-driven rate reached a maximum of
200 spikes/s, which is about twice the maximum BF-driven rate. The rate
decreased gradually with increasing level, becoming inhibited at levels
>55 dB SPL.
The giant cell was labeled with HRP, during which the resting potential slowly rose but remained negative throughout the injection (Fig. 9D). Responses to 200-ms BF tone bursts were collected for RSL curves before and after the HRP injection to assess its impact (Fig. 9E). The HRP injection did not change the cell's SpAc or responses to BF stimuli, thus the confidence is high in associating this morphology to these response properties.
Figure 10A is a camera lucida of a second giant cell (J83095-3-1) with type IV-i unit response properties. The soma, ~25 µm in size, is located in the deep layer just below the fusiform cell layer. Several dendrites emanate from the soma in various directions, some extending into the molecular layer while others project to the deep layer. The axon originated from a dendrite rather than from the soma. Axon segments were found in the DAS.
|
Figure 10B shows examples of this cell's intracellular
responses to various stimuli. Responses to BF tones (8.79 kHz) were excitatory only at low levels (e.g., 8.79 kHz, 15 dB SPL). Strong depolarization and after-stimulus hyperpolarization were absent for BF
excitatory stimuli. At higher levels, however, the responses consisted
of onset spikes followed by sustained hyperpolarizations (e.g., 8.79 kHz, 40 dB SPL). When stimulated by either BF 0.7 octave tone bursts
or noise bursts, the responses were inhibitory at low levels (e.g.,
5.41 kHz, 15 dB SPL and noise, 30 dB SPL), and excitatory at high
levels (e.g., 5.41 kHz, 65 dB SPL and noise, 70 dB SPL). The inhibitory
responses for these stimuli at the lower sound levels were similar to
those at BF except that the inhibition seemed to last longer especially
for the noise stimuli. The excitatory responses for the non-BF stimuli,
however, were very different from those at BF. After an initial onset
spike, a pause appeared, during which the membrane was hyperpolarized. This was followed by a burst of spikes with increasing interspike intervals. Such a discharge pattern would give rise to a pauser peristimulus time histogram. When the stimulus was completed, the
membrane strongly hyperpolarized for ~100 ms. Not shown are responses
to BF +0.7 octave tones, which were similar to the inhibitory responses
at BF.
The RSL curves shown in Fig. 10C are highly nonmonotonic.
The maximum driven rate of 122 spikes/s for BF tones was achieved within a narrow (5-15 dB SPL) excitatory region. The inhibition at
higher levels was so strong that the driven rate was zero between 30 and 60 dB SPL. Some spikes occurred at still higher levels. Increases
in driven rate at high levels were larger for the BF 0.7 octave tones
and for noise, where the rate gradually exceeded SpAc and became
excitatory. This nonmonotonic RSL curve for noise has not been seen
before for type IV units. This giant cell therefore is classified as a
variant of a type IV-i unit, which generally is inhibited by noise at
all high levels.
The cell was labeled lightly by a brief injection of neurobiotin. Figure 10D shows the membrane voltage monitored during the injection. Approximately 5 s after the start of the injection, the electrode suddenly lost contact with the cell, as indicated by the sudden upward voltage shift. The labeled cell was most likely the same as the one from which the data were recorded because the membrane potential was stable during the initial 5 s, the injection was terminated immediately after the cell was lost, and it was the only cell labeled.
Cartwheel cells with weak acoustic responses
Figure 11A shows a camera lucida reconstruction of a cartwheel cell (Jo1493-8-1) with weak acoustic responses. The round cell body was in the fusiform cell layer, and its heavily spinous and recurving dendrites were confined to the fusiform and molecular layers. The axon originated from the cell body and ramified (not shown in the drawing) locally in the molecular and fusiform layers.
|
The cartwheel cell fired complex APs in combination with simple APs (Fig. 11B). Complex APs consist of bursts of simple APs, that usually increase in width and decrease in amplitude, superimposed on a slow, transient depolarization, lasting ~10-30 ms. Complex APs vary in appearance. For example, the depolarization could be as small as 10 mV (e.g., 1.23 kHz, 65 dB SPL) or as large as 30 mV (e.g., 2 kHz, 60 dB SPL), and the number of bursting spikes in a complex from this cell ranged from three (e.g., noise, 50 dB SPL) to five (e.g., 2 kHz, 60 dB SPL). Complex APs appeared randomly and were not acoustically responsive. Simple APs from this cartwheel cell were broader than those from pyramidal cells (average of 0.7 ms in half-size width) and had no undershoots (e.g., 2 kHz, 80 dB SPL). Simple APs outnumbered complex APs and responded weakly to acoustic stimulation at high levels, and some even rode on small (<5 mV) depolarizations (e.g., 2 kHz, 60 dB SPL).
The RSL curves in Fig. 11C clearly indicate weak acoustic responses. The threshold for BF tones (2.00 kHz) was 35-40 dB, and the maximum BF-driven rate was 67 spikes/s, about half the lowest maximum rate for pyramidal cells. The receptive field was broadly tuned and it was difficult to find BF; i.e., for tonal stimuli spanning BF ±2 octaves, there were little differences in thresholds and maximum driven rates. SpAc was near zero, and there were virtually no responses to broadband noise.
The cell was labeled with HRP. During the labeling process, the
membrane potential experienced some fluctuations (Fig. 11D). The changes, however, were small and at all times during the injection the membrane potential was up to 50 mV, indicating that the electrode remained intracellular and thus the confidence of associating this
cell's morphology to the recorded physiology is high. After labeling,
however, the cell became silent and completely unresponsive.
A digitized image of another cartwheel cell (J91895-05-01) is shown in Fig. 12A. The round cell body was located in the fusiform cell layer. Thick, heavily spinous dendrites ramified within the molecular layer, some in a recurving pattern. The axon arose from the cell body and branched in the fusiform cell layer. The axon profile is not visible in this section because it is out of focus.
|
This cartwheel cell is similar to the previous cartwheel cell, Jo1493-8-1, in that it also fired both simple and complex APs. Figure 12B shows several examples of simple and complex APs (notice the time scale is longer in these panels). The large and overshooting simple APs had no undershoots and most of them rode on a small, transient depolarization (~10 mV, 15 ms). The complex APs, on the other hand, consisted of a burst of two to three simple spikes superimposed on larger depolarizations (20 ~ 40 mV, 30 ~ 50 ms). The size of successive simple spikes in a burst consistently decayed as the depolarization developed. The peak of the depolarization usually occurred after a burst of simple spikes (e.g., 5.48 kHz, 70 dB SPL), but also could coincide with the last spike in a burst (e.g., noise, 70 dB SPL). Occasionally, there appeared to be a duplex of complex APs, in which two complexes partially overlapped each other (e.g., 5.48 kHz, 25 dB SPL and 3.37 kHz, 5 dB SPL).
This cell had a low average SpAc rate (12 spikes/s) and responded
weakly to acoustic stimulation. The RSL curve for BF tones (5.48 kHz)
showed a threshold between 25 and 30 dB SPL and a maximum driven rate
of only ~60 spikes/s (Fig. 12C). BF tones usually elicited simple APs, although sometimes, complex APs were generated. At BF 0.7
octave tones and broadband noise, the responses were even weaker with
thresholds
5-10 dB higher and driven rates <50% of those at BF and
comparable level. For BF +0.7 octave tones, the cell was unresponsive.
This cell was labeled by neurobiotin. As seen in Fig. 12D, the cell's membrane potential remained negative throughout the current injection, rising slowing with time. Hence the confidence that the recorded responses are from this cartwheel cell is high. After the injection, the cell became silent and unresponsive, although the resting potential showed no further change.
Electric response properties of identified DCN neurons
Although responses of the identified DCN neurons to acoustic stimulation had a variety of different patterns, as shown in the preceding text, the responses of these cells to electric stimulation were less diverse both within and across cell type. Therefore the presentation of the responses to electric current is organized differently from the acoustic responses. Thus one example of intracellular current responses, the RCL, and the I-V curves derived from these, are presented for each cell type.
Figure 13A shows intracellular responses to 50-ms current pulses from a fusiform/pyramidal cell, a giant cell, and a cartwheel cell. The sudden shift in voltage seen at stimulus onset and offset is due to the voltage drop across the recording electrode. As a general feature, all identified cells were excited by depolarizing (positive values) currents and inhibited by hyperpolarizing (negative values) currents. The larger the current level, the stronger the excitation (inhibition) would be. Only simple spikes were elicited from fusiform/pyramidal cells and giant cells responding to depolarizing current pulses (Fig. 13A, left and middle). Cartwheel cells differed from these other two cell types in that high levels of depolarizing current elicited mainly complex APs (Fig. 13A, right). The response latency shortened and the shapes of the complex APs were distorted at higher current levels. Such distortion would reduce the size of the APs, making them more difficult to detect. There were no after-stimulus hyperpolarizations or anode break excitations from any of the cells. Sag phenomena was observed in only one cell, a fusiform/pyramidal cell with type I/III unit response properties (Jo1895-9-1, not shown here).
|
Shown in Fig. 13B are the RCL curves for these three
cells. The RCL curves from the fusiform/pyramidal cell and giant cell are qualitatively similar to each other: the driven rates increased monotonically with increasing current levels. The threshold to current
(I) was ~0.07 nA for all cells (this was the
smallest depolarizing current used), and the driven rate at 1.0 nA
ranged from 140 to 380 spikes/s (also see Tables 2 and 3). Cartwheel cells, on the other hand, had nonmonotonic RCL curves as a result of
shrinking complex-APs at high current levels (see Table 4). Their
I-V curves, however, were indistinguishable from other cell types (insets in Fig. 13B). In fact, the I-V
curves from all identified cells were approximately linear in the range
from
10 to
50 mV below resting potential with input resistance
ranging from 16 to 117 M
.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell incidence
Most of the identified cells in this study are fusiform cells
(68%), followed by cartwheel cells (21%) and giant cells (10%), a
7:2:1 ratio. By comparison, Schwartz and her colleagues found in a
morphological study of gerbil nongranule DCN cells, 4% fusiform cells,
20% cartwheel cells, and only 1% giant cells, a 4:20:1 ratio
(personal communication). This anatomic study explains why encountering
giant cells is such a rare event. There is still, however, a
discrepancy in the ratio of fusiform/pyramidal to cartwheel cells.
Complex-spiking cells (intracellular contact) and bursting-like units
(extracellular contact) in fact were encountered most frequently; however, they were often either injured or lost. Although cartwheel cells are the second most common DCN cell type, second only to granule
cells in gerbil (see preceding text) and rat (Wouterlood and
Mugnaini 1984), their small size perhaps makes them more prone to damage by impaling microelectrodes. This may be why relatively few
cartwheel cells were marked in this study.
Associations between morphology and physiology
Most of the identified gerbil fusiform/pyramidal cells are type
III units, although some of them are type III-i units, type I/III
units, and even type IV-T units. Identified giant cells are type IV
units of a sort. Finally, cartwheel cells are difficult to classify
according to the response map scheme because they have weak acoustic
response properties (higher thresholds and lower driven rates). The
confidence of these associations between cell morphology and physiology
is high for all of these cells, as demonstrated by the examples in the
preceding text. By providing records of the cells' membrane potential
while marking the cells and, in some cases, providing acoustic
responses both before and after cell marking, we are assured that the
recovered cells are, in fact, the cells from which we recorded.
Although the number of each cell type is limited, especially for giant
cells, these results most likely reflect the general features of these
cell populations. First, the distribution of intracelluarly recorded type III units, type IV-like units, and complex-spiking cells with weak
acoustic responses from a much larger sample of cells (49, 9, and 11%,
see Table 1) is similar to the distribution of the identified
fusiform/pyramidal cells, giant cells, and cartwheel cells (68, 10, and
21%). Second, the response characteristics of cartwheel and giant
cells are fairly consistent. There is a diversity of response patterns
associated with the fusiform/pyramidal cells, and this may be a
reflection of the sensitivity of these neurons to particular
assortments of excitatory and inhibitory inputs each cell receives.
Although two fusiform/pyramidal cells were type I/III units and one was
a type IV-T unit, these response types resemble type III units in
several ways. What is remarkable is that the fusiform/pyramidal cells
did not turn out to be type IV units. This was quite surprising because
in cats, type IV units are associated with the fusiform and giant cell
axons of the DAS (Young 1980). Davis et al.
(1996)
discuss various reasons why type IV units are less
prevalent, and type III units are more prevalent in gerbils, compared
with cat.
Finally, other physiological features of these intracellularly recorded
type III units, type IV units, and complex-spiking cells with weak
acoustic responses were similar to those of the fusiform/pyramidal
cells, giant cells, and cartwheel cells, respectively. These features
include membrane behavior, spontaneous activity, thresholds, and
maximum discharge rate in response to acoustic/electric stimuli (e.g.,
compare Tables 4-6 of Ding and Voigt 1997 with Tables
2-4 in this study).
Similarities and differences in responses across the cell types
All three cell types in this study share certain membrane
behaviors in response to acoustic and electric stimulation. For example, when responding to acoustic stimuli, all cell types exhibited membrane depolarization during excitation and/or hyperpolarization after excitation or during inhibition. In response to electric stimulation, all cell types were excited by depolarizing current and
inhibited by hyperpolarizing current. Excitation thresholds (I) were about the same for all cells (see
Table 2-4).
There are also major differences across the cell types. None of the
cartwheel cells and only one fusiform/pyramidal cells exhibited type IV
or type IV-T unit response properties. Likewise, no giant cells fired
complex APs or had type III, I/III, or IV-T unit responses. A single
fusiform/pyramidal cell, however, did have complex APs, a feature
common to all identified cartwheel cells. The cell, however, had
relatively strong and classifiable (type III) response patterns,
undershooting simple APs, and small complex APs (see Fig.
5B), whereas the cartwheel cells had weak and unclassifiable
acoustic responses, simple APs without undershoots, and fired larger
complex APs (see Figs. 11B and 12B). Overall, the preceding results suggest that different cell types are likely to have
distinct acoustic responses characteristics. Parham and Kim
(1995) reported that 20% of extracellularly recorded
bursting units had strong acoustic responses and these were found
deeper than the nonresponsive bursting units. Extracellularly recorded (AC-coupled) complex spikes from either cartwheel cells or fusiform cells would look like bursts of action potentials without the slow
depolarization. Perhaps bursters with strong acoustic responses correspond to complex-spiking fusiform cells and nonresponsive bursters
correspond to cartwheel cells.
Cartwheel cells also respond differently to electric stimulation than the other cell types. That is, cartwheel cells tended to discharge nonmonotonically to increasing current levels (due to distorted spikes of the complex APs at high depolarizing current levels), whereas fusiform/pyramidal cells and giant cells responded monotonically. Although a nonmonotonic RCL relationship for cartwheel cells may simply reflect the membrane properties that give rise to complex APs, for most fusiform/pyramidal cells and giant cells, a monotonic RCL curve suggests that the inhibitory behaviors of these cells to acoustic stimuli are likely the result of local intrinsic neural circuitry rather than simply the effect of membrane properties.
Similarities and differences within individual cell types
MORPHOLOGY OF FUSIFORM/PYRAMIDAL CELLS.
All identified fusiform/pyramidal cells are located in the fusiform
cell layer. Most of these cells have a typical fusiform-shaped cell
body, although on two occasions a pyramidal shape (see Fig. 1A) and a round shape (see Fig. 4A) would be more
descriptive. Apical dendrites are more plentiful than basal dendrites.
They reach the surface of the DCN, close to the ependyma, and spines are found along parts of the apical dendrites distal to the cell body.
Fewer basal dendrites are seen, and in many cases only a piece of the
less-branched trunk is visible. Such differences in the appearances of
apical and basal dendrites also were seen in fusiform/pyramidal labeled
by Golgi impregnation (Schwartz et al. 1993), Lucifer
yellow (Manis 1990), biocytin (Zhang and Oertel
1994
), and HRP (Smith and Rhode 1985
). Axons,
when identified, do not have local collaterals. In most cases, axons
can be traced directly to the DAS, some even farther (e.g., Fig.
3A), or found in segments in the DAS. The lack of local
axonal collaterals of gerbil fusiform/pyramidal cells is consistent
with results from guinea pig (Manis 1990
) and mouse
(Zhang and Oertel 1994
) but contrary to results from cat
(Smith and Rhode 1985
).
MEMBRANE BEHAVIOR OF FUSIFORM/PYRAMIDAL CELLS IN ACOUSTIC/ELECTRIC
RESPONSES.
Most of the identified fusiform/pyramidal cells fired simple APs with
undershoots. This is consistent with reports by Zhang and Oertel
(1994) and Manis (1990)
, who studied fusiform
cells in slice preparations. When excited acoustically at high levels, most of our fusiform/pyramidal cells exhibited a small membrane depolarization, which usually was followed by a long-lasting
after-stimulus hyperpolarization (e.g., Fig. 4B). Such
membrane behavior in response to acoustic stimuli also was reported in
fusiform cells of anesthetized cats (Rhode and Smith
1986b
; Smith and Rhode 1985
).
After-stimulus hyperpolarization also was found in other
intracellularly recorded units after responses to large depolarizing
current (Ding and Voigt 1997
), and may be a
self-regulatory mechanism of the membrane. After-stimulus
hyperpolarization, however, could also occur without an obvious
preceding depolarization and strong excitation (e.g., Fig.
1B). Therefore this behavior also could be the result of some strong, sustained, and/or long-latency inhibitory inputs on these
cells. When inhibited at off-BF frequencies, fusiform/pyramidal cells
often were hyperpolarized during the stimulation. The sizes of these
hyperpolarizations, however, were different among these cells, perhaps
indicating various strengths of the side-band inhibition. It is
interesting to note that neither of the type I/III fusiform/pyramidal cells exhibited hyperpolarizations at off-BF frequencies nor did they
have an after-stimulus hyperpolarization after excitation (see Fig.
6B and 7B). This phenomenon was common
for all unmarked intracellularly recorded type I/III units as well
(Ding and Voigt 1997
). It is unclear whether this was
due to a disturbance in membrane regulatory mechanisms by the recording
electrode or due to weakened inhibitory inputs onto cells that would
otherwise have type III response properties.
ACOUSTIC RESPONSE TYPES OF FUSIFORM/PYRAMIDAL CELLS.
The majority of recorded fusiform/pyramidal cells had type III unit
response properties. Two of these cells also exhibited unique responses
to broadband noise (see Figs. 3 and 4), which identifies them as type
III-i units, a subclass of type III units recently found in decerebrate
gerbils (Davis et al. 1996). Of the remaining cells, two
had type I/III unit and one had type IV-T unit response properties.
Type I/III units have very low SpAc. At off-BF tonal stimuli, there
were no signs of membrane hyperpolarization (Figs. 6B and
7B) (see also Ding and Voigt 1997
). Side-band
inhibition, therefore could not be detected. Other than that, however,
their tuning curves and responses to noise cannot be distinguished from
type III units. In an intracellular recording study, a type I/III unit
could possibly be derived from a type III unit. For example, if the
impaling electrode alters the membrane properties and results in both a
reduction in SpAc and strength of inhibition, then a type III unit
would appear to be a type I/III unit.
ELECTRIC RESPONSES OF FUSIFORM/PYRAMIDAL CELLS.
Fusiform/pyramidal cells respond to current injection monotonically.
These results are consistent with other studies of this cell type in
slice preparation (e.g., Manis 1990 in guinea pig; Hirsch and Oertel 1988a
,b
; Oertel and Wu
1989
; Zhang and Oertel 1994
in mouse). The mean
slope of the RCL curves for our fusiform/pyramidal cells, 258 ± 70 spikes/s/nA, is greater than that reported by Manis
(1990)
but comparable with Zhang and Oertel's
(1994)
results.
MORPHOLOGY OF GIANT CELLS.
Both giant cells were found in the deep DCN. They have relatively large
somata (>25 µm) and multipolar dendrites extending in many
directions. The giant cell axons, like those of fusiform/pyramidal cells, were seen in the DAS. In one case, the axon was traced out of
the DCN through the DAS without DCN collaterals (Fig. 9A). The morphology of these cells is similar to those labeled by biocytin in mouse slice preparations (Zhang and Oertel 1993b).
MEMBRANE BEHAVIOR OF GIANT CELLS IN ACOUSTIC RESPONSES.
Although one of the giant cells (J22394-11-1, Fig. 9) had a
high resting potential (47 mV) and smaller APs (34 mV), its membrane behavior in response to acoustic stimulation was similar to that of the
other giant cell (J83095-3-1), which had a more normal resting potential and APs. Both cells fired only simple APs and showed
sustained hyperpolarizations at high levels of BF-tone bursts where the
cells were inhibited. Neither cell had strong membrane depolarization
during BF-tone excitation.
RESPONSE TYPES OF GIANT CELLS.
Although both giant cells exhibited type IV unit responses to tonal
stimuli, their noise responses were not typical of classic type IV
units. One cell (J22394-11-1) was inhibited by noise at high
levels and is therefore a type IV-i unit (Davis et al.
1996). The other cell (J83095-3-1), however, had a
novel noise response that has not been described before. It was
inhibited by noise only over a range of levels and as such showed a
partial similarity to type IV-i units. Despite this difference in noise
responses, the overall type IV unit character of both giant cells is
consistent with the hypothesis that type IV responses are from some DCN
projection cells (Young 1980
).
MORPHOLOGY OF CARTWHEEL CELLS.
Labeled cartwheel cells were located either in the molecular layer or
the fusiform cell layer. Cell bodies were usually round, and dendrites
were confined within the two layers. A characteristic feature of rat
cartwheel cells, recurving dendrites, were seen in all such identified
cells (Wouterlood and Mugnaini 1984). Heavy dendritic
spines, another cartwheel cell feature, were observed in two darkly
labeled cells (both shown in the preceding text). Axons often
originated from the cell body and branched within the molecular layer
and/or fusiform cell layer. These observations are consistent with the
general descriptions of DCN cartwheel cells from other studies (e.g.,
Brawer et al. 1974
; Manis et al. 1994
;
Oertel and Wu 1989
; Schwartz et al. 1993;
Wouterlood and Mugnaini 1984
; Zhang and Oertel
1993a
), although Biocytin, used in one of the studies, usually
does not label dendritic spines very well (Zhang and Oertel
1993a
). One interesting finding by Manis et al.
(1994)
is that the recurving pattern of dendritic branches is
not evident in guinea pig cartwheel cells.
AP FEATURES AND MEMBRANE BEHAVIOR OF CARTWHEEL CELLS.
All identified cartwheel cells discharged a combination of simple and
complex APs. Simple APs from cartwheel cells were the slowest (widest
width, average ~0.7 ms) of all cell types and had no undershoots.
There are variations in the profile of complex APs, such as the number
of bursting simple spikes in a complex and the size and duration of the
depolarization. These variations occurred not only from cell to cell
but also within the same cell. The duration and firing pattern of
cartwheel cells are Na+ and
Ca2+ dependent (Golding and Oertel
1996). Whether spontaneously generated or in the
presence of acoustic stimulation, complex APs were outnumbered by
simple APs. The general profile of gerbil cartwheel cell complex APs is
similar to that of mouse cartwheel cells (e.g., Zhang and Oertel
1993a
) and guinea pig (Manis et al. 1994
). There
are, however, some differences in the physiological features of
cartwheel cells among these studies. For example, Manis et al. reported
that only a small portion (3/29) of the complex-spiking (cartwheel)
cells also fired simple APs, whereas simple spikes were common in
cartwheel cells of Zhang and Oertel (1993a)
,
Golding and Oertel (1997)
, and in this study. Zhang and
Oertel also reported that many simple spikes had double undershoots,
which were absent in our cartwheel cells.
RESPONSES OF CARTWHEEL CELLS. Cartwheel cells responded weakly to acoustic stimuli without exception. BF thresholds were higher and the maximum BF rates were ~50% lower than those of most fusiform/pyramidal cells (Tables 2 and also 4). Usually only simple APs responded to acoustic stimuli. Complex APs appeared randomly and did not seem to be driven.
When depolarizing current pulses of low levels were injected, many cartwheel cells responded with simple spikes only. Depolarizing current pulses at high levels, however, triggered complex APs in all cases. The distortion in APs at high current levels is the main reason for the nonmonotonic RCL curves. Strong depolarizing current pulse stimuli also elicited complex APs from cartwheel cells in mouse slice preparations (Zhang and Oertel 1993aARE ALL COMPLEX-SPIKING, WEAK ACOUSTIC CELLS CARTWHEEL CELLS?
The four identified cartwheel cells were all complex-spiking and
responded weakly to acoustic stimuli. Of the seven other complex-spiking cells, which are reported in a separate paper (Ding and Voigt 1997), only one was a type III unit
(J61793-8-), with relatively low threshold and high driven
rate. The other six units had weak acoustic responses similar to those
of the identified cartwheel cells. For example, there were no
significant differences in resting potential, AP size, SpAc, thresholds
and driven rates to acoustic/electric stimuli, and input resistances, although on average the AP size is smaller, SpAc and current-driven rates (at 1.0 nA) are higher for the six complex-spiking units (compare
Table 4 with Table 6 of Ding and Voigt 1997
). These differences are probably due to `less healthy` membranes of some of
these six units, which could not be held long enough for a successful
marking. An increased discharge rate was common for many injured units.
Functional role of the DCN
Investigators have been speculating for a long time that the DCN
performs sophisticated information processing, partly because of the
complexity of its architecture and neural circuitry. For example,
Rhode and Kettner (1987) pointed out that DCN neurons are better suited for encoding spectral rather than temporal
information. Nelken and Young (1994)
suggested that DCN
neurons may be responsible for extracting the notch frequencies and
peaks of the pinna transfer functions. Young et al.
(1995)
also found that electric stimulation in the
somatosensory spinal nuclei can inhibit spontaneous activity of DCN
acoustic units, suggesting that DCN neurons also may be involved in
coordinating or regulating some audio-somatosensory functions, such as
pinna movement toward a sound source.
Although the results of this study do not directly resolve the
functional role of the DCN in the central auditory pathway, they have
demonstrated that morphologically distinct DCN cells can have different
responses to acoustic stimuli and thus may play different roles in
processing auditory information. The two distinct response patterns,
type III and type IV, have been shown to be associated with the
projection (fusiform/pyramidal and giant) neurons. Besides projecting
to the contralateral inferior colliculus, as fusiform/pyramidal cells
do, giant cells also project to the contralateral CN (Cant and
Gaston 1982). It is possible that giant cells extract specific
signal components needed for coordinated processing between the two
sides of CN, whereas the majority of fusiform/pyramidal cells send
other processed information about the acoustic environment to higher stages.
This study also has revealed the uniqueness of DCN cartwheel cells. The
weak acoustic responses of these cells is consistent with a role for
the DCN in processing multisensory information. Indeed, somatosensory
inputs to DCN granule cells have been reported (Wright and Ryugo
1996), and these have been stimulated electrically to active
the granule-cell associated circuitry of the DCN (Davis and
Young 1997
; Young et al. 1995
). Cartwheel cells
are inhibitory interneurons. Their complex APs provide a unique way to
inhibit their targets, including fusiform/pyramidal cells. The
processing of nonauditory information therefore may undergo a different
course than the processing of auditory information.
Because of the diversity of response types associated with fusiform/pyramidal cells and our failure, to date, to identify the neurons with either type II or classical type IV unit response properties, it is clear that additional in vivo experimentation is required.
![]() |
ACKNOWLEDGMENTS |
---|
Thanks are due to P. Patterson for help in preparing the histology and to P. Patterson and K. Hancock for help in producing the figures. Dr. Alice Berglund is thanked for assistance in identifying some of the neurons. K. Hancock also is thanked for comments on earlier drafts of this paper.
This work originally was seeded by grants to H. F. Voigt from the National Science Foundation (IBN-8420495) and the Deafness Research Foundation. Continued support was provided by Grants DC-01099 and DC-00310 from the National Institute on Deafness and Other Communication Disorders and by the College of Engineering at Boston University.
Present addresses: J. Ding, Guidant/CPI, 4100 Hamline Ave. North, MS E207, St. Paul, MN 55112; T. E. Benson, 5 Washington St., D5, Reading, MA 01867.
![]() |
FOOTNOTES |
---|
Address for reprint requests: H. F. Voigt, Dept. of Biomedical Engineering, Boston University, 44 Cummington St., Boston, MA 02215-2407.
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.
1 Before December 1994, 50-ms (acoustic) stimuli were used. In December 1994 the experimental system was updated and longer data records could be acquired. The stimulus duration and interstimulus interval were increased to 100 ms and 500 ms, respectively.
Received 6 November 1998; accepted in final form 7 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|