Department of Zoology, University of Texas at Austin, Austin, Texas 78712
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Klug, Achim, Eric E. Bauer, and George D. Pollak. Multiple Components of Ipsilaterally Evoked Inhibition in the Inferior Colliculus. J. Neurophysiol. 82: 593-610, 1999. The central nucleus of the inferior colliculus (ICc) receives a large number of convergent inputs that are both excitatory and inhibitory. Although excitatory inputs typically are evoked by stimulation of the contralateral ear, inhibitory inputs can be recruited by either ear. Here we evaluate ipsilaterally evoked inhibition in single ICc cells in awake Mexican free-tailed bats. The principal question we addressed concerns the degree to which ipsilateral inhibition at the ICc suppresses contralaterally evoked discharges and thus creates the excitatory-inhibitory (EI) properties of ICc neurons. To study ipsilaterally evoked inhibition, we iontophoretically applied excitatory neurotransmitters and visualized the ipsilateral inhibition as a gap in the carpet of background activity evoked by the transmitters. Ipsilateral inhibition was seen in 86% of ICc cells. The inhibition in most cells had both glycinergic and GABAergic components that could be blocked by the iontophoretic application of bicuculline and strychnine. In 80% of the cells that were inhibited, the ipsilateral inhibition and contralateral excitation were temporally coincident. In many of these cells, the ipsilateral inhibition suppressed contralateral discharges and thus generated the cell's EI property in the ICc. In other cells, the ipsilateral inhibition was coincident with the initial portion of the excitation, but the inhibition was only 2-4 ms in duration and suppressed only the first few contralaterally evoked discharges. The suppression was so slight that it often could not be detected as a decrease in the spike count generated by increasing ipsilateral intensities. Twenty percent of the cells that expressed inhibition, however, had inhibitory latencies that were longer than the excitatory latencies. In these neurons, the inhibition arrived too late to suppress most or any of the discharges. Finally, in the majority of cells, the ipsilateral inhibition persisted for tens of milliseconds beyond the duration of the signal that evoked it. Thus ipsilateral inhibition has multiple components and one or more of these components are typically evoked in ICc neurons by sound received at the ipsilateral ear.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A major focus of auditory research has been on how
acoustic information is encoded in the central nucleus of the inferior colliculus (ICc) (e.g., Aitkin 1986; Brugge
1992
; Irvine 1992
; Oliver and Huerta
1992
; Pollak and Park 1995
; Pollak et al.
1986
; Yin and Kuwada 1984
). The ICc is of
particular interest because it is a nexus in the ascending auditory
pathway that receives and integrates information from a large number of
lower auditory nuclei (Adams 1979
; Aitkin
1986
; Beyerl 1978
; Brunso-Bechtold et al.
1981
; Oliver and Huerta 1992
; Ross et al.
1988
; Roth et al. 1978
; Vater et al.
1992b
; Zook and Casseday 1982
) and
provides the principal ascending input to the medial geniculate body.
The projections that innervate the ICc are both excitatory and
inhibitory. The inhibitory innervation is both glycinergic and
GABAergic and is at least as prominent as the excitatory innervation of
the ICc (Fubara et al. 1996
; Gonzalez-Hernandez
et al. 1996
; Saint Marie et al. 1989
;
Shneiderman and Oliver 1989
; Shneiderman et al.
1988
; Winer et al. 1995
).
The projections to the ICc originate from lower centers that are driven
by the contra- or ipsilateral ears or by both ears. Thus many, if not
most, ICc neurons are involved in the processing of binaural stimuli
(Irvine et al. 1995; Klug et al. 1995
;
Park and Pollak 1993b
; Park et al.
1992
; Ross and Pollak 1989
; Semple and
Kitzes 1987
; Wenstrup et al. 1985
; Yin et
al. 1983
, 1984
). In almost all ICc cells, stimulation of the
contralateral ear evokes a mixture of excitation, which drives ICc
cells, and inhibition, which shapes the pattern of the discharge train
(Le Beau et al. 1996
; Park and Pollak
1993a
; Vater et al. 1992a
). In many ICc neurons,
sound presented only to the ipsilateral ear evokes no discharges but
when presented simultaneously with contralateral sound, suppresses the
contralaterally evoked discharges (Fuzessery et al.
1990
; Irvine and Gago 1990
; Irvine et al.
1995
; Semple and Aitkin 1979
; Semple and
Kitzes 1985
; Wenstrup et al. 1988a
). Cells with these properties are referred to as excitatory-inhibitory (EI) neurons and are of particular interest because their spike counts
vary with interaural intensity disparities (IIDs), the principal cue
animals use to localize high-frequency sounds. Thus the encoding of
IIDs by the population of EI cells in the ICc is thought to be an
important feature of the nervous system that allows animals to
associate a sound source with its location in space (Aitkin
1986
; Fuzessery and Pollak 1985
;
Fuzessery et al. 1990
; Pollak and Casseday
1989
; Wenstrup et al. 1988a
,b
).
The projections that impart binaural properties to ICc neurons,
including EI properties, are of two principal types. One type of
projection is from a lower nucleus that is itself binaural, and thus
the binaural property of the target cell in the ICc actually is created
in that lower nucleus and imposed on the ICc cell through an excitatory
projection. An example is the crossed excitatory projection from the
lateral superior olive (LSO) to the ICc. The LSO is the initial site of
binaural convergence that produces EI neurons (Caird and Klinke
1983; Cant and Casseday 1986
;
Harnischfeger et al. 1985
; Moore and Caspary
1983
) and many ICc cells apparently derive their binaural EI
property through this projection (Klug et al. 1995
;
Park and Pollak 1993b
, 1994
; Shneiderman and
Henkel 1987
; Vater et al. 1992a
). The second
type of projection is from at least two lower nuclei, where one nucleus
is driven only by stimulation of one ear and the other nucleus is
driven only by the opposite ear. The projections from the two lower
nuclei converge on an ICc cell to create the binaural property in the
ICc itself. An example is the convergence of excitatory projections
from a lower nucleus (e.g., cochlear nucleus) driven only by the ear contralateral to the ICc and a GABAergic inhibitory projection from the
dorsal nucleus of the lateral lemniscus (DNLL) that is driven by
stimulation of the ear ipsilateral to the ICc (Faingold et al.
1993
; Kidd and Kelly 1996
; Klug et al.
1995
; Li and Kelly 1992
; Park and Pollak
1993b
, 1994
; Vater et al. 1992a
; Yang and Pollak 1994a
,b
). The convergence of these projections then
creates an EI cell in the ICc the binaural properties of which are
virtually indistinguishable from the EI properties that are created in
the LSO and imposed on the ICc cell.
Intracellular, pharmacological, and reversible lesion studies provide
proof that EI neurons in the ICc actually are formed by the two types
of projections described in the preceding paragraph. Intracellular
studies using sharp electrodes or whole cell patch-clamp electrodes
showed that some EI cells in the ICc receive inhibitory innervation
evoked by the ipsilateral ear (Covey et al. 1996; Kuwada et al. 1997
; Nelson and Erulkar
1963
; Pedemonte et al. 1997
), whereas
pharmacological studies showed that many, but not all, EI cells in the
ICc could be transformed into monaural cells when inhibition was
blocked by antagonists specific for GABAergic or glycinergic receptors
(Faingold et al. 1989
; Klug et al. 1995
; Park and Pollak 1993b
, 1994
; Vater et al.
1992a
). Further supporting the role of ipsilateral inhibition
in the formation of EI properties are the results from studies in which
the binaural properties of ICc neurons were transformed when the
GABAergic input from the DNLL was inactivated reversibly
(Faingold et al. 1993
; Kidd and Kelly
1996
; Li and Kelly 1992
).
The studies cited in the preceding text show that ipsilaterally evoked inhibition at the ICc is not uncommon and is functionally important. Besides these general features, however, little is known about the specific features of ipsilaterally evoked inhibition; how its threshold, latency, and duration compares with the excitation evoked by stimulation of the contralateral ear in the same cell. These are, we believe, important considerations because a more detailed evaluation of the patterns of excitation and inhibition evoked by each ear will provide a better understanding of how the diversity of documented ICc response properties are created and thus provide new insights into the overall strategies that are used by the auditory system for processing acoustic information.
It was for these reasons that we conducted detailed studies of
responses evoked by stimulation of the contralateral and ipsilateral ears in the ICc of Mexican free-tailed bats. We chose this bat as a
subject because it uses a variety of acoustic signals for both
echolocation and social communication and has a relatively hypertrophied auditory system, making it well suited for studies of the
brain's processing of acoustic cues. Moreover, several laboratories
have used Mexican free-tailed bats in behavioral studies
(Balcombe 1990; Balcombe and McCracken
1992
; Gelfand and McCracken 1986
; Schmidt
and Thaller 1994
; Simmons et al. 1978
, 1979
) as
well as anatomic (Grothe et al. 1994
) and
neurophysiological studies (Grothe et al. 1997
;
Oswald et al. 1999
; Park 1998
;
Park et al. 1996
, 1998
; Pollak et al. 1977a
,b
,
1978
) of their brain stem auditory nuclei.
In another report, we comment on features of excitation and inhibition evoked only by stimulation of the contralateral ear in ICc cells of the Mexican free-tailed bat (unpublished results). Here we focus on features of inhibition evoked by stimulation of the ipsilateral ear. We show that stimulation of the ipsilateral ear evokes a complex set of inhibitions in the vast majority of ICc cells that vary in latency, duration, or both features. Furthermore we show that some forms of inhibition act to create EI properties in the ICc, whereas the latencies and durations of other inhibitions are unsuited for the suppression of contralaterally evoked discharges and have different functional consequences.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surgical procedures
Fifteen Mexican free-tailed bats, Tadarida brasiliensis mexicana, were used in this study. Before surgery each animal was anesthetized with methoxyflurane inhalation (Metofane, Pitman-Moore). The hair on the head was removed with a depilatory, and the head was secured in a head holder with a bite bar. The muscles and skin overlying the skull were reflected, and lidocaine (Elkins-Sinn) was applied topically to all open wounds. The surface of the skull was cleared of tissue, and a foundation layer of cynoacrylate and small glass beads was placed on the surface. A small hole then was drilled around the center portion of the inferior colliculus using the landmarks visible through the skull for orientation.
The bat was transferred to a heated recording chamber, where it was
placed in a restraining cushion constructed of foam molded to the
animal's body. The restraining cushion was attached to a platform
mounted on a custom made stereotaxic instrument (Schuller et al.
1986). A small metal rod was cemented to the foundation layer
on the skull and then attached to a bar mounted on the stereotaxic instrument to ensure a uniform positioning of the head. A ground electrode was placed between the reflected muscle and the skin. Recordings were begun after the bats recovered from the anesthetic. The
bats typically laid quietly in the restraining cushion and showed no
signs of pain or discomfort. Supplementary doses of the neuroleptic
Vetamine (Mallinckrodt Veterinary) were given if the bat appeared to be
in discomfort.
Using visual landmarks viewed with an operating microscope, the electrode then was positioned on the surface of the brain on top of the inferior colliculus. Subsequently, the electrode was advanced from outside of the experimental chamber with a piezoelectric microdrive (Burleigh 712 IW). Neurons were sampled at depths between 400 and 1,600 µm from the surface of the colliculus and were in the central nucleus of the inferior colliculus. All experimental procedures were in accordance with a protocol approved by the University of Texas Institutional Animal Care Committee.
Electrodes
In all experiments piggyback multibarrel micropipettes were used
for recordings and iontophoresis of drugs (Havey and Caspary 1980). Single-barrel micropipettes were pulled to a tip
diameter <1 µm and blunted under microscope observation so that the
tip diameter was between 1 and 2 µm. A multibarrel electrode was
pulled from a five-barrel blank (H configuration, A-M Systems), and the tip was blunted so that the tip diameter of the multibarrel array was
15-20 µm. The single-barrel pipette was attached to the five-barrel pipette under microscopic observation and glued with cyanoacrylate so
that the tip of the single-barrel pipette protruded ~10-15 µm from
the broken tip of the five-barrel pipette. The single-barrel micropipette was used for recording and filled with buffered 1 M NaCl
and 2% Fast Green (pH 7.4). Electrode impedances ranged from 5 to 15 M
. Fast Green was used to enhance the visibility of the electrode
for placement in the small hole made in the skull. One barrel of the
five-barrel pipette was the balancing (sum channel) barrel, which also
was filled with buffered 1 M NaCl and 2% Fast Green (pH 7.4). The
remaining four ejection barrels were filled with different drugs as
follows: GABA (500 mM in dH2O, pH 3.5-4.0, Sigma),
bicuculline methiodide (10 mM in 0.165 M NaCl, pH 3.0, Sigma),
strychnine hydrochloride (10 mM in 0.165 M NaCl, pH 3.0, Sigma), and a
cocktail of glutamate (glu) and aspartate (asp) (each 500 mM, pH 8.0, Sigma).
The drug and balancing barrels were connected via silver-silver chloride wires to a six-channel microiontophoresis constant current generator (Medical Systems Neurophore BH-2) that was used to generate and monitor ejection and retention currents. The sum channel that connected to the balancing barrel was employed to balance current in the drug barrels and reduce current effects. The recording barrel was connected by a silver-silver chloride wire to a Dagan AC amplifier (model 2400) for analysis of single-unit activity.
Acoustic stimuli and data acquisition
Sine waves from a Wavetek function generator (model 136) were
shaped into tone bursts with an analog switch (Restek Model 15). The
tone bursts were 20 ms in duration and had 0.5-ms rise-fall times.
Stimuli to both ears were delayed by 20 ms from the start of data
acquisition, using a Binaural Pulse Delay (Restek Model 110). Stimuli
were presented at a rate of four per second; the rate was controlled by
a Restek Model 45 real-time clock, which also timed spike events for
the peristimulus time (PST) histograms. Tone-burst frequency was
monitored by a frequency counter. A 24-bit digital interface NuBus card
(National Instruments DIO-24) and a digital distributor (Restek model
99) connected a Power Macintosh 8500/120 computer to the Restek
equipment and a two-channel digital attenuator (Wilsonics, model PATT).
The output of each independently controlled channel of the attenuator
was sent to a 1/4-in Brüel and Kjaer (B&K) microphone
biased with 200 V DC and driven as speakers. At the start of each
experiment, speakers were inserted into the funnels formed by the
bat's pinnae and positioned adjacent to the external auditory meatus.
The pinnae were folded onto the housing of the microphones and wrapped
with Scotch tape. The acoustic isolation with this arrangement was 40 dB.
Only well-isolated spikes with a high signal-to-noise ratio were studied. Action potentials were fed to a window discriminator (Frederick Haer and Co.) and then to the real-time clock. The Macintosh 8500 computer read the spike data from the real-time clock and generated PST histograms in real time. All stimuli were presented in a pseudorandom order.
When glu/asp was not applied, PST histograms were generated from the spikes evoked by 20 presentations of each stimulus. A larger number of stimulus presentations were used to generate the PST histograms obtained while background activity was evoked by glu/asp. Because the PST histograms were monitored in real time, we observed the build-up of both tone-evoked and background activity and could visualize whether or not an inhibitory gap was generated by repeated presentations of the tone burst. Tone bursts were presented repeatedly until a clear gap was evident in the background activity or when it was apparent that there was no gap in the background activity. Thus a variable number of stimuli was presented during the application of glu/asp to generate each PST histogram, where the number of stimulus presentations was always larger than the 20 tone-burst presentations that were used to generate the PST histograms in the control or predrug condition.
Iontophoresis of drugs and measurement of inhibition
When drugs were not being ejected, a retention current of 15-30 nA of appropriate polarity was applied to the drug barrel to prevent leakage of drugs. When drugs were ejected, the current polarity was reversed for that drug barrel. For each neuron, GABA first was applied iontophoretically, and the ejection current was increased progressively until the neuron was inhibited completely. The purpose of applying GABA was to ensure that drug ejection could influence the discharges of that neuron. If GABA could not completely inhibit the neuron, or if current effects could be observed, recordings were stopped immediately and the electrode was replaced.
Inhibition evoked by stimulation of the ipsilateral ear was visualized as a stimulus locked suppression of background activity. Because ICc cells had little or no spontaneous activity, we evoked background activity by iontophoresing the excitatory transmitters glu and asp onto the cell from which we were recording. Glu and asp were applied at ejection currents of 5-10 nA (electrode negative) at first, and ejection currents were adjusted until the desired background level of activity was achieved. Although high rates of 20-40 spikes/s could be evoked in a few cells, only low rates of 4-10 spikes/s could be evoked in the majority of cells regardless of the current used to eject the excitatory transmitters. We point out that due to the low rates of the glu/asp evoked background activity, the histograms often had short (2-4 ms), random gaps. To ensure that the gap we measured was indeed a stimulus evoked inhibition and not a random gap in the background activity, tone bursts at several intensities were presented. We noted the lowest intensity at which the gap was evoked and defined this as the "threshold for inhibition." The gap then had to be evoked at higher intensities in the same temporal slot as it was at the threshold intensity. In addition, we often generated histograms at a particular ipsilateral intensity more than once, and in all cases the gap appeared in the same time slot and with approximately the same duration in each histogram. Latency was defined as the time between the beginning of the stimulus and the initial appearance of the gap in background activity. Because of the presence of random gaps, the measurements of latency were approximate and have an error of about ±2 ms.
It should be noted that the analysis of gap durations does not quantitatively assess inhibitory strength. Thus the presence of a gap may indicate an inhibition just strong enough to suppress completely the glu/asp discharges or it may indicate an inhibition that was considerably stronger. Moreover it did not identify periods during which the strength of the inhibition may have been sufficient to reduce the background rate but was not strong enough to completely suppress discharges. Unfortunately, there is no satisfactory way of evaluating changes in background spike rate quantitatively because background rates in most cells were not only low but also tended to be highly variable throughout each histogram. With these constraints in mind, we feel our evaluations are justified because the gaps in background activity were clearly stimulus locked, were repeatable and robust, and represent a conservative estimate, rather than an overestimate, of both the threshold and duration of the evoked inhibition.
The roles of GABAergic and glycinergic innervation in producing the ipsilateral inhibition were evaluated by the application of bicuculline, an antagonist specific for GABAA receptors, or strychnine, an antagonist of glycine receptors. The ejection current used for each neuron was determined by initially ejecting the drug at a low current (10 nA, electrode positive) while obtaining rate-level functions by presenting tone bursts of increasing intensity to the contralateral ear. During the application of each drug, rate-level functions were taken repeatedly until the shape of the function and maximum spike count stabilized. The ejection current then was increased, and the procedure was repeated until the maximum spike count no longer increased. Final currents for bicuculline and for strychnine ranged from 10 to 80 nA. The antagonists then were applied to the cell concurrently with glu/asp while tone bursts were presented to the ipsilateral ear.
At the end of the experimental protocol, all drugs were turned off, and the cell was allowed to recover. During recovery, rate-intensity functions were taken every few minutes to assess the level of recovery. Cells were considered completely recovered when the spike counts and spike patterns were similar with those observed in the predrug condition.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General response properties
The effects of both binaural and ipsilateral monaural stimulation were studied in 97 ICc neurons in the Mexican free-tailed bats. Acoustic signals, whether presented to the ipsilateral or contralateral ears, were 20-ms tone bursts set at each neuron's best excitatory frequency (BEF). BEFs were determined with contralateral stimulation and ranged from 17 to 50 kHz. Contralateral stimulation always evoked discharges and, in the majority of cells, the excitation was followed by an inhibition (Fig. 1B). In 56% of the units (n = 54), contralateral signals evoked only one or several spikes at the onset of the tone burst (phasic pattern), whereas 44% (n = 43) discharged throughout the duration of the tone burst (sustained pattern). The features of inhibition evoked by contralateral stimulation in these neurons will be described in a future report (unpublished results). Signals presented monaurally to the ipsilateral ear evoked only inhibition (Fig. 1C) or no response at all.
|
The binaural property of each cell initially was determined from its
IID function. IID functions were generated by driving the cells with
BEF tone bursts presented to the contralateral ear at an intensity that
was fixed at 10-20 dB above the cell's threshold (the lowest
intensity that evoked stimulus locked discharges) while varying the
sound intensity of the same frequency at the ipsilateral ear, from 10 dB below to 20-30 dB above the contralateral intensity. The vast
majority (92%, 89/97) of cells were EI, in that ipsilateral
stimulation suppressed contralaterally evoked spike counts by 40%.
However, the degree of spike suppression differed among the population.
Cell 1 in Fig. 2A,
for example, was a strongly suppressed EI cell, as sound to the
ipsilateral ear completely suppressed all contralaterally evoked
spikes. Cell 2 is an example of a weakly suppressed EI cell,
whereas Cell 3 appeared to be monaural because ipsilateral
stimulation had no effect on the contralaterally evoked spike count. In
74% of the neurons, ipsilateral stimulation suppressed contralaterally
evoked spike counts by
80%, and the discharges in 55% of the cells
could be suppressed completely (Fig. 2B). In the following
sections, we first describe features of ipsilaterally evoked inhibition in the ICc and then evaluate the degree to which that inhibition was
responsible for the spike suppression seen in each cell's IID
function.
|
Ipsilaterally evoked inhibition was evaluated by generating background activity
In 86% (83/97) of ICc cells, ipsilateral stimulation evoked inhibition that was seen as a prominent gap in the background activity evoked by glu/asp. Examples are shown in Figs. 1 and 3. In the remaining 14 cells (14%), no inhibition was evoked by ipsilateral signals at any of the intensities that we presented. Of interest is that some cells that did not exhibit ipsilateral inhibition were, nevertheless, strongly suppressed EI cells (Fig. 11). The features of these cells are consistent with the idea that the ipsilaterally evoked spike suppression was due to an inhibition in a lower binaural center, most likely the LSO, and the resulting binaural properties then were relayed to the ICc cell via an excitatory projection.
|
Features of the ipsilaterally evoked inhibition
Three features of ipsilaterally evoked inhibition were evaluated in greater detail: the threshold of inhibition and its relation to the excitatory threshold, the dependence of inhibitory duration on intensity, and the latency of the inhibition.
THRESHOLD OF INHIBITION.
The threshold of the inhibition was defined as the lowest ipsilateral
sound intensity that produced an obvious gap in the background of
discharges evoked by glu/asp (Fig. 3, 20 dB SPL). Among the 83 cells
that displayed ipsilaterally evoked inhibition, thresholds ranged from
10 dB SPL to +50 dB SPL, although the thresholds of most cells (77%)
were between 10 and 30 dB SPL (Fig. 4A).
|
DURATION OF INHIBITION. The duration of ipsilateral inhibition was variable in each cell and depended on stimulus intensity. Representative examples are shown in Fig. 5. In 69% of the cells (57/83), the duration of inhibition increased monotonically with intensity. In 30 of these cells (36%), the inhibitory duration increased progressively with intensity and the maximum duration was evoked at the highest sound intensity tested (e.g., Figs. 3 and 5A). In 27 other cells (32%), the duration increased with intensity and reached a maximum value, which then plateaued with further intensity increments (Fig. 5B). In 26 cells (31%), however, the duration of the inhibition was nonmonotonically related to ipsilateral sound intensity (e.g., Figs. 14C and 5, C and D). In these cells, inhibitory duration at first increased with intensity, reached a maximum at some intermediate intensity, and then decreased in duration at even higher intensities.
|
|
LATENCY OF INHIBITION. We measured latencies, defined as the time between the beginning of the stimulus and the initial appearance of the gap in background activity, during a 30- to 50-dB intensity range in 83 cells. In most cells, latency decreased with increasing ipsilateral intensity and reached a minimum at the highest intensity presented. However, in some cells the relationship between latency and stimulus intensity was nonmonotonic. In those cells, minimum latency, the shortest latency evoked over this range of intensities, was achieved at an intensity lower than the most intense presented. The distribution of minimum latencies in our sample is shown in Fig. 7. The average minimum latency for the 83 cells was 9.3 ms. The minimum latencies ranged from ~5 to 29 ms, although the majority of cells (76%) had minimum latencies <12 ms. As can be seen in Fig. 7, there was no significant difference between the minimum latencies of phasic and sustained cells (t-test, P > 0.05).
|
Blocking inhibitory transmitters reduced or eliminated ipsilaterally evoked inhibition
Blocking GABAergic and glycinergic innervation eliminated most or all of the ipsilaterally evoked inhibition that we observed. We evaluated the contributions of the two inhibitory transmitters by iontophoresing either bicuculline or strychnine alone or both bicuculline and strychnine simultaneously onto the cell from which we were recording. When only GABAergic inhibition was blocked by bicuculline or glycinergic inhibition blocked by strychnine, the duration of ipsilateral inhibition was reduced in each of the 24 cells tested. Strychnine alone was tested in four cells and reduced the inhibitory period in each cell but never eliminated inhibition. Bicuculline eliminated inhibition in 2 of 20 neurons, as indicated by the presence of glu/asp-evoked discharges in the time slot in which glu/asp discharges were suppressed completely in the predrug condition. An example is shown in Fig. 8A. In the remaining 18 neurons, bicuculline reduced the duration of the inhibition but did not eliminate it (Figs. 8B and 9, A and B).
|
|
In some cells initially tested with bicuculline, we also evaluated whether the reduction in the inhibitory period could be reduced further by the addition of strychnine. Thus in 11 cells in which bicuculline did not completely eliminate inhibition, we first applied bicuculline and then applied strychnine together with bicuculline. In 4 of 11 cells, the additional application of strychnine eliminated the inhibition that was not removed by bicuculline (Fig. 9A). In seven other cells, strychnine further reduced the ipsilateral inhibition but did not eliminate it (Fig. 9B). These results show that the ipsilateral inhibition in many ICc neurons can have multiple components that are glycinergic and/or GABAergic.
Influence of the ipsilaterally evoked inhibition on IID functions
We turn next to the principal question addressed by these studies: the degree to which ipsilateral inhibition at the ICc produced the suppression of contralaterally evoked discharges that was reflected in the neuron's IID function. This question is of interest because contralaterally evoked discharges could be inhibited by ipsilateral stimulation either in the ICc or in a lower nucleus, as mentioned previously. If the spike suppression occurs at the ICc, then the cell should not only exhibit ipsilateral inhibition but the inhibition at the ICc also should be temporally coincident with the contralaterally evoked excitation. It was for this reason that we compared the latencies of contralaterally evoked excitation and ipsilaterally evoked inhibition in individual ICc cells. The excitatory latency was measured from the first discharge evoked by an intensity 10-20 dB above the cell's excitatory threshold, the intensity used to generate the cell's IID function. Each cell's excitatory latency at this intensity then was compared with the minimum latency of ipsilateral inhibition.
The comparison for 83 cells is shown in Fig.
10, where positive latencies indicate
that the inhibition had a shorter latency than the excitation while
negative latencies indicate that inhibition had a longer latency than
the excitation. In ~80% (66/83) of the cells, the latency of
inhibition was equal to or shorter than the excitatory latency (Fig.
10). Presumably, the inhibition and excitation were temporally
coincident in these cells, and therefore the inhibition at the ICc
could have suppressed the excitatory responses. In the other 20%
(17/83) of the cells, the inhibitory latency was 3 ms longer than the
excitatory latency. The significance of latency mismatches, where the
inhibitory is longer than the excitatory latency, is that in cells with
small latency mismatches, the ipsilateral inhibition could not have
suppressed all contralaterally evoked discharges, whereas the
ipsilateral inhibition in cells with substantial latency mismatches
could not have suppressed any of the discharges. In the following
sections, we consider in greater detail how the latencies and
thresholds of ipsilateral inhibition and contralateral excitation
influenced or, in some cases, failed to influence the cell's IID
function.
|
EI cells in which the binaural properties are formed in the inferior colliculus
The similar latencies of ipsilateral inhibition and contralateral excitation in most ICc cells suggests that in those cells the suppression of contralateral discharges occurred at the ICc. Here we show that not only were the latencies of excitation and inhibition coincident, but also that the threshold of inhibition, determined with monaural, ipsilateral stimulation, corresponded to the lowest ipsilateral intensity that suppressed contralaterally evoked discharges in the IID functions and that blocking inhibition at the ICc rescued discharges that had been suppressed previously by ipsilateral stimulation.
Before turning to cells that were inhibited at the ICc by ipsilateral stimulation, we show, for purposes of comparison, data from a cell in which there was no ipsilateral inhibition at the ICc, but the contralaterally evoked discharges of which were suppressed strongly by ipsilateral stimulation (Fig. 11, A and B). As mentioned previously, these features suggest that the ipsilateral inhibition of discharges occurred in a lower nucleus, most likely the LSO, and the binaural response properties then were imposed on the ICc cell through an excitatory projection from the lower nucleus. This interpretation is strengthened further by the results obtained with bicuculline and strychnine (Fig. 11, C and D). When glycinergic and GABAergic inhibition were blocked at the ICc, the response magnitude of the cell increased but the spike suppression caused by ipsilateral simulation was not eliminated (Fig. 11C), presumably because the spike suppression occurred in a lower nucleus.
|
This failure to influence ipsilateral spike suppression with bicuculline and strychnine in EI neurons that had no ipsilateral inhibition is in marked contrast to the effects of blocking inhibition in EI cells that displayed pronounced ipsilateral inhibition that was coincident with the excitation. When binaural stimuli were presented in these latter cells, the drugs eliminated much of ipsilateral suppression of contralaterally evoked spikes, thereby supporting the hypothesis that the suppression of contralateral discharges in these cells was caused by ipsilateral inhibition at the ICc.
An example is shown in Fig. 12. Figure 12A shows that ipsilateral signals of 10 dB SPL caused a partial suppression of contralateral discharges. Increasing the ipsilateral intensity to 20 dB SPL caused a strong suppression and discharges were completely suppressed by 30 dB SPL ipsilateral signals. Figure 12B shows that the features of the ipsilateral inhibition were appropriate for suppressing the contralateral discharges. Note first that the latencies of the contralaterally evoked excitation and ipsilaterally evoked inhibition (Fig. 12, A and B, arrows and dotted line) were temporally coincident. Second, Fig. 12B shows that the threshold of the ipsilateral inhibition, as revealed by the appearance of a gap in background activity, was ~10 dB SPL, the same ipsilateral intensity that initially caused discharge suppression with binaural stimulation in Fig. 12A. Third, Fig. 12D shows that the ipsilateral inhibition was eliminated by the application of bicuculline and strychnine, as indicated by the failure of ipsilateral signals presented monaurally to evoke a gap in the background discharges. Finally, Fig. 12C shows that the contralateral discharges that were previously suppressed strongly or completely by ipsilateral stimulation were largely rescued after inhibition was blocked by bicuculline and strychnine. Thus this cell's IID sensitivity seems to be formed, at least in a large part, by ipsilaterally evoked inhibition at the ICc.
|
A feature in Fig. 12C that might seem curious is that the higher ipsilateral intensities suppressed the later discharges evoked by the contralateral signals even though ipsilateral inhibition at the ICc seems to have been eliminated completely by bicuculline and strychnine as shown in Fig. 12D. Our interpretation is that ipsilateral stimulation evoked an inhibition in a lower nucleus that suppressed the later but not the earlier discharges.
Ipsilateral inhibition was not always discernible from gaps in background activity
In recent studies, Park et al. (Oswald et al. 1999;
Park et al. 1998
) report on a subtle form of
ipsilaterally evoked inhibition at the ICc that affects only the
initial 2-4 ms of the contralaterally evoked discharge train. This
brief inhibition, however, was not apparent in our evaluations of
stimulus locked gaps in the glu/asp evoked background activity. The
reason is there were often gaps of a few milliseconds that were
scattered throughout the background discharge pattern. Thus when a
small gap that preceded the contralaterally evoked discharge train
appeared in our records, we were uncertain as to whether the gap was
actually an inhibition or simply a random gap. Furthermore the
influence of this brief inhibition was often not apparent in IID
functions, especially in cells with sustained discharge patterns. With
20-ms tone bursts, for example, the sustained discharge evoked by
contralateral stimulation generated a high spike count. When
ipsilateral signals were introduced, the brief inhibition at the ICc
only suppressed a few spikes at the very beginning of the discharge
train. But whatever change in spike count resulted from the inhibition,
it was less than the random variations in spike counts that normally
occur when more than one histogram, generated by the same stimulus, is
obtained. Thus the minor spike suppression caused by this brief
inhibition could not be discerned from the histograms obtained with
increasing ipsilateral intensities (Fig.
13A, left).
|
The reality of the inhibition, however, became apparent when the time
scale of the PST histograms was expanded, as shown in Fig. 13A,
right. Moreover, blocking GABAergic and glycinergic inhibition eliminated the brief inhibition (Fig. 13B). These features
are consistent with those reported by Park et al. (Oswald et al.
1999; Park et al. 1998
) and confirm that the
brief inhibition occurs at the ICc and not in a lower nucleus.
We point out that this inhibition was not included in the previous sections, which considered only the inhibition identified from gaps in background activity. However, when we evaluated the expanded histograms, we found that many (28/97, 29%) EI cells had this early, brief inhibition. Additionally, some of these neurons also had a longer lasting inhibition that was apparent from a gap in background activity. The cell in Fig. 14, for example, had a pauser-like discharge pattern to contralateral tone bursts. The brief ipsilaterally evoked inhibition suppressed a few spikes in the early portion of the discharge train (Fig. 14B), whereas a longer-latency ipsilaterally evoked inhibition, that was obvious as a gap in background activity, suppressed spikes in the later portion of the discharge train (Fig. 14C). Cells of this type further illustrate that ICc cells can be influenced by multiple forms of ipsilaterally evoked inhibition, each of which is evoked with a different duration and latency.
|
Ipsilateral inhibition did not suppress contralaterally evoked discharges in some cells
The neurons considered in the preceding section had ipsilaterally evoked inhibitory latencies that were coincident with latencies of the contralaterally evoked excitation. However, the inhibitory and excitatory latencies were not coincident in all ICc cells. Indeed, one of the surprising results of this study is that in some cells, the latencies of ipsilateral inhibition were so long that the inhibition began after the contralaterally evoked discharge trains had largely ended or ended completely. Latency mismatches between excitation and inhibition were seen in both strongly inhibited EI cells and in cells that were weakly inhibited or not inhibited at all by stimulation of the ipsilateral ear.
A strongly inhibited EI cell with a large latency mismatch is shown in Fig. 15. The PST histograms in the top panel show that discharges evoked by contralateral signals were suppressed completely by a 30-dB SPL ipsilateral signal. The bottom panel shows that ipsilateral signals of 20-30 dB SPL produced a gap in the glu/asp-evoked background and thus the ipsilateral signal evoked inhibition in the ICc cell. The inhibition's latency was so long that the inhibition was initiated only after the excitatory response had ended. Consequently, the ipsilateral inhibition could not have suppressed the contralaterally evoked discharges at the ICc; rather the suppression of the contralaterally evoked discharge must have occurred downstream, presumably in the LSO.
|
An example of a weakly suppressed cell that received ipsilateral
inhibition with a long latency is shown in Fig.
16. Notice that the spike counts
elicited by a 10-dB SPL contralateral signal were hardly suppressed by
ipsilateral intensities of 10-20 dB SPL and only moderately suppressed
at 30 dB SPL. Nevertheless, ipsilateral signals presented by
themselves at 10-30 dB SPL, evoked prominent and long-lasting
inhibitions (Fig. 16B). The latencies of the inhibition,
however, were longer than the latency of excitation and thus the
inhibition only overlapped with a few of the later spikes of the
contralaterally evoked discharge train. Although the ipsilateral
inhibition may have suppressed a few of the later contralaterally
evoked discharges, its long latency prevented it from suppressing the
major portion of the discharge train. Thus a prominent inhibition was
evoked by stimulation of the ipsilateral ear, but that inhibition did
not create the binaural property of this EI cell. Of the eight weakly
inhibited cells the contralateral spike counts of which were suppressed
by 40%, five cells had ipsilaterally evoked inhibition with
latencies that were longer than the excitatory latencies.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study has shown that stimulation of the ipsilateral ear evokes an inhibition in the vast majority (86%) of ICc cells. The inhibition is often complex and can have multiple components that are glycinergic and/or GABAergic. Each component of the ipsilateral inhibition can vary markedly in latency, duration, and threshold. Additionally, inhibition has a persistence in most cells and continues to exert its influence for many milliseconds after the stimulus has ended. In the following text, we first discuss how these findings compare with those from previous studies and then turn to some of the functional implications of the various inhibitory components evoked in the ICc by stimulation of the ipsilateral ear.
Prevalence of ipsilaterally evoked inhibition
Although ipsilaterally evoked inhibition at the ICc has been
reported in studies from this laboratory (Klug et al.
1995; Park and Pollak 1993b
) and other
laboratories (Covey et al. 1996
; Faingold et al.
1989
; Kidd and Kelly 1996
; Kuwada et al.
1997
; Li and Kelly 1992
; Vater et al.
1992a
), we nevertheless were surprised to find that 86% of ICc
cells were inhibited by stimulation of the ipsilateral ear. In our
previous studies of the mustache bat ICc, we found that blocking
GABAergic and glycinergic inhibition reduced or eliminated ipsilateral
inhibition in ~50% of the EI cells (Klug et al.
1995
), and it was our expectation that about the same
percentage of cells would exhibit ipsilateral inhibition in the ICc of
Mexican free-tailed bats. Although species differences might account
for some of the disparity of the results among these studies,
differences in methodology seem more relevant. In previous studies
(Klug et al. 1995
), we focused on IID functions, i.e.,
how the spike counts evoked by a contralateral signal were suppressed
as the ipsilateral intensity increased, and then evaluated whether or
not the cell's IID function changed when inhibition was blocked. Such
evaluations only reveal the ipsilateral inhibition that is coincident
with contralaterally evoked discharges and thereby can suppress
contralaterally evoked discharges. Those studies almost surely missed
or underestimated ipsilateral inhibition in onset cells whose latencies
of ipsilateral inhibition were longer than latencies of excitation
(e.g., Fig. 15), and in cells that received only a brief, short latency
inhibition of the sort shown in Fig. 13. About 20% of the cells in our
sample of ICc cells in the Mexican free-tailed bat had such mismatched latencies of excitation and inhibition, and thus had we evaluated ipsilateral inhibition in the mustache bat as we did in the present study, the percentages of ICc cells in the mustache bat that are inhibited by ipsilateral stimulation probably would have been substantially higher and closer to the value that we found in the
present study.
The high percentage of ipsilaterally inhibited cells that we observed
in this study, however, is in close agreement with studies of the rat
ICc reported by Kelly and his colleagues (Kidd and Kelly
1996; Li and Kelly 1992
). They recorded
interaural intensity and time disparity functions from ICc cells
before, during, and after reversibly inactivating the DNLL, the
principal source of ipsilaterally evoked GABAergic input to the ICc.
They found that inactivation of the DNLL changed the binaural functions
in the vast majority of ICc cells. Thus most ICc cells in the rat are inhibited by stimulation of the ipsilateral ear, and in this regard, the studies of the rat's ICc are concordant with the results of this study.
Ipsilateral inhibition has multiple roles in the formation of binaural properties
In addition to the inhibition that is temporally coincident with excitation, and thereby creates EI properties, there are at least three types of more subtle and complex influences of inhibition that are evoked in the ICc by ipsilateral stimulation. The subtleties and complexities are a consequence of the timing and duration of the ipsilateral inhibition and are not always expressed as a marked change in the IID function when inhibition is blocked.
The first type was discovered by Park and his colleagues (Oswald
et al. 1999; Park et al. 1998
) and is the very
brief ipsilaterally evoked inhibition that affects only the initial
2-4 ms of the contralaterally evoked discharge train (e.g., Fig. 13
and 14). The subtlety is that its influence on IID functions is often
not apparent, especially in cells with sustained discharge patterns.
With 20-ms tone bursts, for example, the sustained discharge evoked by
contralateral stimulation generates a high spike count. When
ipsilateral signals are introduced, this brief inhibition at the ICc
only suppresses a few spikes at the very beginning of the discharge
train and results in only a minor reduction or even no apparent
reduction in spike count. The IID functions of these cells would
suggest that they are very weakly inhibited cells or even nonsuppressed cells. Because "long" tones of this type also were used in many previous studies (Klug et al. 1995
; Park and
Pollak 1993b
), the brief ipsilateral inhibition almost surely
was overlooked in many cells. The full influence of the transient
inhibition becomes apparent when very brief stimuli, which evoke only a
few discharges, are used as stimuli (Oswald et al. 1999
;
Park et al. 1998
). When these signals are employed, the
brief ipsilateral inhibition is sufficiently long to completely
suppress the brief discharge train at appropriate IIDs. Thus these
cells express very weakly suppressed EI properties or even
"monaural" properties with "long" stimuli, whereas the same
cells express strongly suppressed EI properties when stimulated with
transient stimuli. Park and his colleagues suggest (Oswald et
al. 1999
; Park et al. 1998
) that such cells encode the location of transient signals, such as the brief FM signals
bats emit for echolocation, through their discharge magnitude, whereas
the location of longer signals, such as communication signals, could
not be encoded by discharge magnitude. Rather the brief ipsilateral
inhibition could change the latency of those IC cells, whereas the
latency change could be a function of IID. Latency may be another way
of coding for location, or those cells may code for other features of
the longer signals.
The second subtle type of ipsilateral inhibition is the inhibition that has a latency longer than the latency of the excitation. In some of these cells, the ipsilateral inhibition begins after the discharge train is well under way (e.g., Fig. 14), whereas in other cells it begins after the end of the discharge train (e.g., Fig. 15 and 16). Moreover, these cells express weakly and strongly suppressed EI properties. In both the weakly and strongly suppressed cells, ipsilateral signals generate a long latency inhibition at the ICc, but that inhibition does not participate in the suppression of contralaterally evoked discharges. Rather the suppression of contralaterally evoked spikes by ipsilateral stimulation must occur in a lower center. The functional significance of the long latency inhibition at the ICc is unclear, but it is important for something other than the coding of IIDs and thus apparently is not relevant for sound localization.
The third type of ipsilateral inhibition the function of which is
more complex than simply suppressing contralaterally evoked discharges
is the persistent inhibition, the inhibition that persists for some
time after stimulus offset. Previous intracellular and patch-clamp
studies showed that persistent inhibition occurs in the ICc
(Covey et al. 1996; Kuwada et al. 1997
),
although its prevalence was not apparent from those studies. Studies of
the precedence effect in the rabbit (Fitzpatrick et al.
1995
) and cat ICc (Litovsky and Yin 1998
;
Yin 1994
) also showed that an initial binaural stimulus
produces a long-lasting suppression of responses to signals that
closely follow the initial signal. However, those studies recorded
single-unit activity with extracellular electrodes and therefore could
not conclusively show that the actual inhibition occurred at the ICc
and not in a lower nucleus. The reversible inactivation studies of the
DNLL, by Kidd and Kelly (1996)
, however, demonstrated
that ipsilaterally evoked persistent inhibition is indeed a feature of
the ICc. They recorded single units from the rat ICc while presenting
clicks to the ipsilateral ear followed by clicks to the contralateral
ear that were delayed by various intervals. The ipsilateral clicks
produced a persistent inhibition that suppressed responses evoked by
the contralateral clicks for periods ranging from 10 to 30 ms, and this
was observed in the majority of ICc cells. When the DNLL was
inactivated, the cells responded to contralateral clicks at shorter
intervals after the ipsilateral click than they did before or after
recovery from DNLL inactivation, showing that the persistent inhibition
was generated in the ICc and not in a lower nucleus.
Persistent inhibition evoked by ipsilateral stimulation is not unique
to the ICc, but it is also a robust feature of EI neurons in the DNLL
(Yang and Pollak 1994a,b
). However, unlike the DNLL, where persistent inhibition is evoked only by ipsilateral signals and
not by contralateral signals, persistent inhibition in the ICc is
evoked in almost all ICc cells by signals presented to the
contralateral as well as the ipsilateral ears. In a future report of
ICc cells in the Mexican free-tailed bat, we show that contralateral
signals evoke an excitation followed by an inhibition that persists for
26 ms, on average, in 85% of ICc cells (unpublished results). These
values are similar to those for ipsilaterally evoked persistent
inhibition, which is evoked in 86% of ICc cells and lasts for 15 ms on
average. Thus persistent inhibition evoked by stimulation of either ear
occurs in the vast majority of ICc cells and is a feature that
apparently sets ICc neurons apart from DNLL neurons or neurons in any
of the other lower nuclei.
Concluding comments
We have shown that stimulation of the ipsilateral ear evokes inhibition in the vast majority of ICc neurons in Mexican free-tailed bats. The inhibition can have multiple components that vary in latency, threshold, and duration. Individual cells can display one or more inhibitory components. Some cells have both a very brief, early inhibition that suppresses the first few discharges of the train and a longer latency inhibition that may or may not suppress discharges evoked by a contralateral signal. In other ICc cells, the ipsilateral inhibition is coincident with contralaterally evoked discharges in both time and duration, but the inhibition has a duration that is many milliseconds longer than the signal that generated it. In short, ipsilateral inhibition at the ICc is prevalent and complex. Moreover, contralateral signals also evoke persistent inhibition (unpublished results). Thus the variety of inhibitory features, evoked by stimulation of the ipsilateral and contralateral ears, are complex but have to be factored into hypotheses concerned with functional activity of the mammalian ICc.
![]() |
ACKNOWLEDGMENTS |
---|
We thank C. Resler for designing and implementing the electronics for data acquisition. We also thank D. Ryugo, M. Burger, L. Hurley, C. Resler, and T. Park for critical comments.
This work was supported by National Institute of Neurological Deafness and Other Communications Disorders Grant DC-00268.
![]() |
FOOTNOTES |
---|
Address reprint requests to: G. D. Pollak
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 25 January 1999; accepted in final form 19 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|