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Afferent connections to the dorsal nucleus of the lateral lemniscus of the mustache bat evidence for two functional subdivisions

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THE JOURNAL OF COMPARATIVE NEUROLOGY 373~575-592 ( 1996)
Afferent Connections to the Dorsal Nucleus
of the Lateral Lemniscus
of the Mustache Bat:
Evidence for Two Functional Subdivisions
LICHUAN YANG, &IN LIU, AND GEORGE D. POLLAK
Department of Zoology, University of Texas at Austin, Austin, Texas 78712
ABSTRACT
The dorsal nucleus of the lateral lemniscus (DNLL) of the mustache bat, Pteronotus
parnellii, was found to consist of two divisions. The neurons in each division were distinguished
by their temporal discharge patterns evoked both by tone bursts and sinusoidal amplitudemodulated (SAM) signals. Neurons in the anterior one-third of the DNLL responded to tone
bursts with an onset discharge pattern and only phase-locked to SAM signals with low
modulation frequencies ( < 300 Hz). Neurons in the posterior two-thirds of the DNLL
responded to tone bursts with a sustained discharge pattern and phase-locked to SAM signals
with much higher modulation frequencies (400-800 Hz). In addition, there was a different
frequency representation in the two divisions. The frequency representation in the posterior
division was from about 15 to 120 kHz, whereas in the anterior division it was only up to 62
kHz.
The physiological differences were further supported by data from experiments that
revealed the sources of afferent projections to the two DNLL divisions. Retrograde labeling
showed that the afferent projections to the two divisions were from different neuronal
populations. Input differences were of two types. Some nuclei projected to one or the other
DNLL division, but not to both. For instance, the ventral nucleus of the lateral lemniscus
projected predominately to the anterior DNLL and provided little or no inputs to the posterior
DNLL, whereas the medial superior olive innervated the posterior but not the anterior DNLL.
Other lower nuclei projected to both DNLL divisions. These include the contralateral cochlear
nucleus, the ipsi- and contralateral lateral superior olives, the intermediate nucleus of the
lateral lemniscus, and the contralateral DNLL. However, the projections to each division of the
DNLL originate from different neuronal subpopulations in each lower nucleus. The functional
implications of these findings are discussed in the context of the possible impacts that the two
DNLL divisions exert on their postsynaptic targets in the inferior colliculus.
1~ Wiley-LL<s, Inc.
Indexing terms: amplitude modulation, temporal discharge patterns, differential projections, auditory
pathway
The dorsal nucleus of the lateral lemniscus (DNLL)
occupies a strategic position in the ascending auditory
pathway. Located immediately below the inferior colliculus,
it receives innervation from a complement of lower nuclei
similar to those that innervate the inferior colliculus, and it
sends strong bilateral projections to the central nucleus of
the inferior colliculus (ICc; Glendenning et al., 1981; Kudo,
1981; Zook and Casseday, 1982, 1985, 1987; Ross et a].,
1988; Shneiderman et al., 1988; Ross and Pollak, 1989;
Hutson et al., 1991; Bajo et al., 1993; Merchan et al., 1994;
Huffman and Covey, 1995; Vater et al., 1995). DNLL
neurons are also distinguished by two additional features:
1) They are largely binaural, and those neurons tuned to
(
1996 WILEY-LISS, INC.
high frequencies are driven by stimulation of the contralatera1 ear and inhibited by stimulation of the ipsilateral ear
(Brugge et al., 1970; Buckthought eta]., 1993; Covey, 1993;
Markovitz and Pollak, 1994; Yang and Pollak, 1994a); and
2) they are strongly immunoreactive for antibodies against
conjugated y-aminobutyric acid (GABA) or glutamic acid
decarboxylase (GAD), the rate-limiting enzyme for the
synthesis of GABA (Adams and Mugnaini, 1984; Thompson
et al., 1985; Moore and Moore, 1987; Roberts and Ribak,
Accepted May 5, 1996.
Address reprint requests to Dr. George D. Polkdk, Department of Zoology,
University of Texas at Austin, Austin, T X 78712.
576
1987; Glendenning and Baker, 1988; Vater et al., 1992a;
Covey, 1993; Vater, 1995; Winer et al., 1995).
The prominent immunoreactivity of DNLL neurons for
GABA suggests that the DNLL exerts a powerful inhibitory
influence on its targets in the ICc. Indeed, Oliver and
Shneiderman (1989) report that in the cat, DNLL projections account for roughly one-third of the inhibitory synaptic contacts in the ICc. The putative inhibitory impact of the
DNLL on its targets in the ICc, suggested by its connections
and neurochemistry, has been confirmed by neurophysiological studies. In several studies, binaural properties of
high-frequency ICc neurons were changed dramatically
either when GABAergic inhibition was blocked by the
iontophoretic application of bicuculline or when the DNLL
was reversibly inactivated with pharmacological agents
(Faingold et al., 1989, 1993; Li and Kelly, 1992; Vater et al.,
1992b; Park and Pollak, 1993; Mug et al., 1995). The
evidence provided by these studies leaves little doubt that
the inhibitory influences of the DNLL play a substantial
role in forming the binaural properties of many ICc neurons.
The DNLL may play an even more pervasive and complex
role in the processing of acoustic information than indicated by the studies cited above. Recent studies show that
DNLL neurons respond in unusual ways to multiple binaural stimuli (Yang and Pollak, 1994a,c). An initial stimulus
that is more intense at the inhibitory ear than at the
excitatory ear evokes a long-lasting inhibition in DNLL
cells. The long duration of the inhibition prevents DNLL
cells from discharging to subsequent stimuli that would
normally be excitatory to them. Thus, whether a binaural
signal can drive DNLL cells depends upon the cell’s immediate history (whether an initial signal received prior to the
second signal evoked an inhibitory response). These response properties, and the way in which they would impact
their targets in the ICc, may well have a profound influence
on the way the auditory system processes the reception of
multiple signals that emanate from various regions of
space.
Although the binaural properties of high-frequency DNLL
neurons are homogenous in that almost all are excitatoryinhibitory (EI), their discharge patterns evoked by tone
bursts presented to the contralateral (excitatory) ear are
more variable. Some EI neurons discharge only at the onset
of a tone burst, whereas others respond with a sustained
discharge whose duration corresponds to the duration of
the tone burst (Aitkin et al., 1970; Covey, 1993; Markovitz
and Pollak, 1993; Yang and Pollak, 199413). Heterogeneity
among the population of DNLL neurons is also indicated by
anatomical studies. Several investigators report that the
DNLL in the cat and rat is composed of several types of
neurons and that each type is localized to a particular
DNLL region (Kane and Barone, 1980; Iwahori, 1986;
Shneiderman et al., 1988; Bajo et al., 1993). The significance of the various cell types, however, is unclear. In a
recent intracellular study of rat DNLL neurons in brain
slices, Wu and Kelly (1995) report that the various cell
types all have similar intrinsic membrane characteristics
and respond in a similar way to sustained depolarizing
currents, suggesting that all of the cell types have a similar
complement of voltage-sensitive channels.
Here we report on new features of the DNLL of the
mustache bat. This animal captures prey and orients in the
dark by emitting loud echolocation calls and listening to the
echoes reflected from objects in its environment (Griffin,
L. YANG ET AL.
1958; Novick and Vaisnys, 1964; Pollak and Casseday,
1989). Its echolocation calls are characterized by a long
constant-frequency component and are terminated by a
brief downward-sweeping frequency-modulated component. The constant-frequency component of each call is
emitted with four harmonics, at 30, 60, 90, and 120 kHz.
The 60-kHz component is always dominant. When a flying
insect crosses its path, the insect’s moving wings impose
both frequency and amplitude modulations on the long
constant-frequency component of the echo reflected from
the insect. It is from these modulation patterns on the
echoes of the 60-kHz constant frequency component that
the bat not only distinguishes its prey from background
objects but also identifies its prey (Goldman and Henson,
1977). Because of the emphasis placed on this component,
60 kHz is overrepresented in every auditory nucleus. This is
one reason that the DNLL of the mustache bat is enlarged
relative to the DNLL of other bats as well as other
mammals (Ross et al., 1988; Markovitz and Pollak, 1993;
Winer et al., 1995).
For the past several years, we have been studying the
physiology of the mustache bat’s DNLL as well as its impact
on its targets in the ICc (Yang and Pollak, 1994a-c). During
the course of our recent studies, we noted that the DNLL is
divided into two functional divisions based on temporal
discharge patterns evoked by tone bursts and more complex
signals. Here we illustrate the two functional divisions and
show that each division receives a unique afferent innervation from cell groups in lower nuclei.
MATERIALS AND METHODS
Surgical procedure
Thirteen mustache bats, Pteronotus parnellii parnellii,
were used in this study. Prior to surgery, each animal was
anesthetized with methoxyflurane inhalation (Metofane,
Pitman-Moore, Inc.), and 0.02 mgig neuroleptic (InnovarVet, Pitman-Moore, Inc.) was injected intraperitoneally.
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, Inc.) 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 was then
drilled around the central portion of the inferior colliculus
using the landmarks visible through the skull for orientation.
The bat was then transferred to a heated recording
chamber where it was placed in a restraining apparatus
attached to 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 uniform
positioning of the head. A ground electrode was placed
between the reflected muscle and the skin. Recordings were
begun after the bat recovered from the anesthetic. Supplementary doses of the neuroleptic were given if the bat
appeared to be in discomfort. These methods have been
approved by the Institutional Animal Care and Use Committee of the University of Texas.
Localization of the DNLL
A change in tonotopic sequence was used to determine
when the electrode had left the ICc and entered the DNLL,
SUBDIVISIONS IN DNLL
as was done in previous studies (Markovitz and Pollak,
1993; Yang and Pollak, 1994a-c). As the electrode was
advanced through the ICc, a repetitive tone burst was
presented to the contralateral ear that evoked background,
multiunit activity. This background activity was more
prominent with the larger-tipped pipettes used for the
injection of tracers, but it was also discernible with the
finer-tipped pipettes used to record single-unit activity. The
best frequency, the frequency to which the background
activity was most sensitive, was then determined every
50-100 IJ-malong the penetration. A reversal in the best
frequency of the background activity at a depth of about
2,200-2,400 pm signaled that the electrode had left the ICc
and entered the DNLL. For the next 400-500 pm, there
was a systematic change in best frequency from low to high.
A second abrupt change in best frequency indicated that the
electrode had left the DNLL and entered the intermediate
nucleus of the lateral lemniscus (INLL). These changes in
best frequency, as well as the tonotopic organization of the
DNLL, served as reliable indicators of electrode location.
Systematic mappings of the best frequencies and temporal discharge patterns of single- or multiunit activity were
conducted in the DNLL of four bats. In these experiments a
single micropipette filled with horseradish peroxidase (HRP;
5% in 0.9% NaCl) was used for making multiple penetrations along the anterior-posterior axis of the DNLL. The
electrode position at each recording site was recorded by
digital positioners attached to the stereotaxic instrument.
In the last penetration of each experiment, a small iontophoretic injection of HRP (1.5 pA for 3 minutes, electrode
positive) was made at a selected site in the DNLL. The
location of the HRP injection site was later used as an
adjustment point to reconstruct the distribution of best
frequencies and temporal discharge patterns in the DNLL.
577
tone bursts and SAM signals were fed to a window discriminator and then to a Macintosh 7100 computer for generating raster displays.
Administration of retrograde tracers
Injections of tracers were made with pipettes having tip
diameters of 5-10 IJ-m that were filled with 10% lysine
fixable biotinylated dextran (BD; 3 kD, pH 7.4) or 10%
lysine fixable dextran conjugated to fluorescein ( 3 kD, pH
7.4) or tetramethylrhodamine ( 3 kD, pH 7.4). The dextrans
were obtained from Molecular Probes Inc. These fluorophores were chosen because they can be applied iontophoretically, they transport well retrogradely, and they fluoresce brightly. The electrode was positioned on the surface
of the inferior colliculus by using visual landmarks viewed
with an operating microscope. Subsequently, the electrode
was advanced from outside of the experimental chamber
with a piezoelectric microdrive (Burleigh 7121W). Injections of dextrans were made by passing positive current
(2.5-5 pA) that was pulsed (5 seconds on, 5 seconds off) for
8-20 minutes. After a 48-hour survival time, the animal
was deeply anesthetized with methoxyflurane inhalation
(Metofane, Pitman-Moore, Inc.). It was then perfused
transcardially with phosphate-buffered saline (PBS; 0.1 M,
pH 7.4) followed by a fixative solution of 4% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer (0.1 M,
pH 7.4). For animals that were injected with dextran
amines conjugated to fluorescein or rhodamine, 1.25%
glutaraldehyde was spared from the fixative solution because it caused autofluorescence in the tissue. After fixation, the brain was dissected out and postfixed for 2 hours.
It was then transferred to a 30%sucrose solution overnight
for cryoprotection. The brain was sectioned on a freezing
microtome at a thickness of 40 pm. With BD injections,
Acoustic stimuli and data acquisition
sections were first incubated in 0.3%Triton X-100 diluted
Acoustic stimuli were presented through %-inchBruel & with PBS for 1 hour. The sections were rinsed three times
Kjaer condenser microphones driven as loudspeakers. Micro- in PBS and then reacted with ABC procedure (Vectastain
pipettes whose tips were broken to a diameter of about 2 ABC Elite kit, Vector Laboratories) followed by diaminobenIJ-mwere used for recording multiunit activity, and micropi- zidine reaction (DAB peroxides substrate kit, Vector
pettes with tip diameters of less than 1.0 pm were used to Laboratories). Sections were then mounted, air-dried,
record the action potentials of single units. When a unit was and counterstained with cresyl violet. With injections of
isolated, its best frequency was determined with tone dextran amines conjugated to fluorescein or rhodamine,
bursts that were 20 msec in duration and had 0.5-msec sections were mounted on the slides without further reacrisefall times. The tone bursts were generated by shaping tion and coverslipped with FITC-Guard mounting medium
a sinewave from a Wavetek function generator (model 136) (TESTOG).
with a custom-made analog switch. The binaural property
Sections from animals that received injections of BD were
was then assessed by driving the cell with a tone burst
examined under brightfield illumination. Sections from
presented to the contralateral ear at 20 dB above the
animals injected with fluorophores were evaluated with
threshold of the best frequency. With the intensity fixed at
the contralateral ear, the same frequency was then pre- epi-illumination using appropriate filter cubes. The fluoressented simultaneously to the ipsilateral ear, and the inten- cence of cells filled after injecting dextran conjugated to
sity of the ipsilateral signal was increased from 10-20 dB tetramethylrhodamine was typically a little more intense
below the intensity at the contralateral ear to 40 dB above than cells filled after injections of dextran conjugated to
that intensity in steps of 10 dB. After determining the fluorescein. Moreover, wavelengths passed by the fluoresneuron’s binaural property, the ipsilateral signal was turned cein cube could also evoke a slight fluorescence in cells
off, and sinusoidal amplitudemodulated (SAM) signals of strongly stained by rhodamine. To ensure that double70-msec duration were presented to the contralateral ear. labeled cells could be distinguished from such bleed-over, in
The SAM signals were created digitally on a Macintosh one experiment we injected both rhodamine- and fluorescein7100 computer. The carrier frequencies of the SAM signals conjugated dextran in the same site. The labeled cells in this
were set at the neuron’s best frequency. A set of SAM case fluoresced intensely when viewed with both cubes.
signals, each with a different modulation frequency, was This served as a standard, and we considered cells as
presented to each unit. The modulation frequencies ranged doubled-labeled only when clear fluorescence was observed
from 100 to 800 Hz in 100-Hz steps. Spikes evoked both by with both cubes.
I’
a
3
579
SUBDIVISIONS IN DNLL
RESULTS
General features of the mustache bat DNLL
As seen in sagittal and transverse sections (Fig. 1 B,C),
the DNLL has well-defined boundaries that distinguish it
from surrounding structures. Dorsally it is separated from
the inferior colliculus by a nearly acellular space filled with
the fibers entering the ventrolateral border of the central
nucleus of the inferior colliculus (ICc). Ventrally it is
demarcated from the intermediate nucleus of the lateral
lemniscus (INLL) by crossing fibers of the lateral lemniscus. Medially and anteriorly it is distinct from the paralemniscal nucleus by the ascending fiber bundles of the lateral
lemniscus. Laterally and posteriorly the DNLL extends
almost to the edges of the brain surface where it is
encapsulated by a thin layer of lateral lemniscal fibers. A
three-dimensional view suggests that the DNLL of the
mustache bat is cone-shaped with a large, rounded posterior region that tapers into a smaller anterior region (Fig.
1D). The anterior-posterior axis is about 850 pm, and the
lateral-medial axis is about 500 pm. The dorsal-ventral
axis is about 450 pm posteriorly and tapers to about 100
pm anteriorly.
In Nissl-stained material the DNLL is organized as
vertically oriented parallel columns of cell somata separated
by 30-50 km of “acellular” spaces (Figs. 1B, C, 2A). Each
column is about 20-30 pm in width and contains one to two
rows of somata. The “acellular” space contains the dendrites and axons of DNLL cells as well as the ascending
fibers of the lateral lemniscus. Figure 2B shows retrogradely labeled cells in the DNLL found after injecting
biotinylated dextran amines (BD) into the 60-kHz region of
both the contra- and ipsilateral ICc. The labeled cells
present a Golgi-like appearance, and in this case the cells
presumably correspond to the 60-kHz representation in the
DNLL. The dendrites of the labeled DNLL cells were
horizontally oriented and traversed the spaces between
columns of somata. Because the ascending fibers of the
lateral lemniscus also travel vertically in these spaces, it
seems that the crossovers at these spaces, between the
dendrites and the ascending lemniscal fibers, provide a
substrate for the DNLL cells to capture the ascending
information from lemniscal fibers.
From evaluations of labeled cells we could discern no
obvious cellular segregations, but rather the neuronal
architecture of the mustache bats’ DNLL presented a fairly
homogenous appearance. Comparison of DNLL filled cells
were made following injections of BD in either the contralateral DNLL or in both the 60-kHz regions of the ipsi- and
contralateral ICc. Labeled neurons were evaluated in terms
of their size, shape of their somata, and their dendritic
arborization. DNLL neurons were classified into one of
three groups: fusiform shaped, round or oval-shaped, and
multipolar (Fig. 2C). The somata of the fusiform cells were
either medium (9-10 Fm) or large (11-13 pm), with their
dendrites projecting laterally (Fig. 2Ca,b). Although we did
not do cell counts, fusiform cells appeared to be slightly
more prevalent in the posterior two-thirds of the DNLL
than in the anterior one-third of the DNLL. The round or
oval-shaped cell type typically had a single primary dendrite
which gave rise to two or more secondary dendrites oriented
in the same direction (Fig. 2Ce,f). The somata of round cells
varied in size from small (5-8 pm) to medium (9-10 pm).
Fewer had large somata (11-13 pm). Round cells of each
size appeared to be distributed throughout the nucleus. The
third type was multipolar cells (Fig. 2Cc,d). The somata of
these cells ranged from medium (9-10 pm) to large (11-13
pm), and they appeared to be slightly more frequent in the
anterior one-third of the DNLL.
Afferent projections to the DNLL
The nuclei that provide afferent inputs to the DNLL were
determined from the locations of retrogradely labeled cells
following injections of either BD or rhodamine-conjugated
dextran that filled almost all of the DNLL. As illustrated in
Figure 3, the injected tracer was confined to the DNLL with
little or no discernable spread to adjacent structures. The
clear retrograde labeling that resulted from these injections
is illustrated in Figure 4 by the cells in the lateral superior
olive (LSO) labeled after an injection of BD or fluoresceinconjugated dextran. Three bats received large injections of
this sort, and the results were similar among the cases. As
shown for one case in Figure 5, labeled cells were found in
the contralateral cochlear nucleus, the ipsilateral medial
superior olive (MSO), the ipsilateral ventral nucleus of the
lateral lemniscus (VNLL), the INLL, and in the contralateral DNLL. A small number of labeled cells was also found
in the ipsilateral paralemniscal nucleus (not shown in Fig.
5) and in some ventral periolivary nuclei. The largest
number of labeled cells, however, were found in the ipsiand contralateral LSOs. This complement of nuclei that
provide afferent input to the DNLL in the mustache bat is
very similar to the nuclei that project to the DNLL in other
bats and other mammals (Glendenning et al., 1981; Kudo,
1981; Shneiderman et al., 1988; Hutson et al., 1991; Bajo et
al., 1993; Merchan et al., 1994; Huffman and Covey, 1995;
Vater et al., 1995). Finally, labeled cells in the ICc were
rare, and those that were found were always in its ventral
portion. This suggests that if the mustache bat’s DNLL
receives any descending projections from the ICc, that
projection is much weaker than the descending projection
from the 1Cc that has been reported in the DNLL of rat and
cat (Carey and Webster 1971; Hutson et al., 1991; Caicedo
and Herbert, 1993).
There are two subdivisions in the DNLL
of the mustache bat
Fig. 1. A: Brain of the mustache bat with the cerebellum partially
removed to reveal the inferior colliculus (IC) and nuclei of the lateral
lemniscus (NLL).Also shown are the auditory nerve (AN)
and cochlear
nucleus (CN).B: Sagittal section stained with cresyl violet showing the
dorsal nucleus of the lateral lemniscus (DNLL) in relation to the
intermediate and ventral nucleus of the lateral lemniscus (INLL and
VNLL). D and P refer to dorsal and posterior, respectively. C: Transverse section showing the nuclei of the lateral lemniscus. D and M refer
to dorsal and medial, respectively. D: Three-dimensional drawing of the
DNLL. D, R, and L refer to dorsal, rostral, and lateral, respectively. The
DNLL is enclosed by dashed lines. Scale bars = 400 Fm in B (corresponds to 1.5mm in A), 300 Km in C, 200 Fm in D.
The DNLL has at least three features that are common at
any locus and which serve to define it as a nucleus. First, as
mentioned previously, we did not observe any obvious
architectonic subdivisions in the DNLL. Second, all of the
neurons that we sampled in the DNLL had the same
binaural property: They were excited by stimulation of the
contralateral ear and inhibited by stimulation of the ipsilateral ear and thus were excitatoryinhibitow or El. The
third common feature was that there was only one tonotopic arrangement in the mustache bat DNLL: Neurons
Figure 2
C
SUBDIVISIONS IN DNLL
Fig. 3. Transverse section through the brain of one bat showing
that the injection of biotinylated dextran was confined largely to the
DNLL. D and L refer to dorsal and lateral, respectively. Scale bar =
300 pm.
tuned to low frequencies were represented dorsally, whereas
neurons tuned to progressively higher frequencies were
represented ventrally. This tonotopic pattern was also seen
in previous studies (Markovitz and Pollak, 1993; Yang and
Pollak, 1994a,b) and is illustrated in Figure 6.
There are, however, two features that distinguish neurons in the anterior one-third of the DNLL from those in
the posterior two-thirds of the DNLL. The first and principal feature is a pronounced difference in temporal discharge
pattern evoked by tone bursts and sinusoidal amplitudemodulated (SAM)signals (Figs. 6, 7). When tone bursts at
each neuron’s best frequency were presented to the contralateral ear, most neurons in the posterior DNLL responded
with a sustained temporal discharge pattern, in which the
discharge train lasted for the duration of the tone burst
(Fig. 6C). In contrast, most neurons in the anterior DNLL
responded with an onset pattern, in which discharges were
evoked only at the beginning of the tone burst (Fig. 6C). As
mentioned above, both onset and sustained neurons were
inhibited by stimulation at the ipsilateral ear, and thus
were EI. Examples of sustained and onset neurons are
shown in Figure 6C. Onset and sustained neurons were
Fig. 2. A: Transverse Nissl-stained section showing cells in the
DNLL (enclosed by dashed lines) organized into vertically oriented
columns. One column is indicated by arrows. B: A row of cells in DNLL
labeled by injections of biotinylated dextran amines into the 6O-kHz
region of both the contra- and ipsilateral central nucleus of the inferior
colliculus (ICc).The labeled cells presumably correspond to the 60-kHz
representation in the DNLL. Notice that neurites of cells are horizontally oriented and cross columns of somata. C: Three main types of
DNLL cells. Fusiform (a,b), multipolar (c,d), and round cells (e,D.
Arrows show axons. Scale bars = 100 pm in A and B, 50 bm in C.
581
often intermingled at the juncture of the two regions, but
one or the other type was dominant at the more posterior
and anterior portions of the DNLL (Fig. 6A, B).
Sustained and onset neurons also responded differently
to SAM signals (Fig. 7). Of the 77 DNLL neurons from
which we recorded, the majority responded to SAM with
discharges that were phase-locked to each cycle of the
amplitude-modulated signal. The difference was that the
discharges of the 45 sustained neurons phase-locked to
modulation frequencies as high as 400-600 Hz, and a few
phase-locked to rates as high as 800 Hz; on the other hand
75% (24132) of onset neurons only phase-locked to low
modulation frequencies, typically below 300 Hz. Twentyfive percent of the onset neurons (8132) did not to phaselock to any of the SAM signals that we presented. The three
neurons in Figure 7 are representative and illustrate the
pronounced disparity in the phase-locking of sustained and
onset neurons to the time variations of SAM signals.
The second feature was a difference in the frequency
representation between the anterior DNLL, the region
dominated by onset neurons, and the posterior DNLL, the
region dominated by sustained neurons. Although both
regions had a similar tonotopic sequence, in which neurons
tuned to low frequencies were located dorsally and neurons
tuned to progressively higher frequencies were located
ventrally, the total range of frequencies represented in the
anterior DNLL was smaller than the range represented in
the posterior DNLL (Figs. 6 and 8, top panel). Specifically,
the anterior DNLL had neurons tuned from about 15 kHz
to about 62 kHz. Neurons tuned to frequencies higher than
about 62 kHz were rarely encountered even in the most
ventral regions of the anterior DNLL (Fig. 8, top panel). In
contrast, the frequency representation in the posterior
DNLL ranged from about 15 kHz to 120 kHz and encompassed the mustache bat’s entire hearing range (Fig. 8, top
panel). A summary figure illustrating the general tonotopic
arrangement of the DNLL and the difference in frequency
representation between the anterior and posterior divisions
is shown in Figure 6D.
In summary, the DNLL is functionally divided into a
smaller anterior division and a larger posterior division, in
which neurons in each division have a different temporal
discharge pattern to tone bursts and SAM signals. However, the neurons in both divisions have the same binaural
property, they have a similar complement of cell types, and
they display a comparable tonotopic organization, although
the posterior division encompasses the bat’s entire hearing
range whereas the anterior division has a more restricted
frequency representation.
Afferent inputs to each of the DNLL divisions
We next determined whether the same set of lower nuclei
provided inputs to both divisions of the DNLL. We did this
in six bats by first mapping the DNLL to identify the two
DNLL divisions based on the discharge patterns of their
neurons evoked by tone bursts. After verifying t h a t 4 e
electrode was in one or the other division, the electrode was
advanced or retracted so that it was 100-200 pm from the
dorsal surface of the DNLL, which was about in the middle
of the dorsal-ventral axis of either the anterior or posterior
subdivision. In four bats BD was injected in only one DNLL
division (in the anterior division in two bats and in the
posterior division in two bats). In two other bats both
divisions of the DNLL were injected; rhodamine-conjugated
dextran was injected into one DNLL division, and fluores-
L. YANG ET AL
582
Fig. 4. Labeled cells in the contralateral lateral superior olive (enclosed by dashed lines) in two bats
following injections of fluorescein conjugated to dextran (left) or biotinylated dextran (right) in DNLL.
Scale bar = 100 pm.
cein-conjugated dextran was injected into the other division. The time between the injections of the two fluorophores was 30-45 minutes. We refer to the experiments
that used two fluorophores as double-labeling experiments.
Examination of the histological sections showed that the
injections were confined to the desired division of the
DNLL.
The main finding is that the two DNLL divisions receive
different afferent projections. The different projections
derive in part from different lower nuclei and in part from
different cell groups of some nuclei that project to both
DNLL divisions. The percentage of cells in each lower
nucleus that were labeled is shown in Figure 9. Figure 10
shows the location of the labeled cells in one case in which
fluorescein-conjugated dextran was injected into one DNLL
division and rhodamine-conjugated dextran was injected
into the other division. Below, we turn first to the nuclei
that project to both DNLL divisions and show that the cell
groups in each nucleus that project to the anterior DNLL
are different from those that project to the posterior DNLL.
We then consider the projections from the VNLL and MSO,
the two nuclei that project to either the anterior or posterior DNLL divisions but not to both divisions.
Nuclei that project to both DNLL divisions
Both DNLL divisions received projections from the contralateral cochlear nucleus, the LSOs on both sides, the ipsilateral
INLL, and from the opposite DNLL (Figs. 9, 10). The projections from the cochlear nucleus were sparse in that only a small
number of cells were labeled in most of the cases. The projections to the anterior DNLL were from the anteroventral
cochlear nucleus and occasionally from the posteroventral
cochlear nucleus. The projections to the posterior division
were divided equally between the anteroventral cochlear
nucleus and the dorsal cochlear nucleus (Fig. 10).
Although the anteroventral cochlear nucleus sent projections to both the anterior and posterior DNLL, it was
unclear from the BD cases whether the same cells in the
anteroventral cochlear nucleus projected to both DNLL
divisions. In the double-labeling experiments, in which
rhodamine-conjugated dextran was injected into one DNLL
division and fluorescein-conjugated dextran was injectec
into the other DNLL division, cells in the anteroventra
cochlear nucleus were labeled either with fluorescein 01
with rhodamine, but none were double labeled (Fig. 10)
This suggests that those cell groups in the anteroventra’
cochlear nucleus that project to the anterior DNLL arc
distinct from those that project to the posterior DNLL.
The projections from LSO were of particular interest
because they provided the heaviest input to both DNLL
divisions. The main result is that different LSO cell groups
projected to the two DNLL divisions. We point out first that
one component of the difference in projections from LSO t c
the two divisions of the DNLL can be explained by a
tonotopic projection system, in which LSO neurons tuned
to a particular frequency send axonal projections to regions
of higher nuclei that represent the same frequency. Thri
tonotopic arrangement of the mustache bat LSO is parti
tioned into three general frequency regions, each of approximately equal size: The dorsolateral limb represents frequencies from about 10 to 59 kHz, the ventromedial limh
represents frequencies from about 63 to 120 kHz, and
sandwiched between these is a central limb that is isofrequency for frequencies within 60-63 kHz-the exact frequency varies with the individual animal (Ross et al., 1988;
Covey et al., 1991).Because the frequency representation in
the anterior DNLL extends only to about 62 kHz, the
ventromedial limb of the LSO, which represents high frequencies, would not be expected to project to the anterior DNLL. On
the other hand, the ventromedial limb of the LSO should
project to the posterior DNLL, which has a prominent representation of high frequencies, from 63 to 120 kHz. The two other
LSO limbs, the dorsolateraland central limbs, should project tc
both the anterior and posterior DNLL, because the twc
DNLL divisions have “low”-frequency representations at
well as 60-kHz representations.
These predicted tonotopic projection patterns were con
firmed. The lower panel of Figure 8 shows the counts oa‘
labeled cells in the contralateral LSOs in four bats. Thew
cells were from selected sections in which all three LSO
SUBDIVISIONS IN DNLL
583
6
Fig. 5. Distribution of cells labeled by a large injection of biotinylated dextran in the DNLL of one bat. The injection was made in the left
DNLL. indicated by shading. The most caudal section is in the lower
right and is labeled 1, and the most rostra1 section is in the top left and
is labeled 6. AVCN, Anteroventral cochlear nucleus; DCN, dorsal
cochlear nucleus; INLL, intermediate nucleus of the lateral lemniscus;
LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid
body; MSO, medial superior olive; N. VII, nucleus of the seventh cranial
nerve; PVCN, posteroventral cochlear nucleus; VNLL, ventral nucleus
of the lateral lemniscus; VIII, auditoly nerve.
limbs were apparent in each section. In two bats, BD was
injected in the anterior DNLL, and in two other bats BD
was injected in the posterior DNLL. Anterior DNLL injections resulted in a large number of labeled cells in the
low-frequency, dorsolateral limbs of the LSOs and in the
central limbs that represent 60 kHz. Virtually no labeled
cells were found in the high-frequency, ventromedial limbs.
In contrast, there were labeled cells in all three limbs of the
LSO following injections in the posterior DNLL. There
were, however, fewer labeled low-frequency (in the dorsolatera1 limb) and more labeled 60-kHz cells (in the central
limb) following posterior DNLL injections than there were
with anterior DNLL injections. This pattern was most
likely due to the difference in frequency representations of
the anterior and posterior injection sites. All injections were
made in the center of the DNLL, midway along its dorsoventral extent. In the posterior DNLL, the central region along
the dorsoventral axis corresponds to the 60-kHz representation (Fig. 6D), and injections there resulted in a large
number of labeled cells in the central limb of the LSO. In
contrast, “low frequencies” are represented in the center of
the anterior DNLL (Fig. 6D), which could explain the large
number of labeled cells in the dorsolateral (“low”-frequency) limb of the LSO following anterior DNLL injections. Thus, the pattern of labeled LSO cells is consistent
with the differential frequency representations in the anterior and posterior DNLLs.
The most surprising result was that different populations
of LSO cells projected to the anterior and posterior DNLL,
and this result was obtained after taking the difference in
L. YANG ET AL.
584
4
R
c
B
P
Onset
Sustained
contrd = 30 dB SPL
contra = 30 dB SPL
= 10 dFl SPL
lpl=
10 dB SPL
,
lpl=20
dB SPL
1p.l
p i = 30 dB SPI.
= 20 dE SPL
Ip;i = 3odB SPL
'2
i p i = 4 0 W SPL
:
\
antcnor DNLL
Ipl=40 dB SPL
,
Tonotopic Organization of DNLL
%
I 1::
'PI
D
60-62kHz
i
postenor DNIL
.
I
lp.1
=so dB SPL
'
ipi = SO dB SPL
I
Ipl=60 dB SPL
70 rns
dB SPL
1p.l = 60
70 rns
Fig. 6 . A. Best frequencies of multiunits recorded in several penetrations along the rostrocaudal axis of the DNLL in one bat. In all cases
low frequencies were represented dorsally and high frequencies ventrally. Note that the best frequencies range from 40.7 kHz up to 112
kHz in the posterior DNLL, whereas the highest best frequency in the
anterior DNLL was only 61.9 kHz. The discharge patterns evoked by
tone bursts at each recording site are listed below the best frequencies.
Open circles indicate sites at which onset responses were recorded, and
solid circles indicate sites at which sustained responses were recorded.
Dorsal and rostra1 are indicated in the upper left. B: Best frequencies of
single units recorded in the more dorsal portions of the DNLL in one
bat. Although units in the more ventral DNLL were not recorded, there
is a clear tonotopic progression from low to higher frequencies. Low
frequencies between 16 and 30 kHz are represented in the dorsal DNLL
along the entire rostrocaudal axis. C: Raster displays of the discharges
from two DNLL units showing that both onset neurons from the
anterior DNLL and sustained units from the posterior DNLL were
excited by stimulation of the contralateral ear and inhibited by stimulation of the ipsilateral ear. The intensities at the contralateral ear were
held constant at 30 dB SPL in both neurons, and intensities at the
ipsilateral ear were varied. The ipsilateral intensities are indicated
below each histogram. Each raster display was generated by 20
repetitions of a binaural stimulus. Tone burst durations were 70 msec.
D: Schematic illustration of tonotopic arrangement of the DNLL
showing the loci of the three major frequency bands constituting the
mustache bat's hearing range: 1) < 60 kHz; 2) 60-62 kHz; and 3 ) > 62
kHz. Also shown is the difference in frequency representation between
the anterior and posterior DNLL.
SUBDIVISIONS IN DNLL
Sustained Neuron
Posterior DNLL
585
Onset Neuron
Anterior DNLL
Onset Neuron
Anterior DNLL
Raster displays of discharges evoked by sinusoidal amplitude
modulated (SAM) signals from a sustained neuron in the posterior
DNLL (left 1 and from two onset neurons in the anterior DNLL (middle
and right). The waveform of either the tone burst or SAM signal is
shown below each raster. The top panels show discharge patterns
evoked by 70-msec tone bursts at the cell's best frequency. The panels
below show the discharges evoked by SAM signals of increasing
modulation frequency, shown at the far left. The carrier frequencies
were a t the unit's best frequency. Note the difference in the phaselocking as the SAM modulation frequency increased. All signals were 70
msec in duration and were 40 dB SPL (sound pressure level is sound
pressure re 0.0002 dynes/cm2).The number at the right corner of each
raster display is the total spike count. The numbers a t the top of each
panel show how many neurons of each type were in our sample. The
best frequencies were 85.2 kHz (left),61.25 kHz (middle),and 60.6 kHz
(right1.
frequency representation of the two DNLL divisions into
account. This segregation of projections was confirmed in
two double-labeling experiments. Although a n occasional
LSO cell was labeled with both rhodamine and fluorescein,
colabeled cells were rare in all cases. The vast majority of
cells were labeled with either rhodamine or fluorescein but
not with both fluorophores. In double-labeled experiments,
unlike the BD experiments described above, the injections
in both DNLL divisions were centered in locations that
represented similar frequencies, between 52 and 56 kHz.
Furthermore, both rhodamine- and fluorescein-labeled cells
in the LSO occurred throughout the dorsolateral limbs,
which encompassed low frequencizs, as well as the enlarged
60-kHz representation in the central limb (Fig. 10).These
results provide strong support for concluding that the LSO
cell groups which innervate the anterior DNLL are separate
from those that innervate the posterior DNLL. However,
we did not evaluate the morphology of the LSO cells labeled
with each fluorophore, and thus are unable to conclude
whether a particular LSO cell type projects to the anterior
DNLL and a different LSO type projects to the posterior
DNLL.
Comparable results were also found in the INLL. This
nucleus, like the LSO, sends projections to both DNLL
divisions. BD injections in either division of the DNLL
resulted in substantial labeling in the INLL (Fig. 9). From
these cases, however, we could not determine whether the
same or different INLL cells projected to the two DNLL
divisions. This question was answered by two cases in
which both rhodamine and fluorescein injections were
made in the two divisions in the same animal. In both
cases the INLL had both rhodamine- and fluoresceinlabeled cells, and like the LSO, there were few doublelabeled cells (Fig. 10).
The DNLL on one side also projected to both DNLL
divisions on the opposite side. Injections of BD in the
anterior division resulted in labeling throughout the opposite DNLL, in both the anterior and posterior regions. The
labeling, however, was largely confined to the dorsolateral
aspects of both DNLL divisions. With injections in the
posterior DNLL, labeling was also found in both DNLL
divisions on the opposite side, although fewer cells were
labeled than with anterior injections. The labeled cells,
moreover, were also largely in the dorsolateral aspects of
L. YANG ET A
586
Anterior Division
Posterior Division
50-
40
A
U
n
30-59
60-64
65-79
c-CN
c-LSO
I-LSO
i-MSO
I-INLL
I-VNLL c-DNLL I-PL
c-CN
c-LSO
i-LSO
I-MSO
i-INLL
I-VNLL c-DNLL I-PL
80-110
Best Frequency (kHz)
Anterior injection
Posterior Injection
U
Q
Q
r
Anterior
c] Posterior
Fig. 8. Top: Distribution of best frequencies recorded from several
penetrations through the anterior and posterior DNLL in one bat.
Recordings were of multiunit activity. Part of these are shown in Figure
6A. Bottom: Number of labeled cells in each of the three limbs from
selected sections of the LSO after injections of dextran in the anterior
(two cases) or posterior divisions (two cases) of the DNLL.
the opposite DNLL. The same pattern of labeling was also
seen with rhodamine- and fluorescein-conjugated dextran:
when each of these tracers was injected into one of the
DNLL divisions in the same animal, there were few if any
doubled-labeled cells, which suggests that separate DNLL
populations project to the anterior and posterior divisions
in the opposite DNLL.
Lower nuclei that project to one or the other
division of the DNLL but not to both
Two nuclei, the VNLL and MSO, provided inputs to only
one of the divisions of the DNLL (Figs. 9, 10). The VNLL
projected almost exclusively to the anterior DNLL, and the
MSO projected almost exclusively to the posterior DNLL.
These differences were seen both with BD injections and
when fluorescent tracers were injected into both DNLL
divisions in the same bat. With injections of BD in the
c-CN
c-LSO
I-LSO
I-MSO
I-INLL
inject ioi
injectic
I-VNLL c-DNLL i-PL
Fig. 9. Histograms illustrating the percentage of labeled cells i
each Lower nucleus after injecting retrograde tracers into the anteric
(A)and posterior (B) division of the DNLL. C: The percentage c
labeled cells in each lower nucleus averaged for four cases. Cells i
nuclei contralateral to the injected DNLL are indicated by c, whered
cells in nuclei ipsilateral to injected DNLL are indicated by i. PL refer
to paralemniscal nucleus.
anterior division, substantial numbers of labeled cells wert
found in the VNLL but few if any in the MSO. Conversely
BD injected into the posterior DNLL resulted in labeling 11
the MSO, but virtually no cells were labeled in VNLL. 11
MSO, labeled cells were found throughout the nucleus anc
thus encompassed much of the tonotopic representation. 11
the more caudal regions, labeled cells were predominately in thl
ventromedial portion of the MSO, where high frequencies ar*
represented (Ross et al., 1988 Covey et al., 1991). In morc
rostral regions, labeled cells tended to be located in the mi&€
gion of the MSO where 60 kHz is represented, and in the moe'
rostral region labeled cells were located dorsally, in the 10%
frequencyrepresentation.The findingthat MSO projects large]!
SUBDIVISIONS IN DNLL
587
Fig. 10. Locations of labeled cell bodies in selected transverse
sections in one double-labeling case in which the dextran conjugated to
fluorescein (green) was deposited in the posterior division, whereas the
dextran conjugated to rhodamine (red) was deposited in the anterior
division of the DNLL. The most caudal section is in the lower right and
is labeled 1,and the most rostral section is in the top left and is labeled
6. Injections were in the left DNLL. Abbreviations are the same as in
Figure 5.
to the posterior DNLL is consistent with the results of a study
by Vater et al. (1995)that traced the efferent projections from
the MSO in the mustache bat.
When the posterior DNLL was injected with fluorescein
and the anterior DNLL with rhodamine, the results were
consistent with those obtained when BD was injected into
one or the other DNLL division (Fig. 10). In these double-
labeling experiments, the injection of fluorescein in the
posterior DNLL resulted in labeled cells in the MSO but not
in the VNLL. Conversely, the injection of rhodamine in the
anterior DNLL labeled cells in VNLL but not in MSO.
Complementary results were obtained when we switched
the tracers, injecting fluorescein in the anterior DNLL and
rhodamine in the posterior DNLL.
L. YANG ET AL.
588
DISCUSSION
There are three main findings in this study. The first is
that the mustache bat DNLL has two divisions: a n anterior
division in which almost all neurons respond with a n onset
discharge pattern to tone bursts and Only phase-lock to
SAM signals with low modulation frequencies, and a posterior division whose neurons respond to tone bursts with a
sustained dischargepattern and Phase-lock to much higher
modulation rates. The second finding is that although the
two DNLL divisions have a common tonotopic Organization, there is a different frequency representation in the
two divisions. The frequency representation in the posterior division is from about 15 to 120 kHz, which encompasses the bat's entire hearing range, whereas the representation in the anterior division is more restricted, and only
represents frequencies up to 62 kHz. The third finding is
that the afferent projections to the two divisions are from
different neuronal populations. Input differences are of two
types, and are schematically illustrated in Figure 11. The
VNLL projects predominately to the anterior DNLL and
provides little or no inputs to the posterior DNLL, whereas
the MSO innervates the posterior DNLL but not the
anterior division. Other lower nuclei, especially the LSO,
INLL, and contralateral DNLL, project to both DNLL
divisions, However, the projections to the two divisions
The neuoriginate from different neuronal subpopu~at~ons~
rons in the LSO, INLL, and the contralateral DNLL that
project to the anterior DNLL do not project to the posterior
DNLL, and the neurons in those nuclei that project to the
posterior DNLL do not project to the anterior DNLL.
A question that emerges with regard to the differential
innervation from the VNLL and MSO is whether this, in
fact, reflects a difference in the topography of the innervais a conse.
tion by the two nuclei, or whether the
quence of damage to the fibers from MSO that selectively
passed through the posterior DNLL and fibers from VNLL
that selectively passed through the anterior DNLL. If these
results were due solely to the uptake of tracers by damaged
fibers rather than to a differential innervation, then the
fibers from both the MSO and VNLL should provide no
collateral innervation to the DNLL. This is unlikely for
several reasons. The first reason follows from the observations by Iwahori (1986).From Golgi-impregnated material
in the rat, he observed that most ascending fibers provide
collateral innervation to the DNLL. The second reason is
that all previous connectional studies found that DNLL
cells receive innervation from both MSO and VNLL. These
studies are discussed below. The third reason follows from
the recent anterograde study by Vater et al. (19951,mentioned previously. They showed that projections from the
mustache bat MSO terminate in the posterior aspect of the
DNLL. Their findings with anterograde tracers are concordant with our results obtained with retrograde tracers: that
only the posterior DNLL is innervated by the MSO. Taken
together, these observations support the conclusion that
the innervation provided by the MSO and VNLL are
topographically segregated in the mustache bat DNLL.
Comparison with previous studies
The principal physiological, neurochemical, and anatomical features of the mustache bat's DNLL are similar to
those that have been described in other mammals. The
binaural properties of DNLL cells have been studied in a
variety of mammals including cat, rat, and in two species of
bats (Brugge et al., 1970; Metzner and Radtke-Schuller,
1987; Buckthought et al., 1993; Covey, 1993; Markovitz
and pollak, 1994;Yang and pollak 1gg4a),All of these
studies have shown that the DNLL dominated by binauand that DNLL
ral
tuned to high frequencies are almost entirely EI. Immunocytochem~ca~
studies in
a variety of species have also shown that the DNLL is
dominated by
that stain intensely with antibodies
directed against either GABA or glutamic acid decarboxy]1984;Thompson et al., 1985;
ase (Adams and
Moore and Moore, 1987;Roberts and Ribak, 1987;Glendenning and Baker, 1988; Vater et al., 1992a, 1995; Covey,
1993;Winer et a]., 1995).Additionally, the DNLL in the rat
(Merchan et al., 1994), cat (Atkin et al., 1970), and
mustache bat (Markovitz and Pollak, 1993) possesses a
pronounced tonotopic organization. The big brown bat is
the only animal for which a tonotopic organization has not
been observed in the DNLL (Covey, 1993).
appear to be
The afferent connections Of the DNLL
there are
Some
among
minor differences. Studies in the cat and rat have shown
that the DNLL receives projections from the contralateral
from the ipsilateral MSO, and strong
bilateral projections from the LSO. In addition it also
receives inputs from the ipsilateral VNLL and INLL and
from the contralateral DNLL via the commissure of Probst
(Glendenninget a]., 1981;Kudo, 1981;Shneiderman et al.,
1988;Hutson et al., 1991;BaJo et al., 1993;Merchan et al.,
1994;Huffman and Covey, 1995;Vater et al., 1995).These
are the same connections that were consistently observed
with large injections of dextran amines in the present
is
Another area Of general agreement
the
types in the DNLL. Previous anatomical studies of the
DNLL in rat and cat report that there are various cell types
in the DNLL and each type is regionally distributed (Kane
and Barone, 1980;Iwahori, 1986;Shneiderman et al., 1988;
BaJo et al., 1993).Most investigators group DNLL cells into
three major types, fusiform, multipolar, and round O r oval
Cells, where each type more or less dominates different
regions o f t h e DNLL. These are the Same general cell types
that we observed in the mustache bat DNLL. Moreover, we
also observed that fusiform cells tend to be slightly more
common in the Posterior DNLL and that multipolar cells
are slightly more common in the anterior DNLL. As
mentioned in the opening section, the functional significance of the three cell types is at the moment unclear. In a
recent intracellular study of rat DNLL neurons in brain
slices, w u and Kelly (1995)report that all three types of'
DNLL cells express similar types of intrinsic membrane
properties.
We point out that our observations of DNLL cell types are
somewhat limited, as a complete description of the neuronal architecture of the DNLL was not the primary goal of
this study. Our evaluations of DNLL cell types, for example.
were made from material sectioned only in the transverse
plane and only from filled cells in the putative 60-kHz
contour of the DNLL. But even with these limitations, our
observations concerning cell types in the mustache bat
DNLL are consistent with those seen in other animals.
There are, however, two differences between the projec.tions that we found and those reported in other animals.
One is the presence of a small projection from the dorsal
cochlear nucleus that we found after injections of retrograde tracer in the posterior DNLL. Whereas inputs from
SUBDIVISIONS IN DNLL
589
Projections to Anterior DNLL
DNLL
d
DNLL
3
VNLL -%
4
Cochlear
Nucleus
Q
7
Cochlea
MNTB
Projections to Posterior DNLL
DNLL /
MNTB
Fig. 11. Schematic diagram showing projections to the anterior and
the posterior divisions of the DNLL. The thickness of the arrows
indicates the strength of the projections. The neuronal population in
each nucleus is indicated by solid and open circles. Notice that the
population of cells in each nucleus that projected to the anterior DNLL
(indicated by open circles) was different from the population that
projected to the posterior DNLL (indicated by solid circles). VNLL in
the top panel is highlighted, and its projection is shown as a dashed line
to indicate axons from VNLL projected only to anterior DNLL and not
to posterior DNLL. In the lower panel, MSO is highlighted, and its
projection is shown as a dashed line to indicate its axons projected only
to the posterior DNLL and not to the anterior DNLL. Abbreviations are
the same as in Figure 5.
590
the anteroventral cochlear nucleus have been reported in
the cat (Glendenning et al., 1981; Shneiderman et al.,
19881, and were seen in this study as well, inputs from the
dorsal cochlear nucleus have been questioned. Of interest
in this regard are observations by Shneiderman et al.
(1988). They never found terminals in DNLL following
injections of anterograde tracers in dorsal cochlear nucleus.
They suggest that the labeled cells in dorsal cochlear
nucleus reported by others (Glendenning et al., 1981) may
have been a consequence of HRP uptake in cut fibers
traveling through the DNLL. We cannot rule out the
possibility that cut fibers contributed to the labeled dorsal
cochlear nucleus cells seen in the present study, but we also
cannot rule out the possibility that the projection is real and
simply represents a small species-specificdifference.
The other difference concerns the presence of descending
projections from the ICc that have been reported in studies
of rat and cat (Carey and Webster, 1971; Hutson et al.,
1991; Caicedo and Herbert, 1993). We found very few
labeled cells in the ICc even after large injections of BD in
the DNLL. These data suggest that the mustache bat
DNLL does not receive significant descending inputs from
the ICc and further suggest that such a descending projection may be a species-specificfeature.
The overall view, then, is that although there may be
some minor species-specific features, the general features of
the mustache bat DNLL are consistent with those seen in
the DNLL of other mammals. What is not clear, and is very
difficult to evaluate, is whether the DNLLs in other mammals are also divisible into the two major subdivisions that
we observed in the mustache bat. One reason is that there
have been only a limited number of neurophysiological
studies of the DNLL in other mammals. Most of those
studies, however, did not map the response properties in
DNLL, nor did they evaluate response properties evoked by
S A M signals. The only exception was the study by Covey
(1993) of the DNLL in the big brown bat. She identified a
region just anterior to the main body of the DNLL that she
called the dorsal paralemniscal area. Although most of the
cells in the dorsal paralemniscal area were GABA positive,
as were the cells in the DNLL, the distinguishing feature of
the dorsal paralemniscal area is that it is dominated by
monaural neurons. Because the neurons in the anterior
DNLL of the mustache bat are all binaural and EI, and
because this region receives similar afferent inputs as the
posterior DNLL, we conclude that anterior DNLL in the
mustache is indeed a division of the DNLL and does not
correspond to the dorsal paralemniscal area.
Functional implications of the differential
afferent connections of the two divisions
of the DNLL
The results presented here provide a detailed picture of
the functional divisions of the mustache bat DNLL and the
afferent connections of each division. Below we discuss
some functional consequences of the afferent connectional
specificity and point out why, for the most part, the results
reported here raise more questions about the functional
impact of the connectional patterns than they answer.
Projections that target only one DNLL division. Given
the pronounced differences in response properties of neurons in the anterior and posterior DNLL, it was not
surprising to find that there are some differences in the
subset of lower nuclei that innervate the two DNLL
divisions. One prominent difference in afferent inputs is
L. YANG ET AL.
that the anterior region receives a substantial projection
from the VNLL, whereas the posterior region receives little
or no innervation from the VNLL. It would seem reasonable to suppose that the prominent projections from the
VNLL to the anterior DNLL play a significant role in
shaping its neuronal response properties. VNLL neurons
stain intensely for antibodies against conjugated glycine
and presumably exert inhibitory influences on their targets
(Park et al., 1991; Winer et al., 1995). Additionally, VNLL
neurons are strikingly similar to octopus cells in the
cochlear nucleus in that they discharge only once at the
onset of a tone burst, and each discharge has a remarkably
constant latency (Covey and Casseday, 1991). As VNLL
cells respond with only one initial discharge, the inhibitory
effect of that discharge probably has only a minor influence
on the initial phasic discharges of anterior DNLL neurons
evoked by tone bursts. Furthermore, the single discharges
from VNLL cells probably play no role in suppressing the
discharges to the later portions of the tone burst of anterior
DNLL neurons. With SAM signals, however, the VNLL
could provide repetitive bursts of inhibition to the anterior
DNLL that are tightly locked to the phase of each cycle of
the SAM signal. If this is the case, then it might well be that
the glycinergic inhibition provided by the VNLL could
contribute to the shaping of the responses of its DNLL
targets to SAM signals having different modulation rates.
In other words, it seems reasonable to assume that inhibition from VNLL plays at least some role in limiting the
phase-locking to SAM signals of higher modulation rates in
the anterior region of the DNLL.
The MSO is the other projection that targeted only one
DNLL division, in this case the posterior division. We point
out that the MSO in the mustache bat is largely monaural,
receiving inputs predominately from the contralateral cochlear nucleus and almost no inputs from the ipsilateral
cochlear nucleus (Covey et al., 1991; Grothe et al., 1992).
Additionally, Grothe (1994) showed that MSO neurons in
the mustache bat respond phasically to tone bursts and
phase-lock only to SAM signals with low modulation rates.
Paradoxically, although we found that the MSO projects
selectively to the posterior DNLL, the response properties
of MSO neurons are similar to those we observed in the
anterior DNLL, rather than to those of the posterior
DNLL. Further complicating interpretations of MSO projections are the results from recent studies which indicate that
the mustache bat MSO has GABAergic, glycinergic, and
excitatory principal cells, all of which may project to the ICc
and presumably to the DNLL as well (Vater et al., 1995;
Winer et al., 1995). In previous reports we pointed out that
the roles of either excitatory or inhibitory MSO neurons in
shaping the responses of their DNLL targets is unclear
(Yang and Pollak, 1994a-c). Unfortunately, a more detailed
view of the projections from MSO to DNLL provides no
additional insights into this question.
Projections that target both DNLL divisions. The finding that both DNLL divisions receive strong bilateral inputs
from the LSO was expected but also had some unexpected
features. On the one hand, we anticipated finding prominent inputs from the LSO as they are one of the primary
projections found in every previous connectional study of
the DNLL, and their binaural, EI properties are consistent
with the EI response properties of DNLL neurons. EI
properties are first created in the LSO (Boudreau and
Tsuchitani, 1968; Caird and Klinke, 1983; Moore and
Caspary, 1983; Covey et al., 19911, and our previous studies
SUBDIVISIONS IN DNLL
suggest strongly that the EI properties in the majority of
DNLL neurons are imposed upon DNLL neurons by a
well-established excitatory projection from the contralatera1 LSO (Yang and Pollak, 1994a,c). Furthermore, glycinergic inhibitory inputs from the ipsilateral LSO and
GABAergc inputs from the other DNLL are consistent
with other more complex properties evoked in DNLL
neurons by several stimuli that follow each other in close
temporal succession (Yang and Pollak, 1994~).These features were seen in DNLL neurons that responded with
onset discharges as well as those that responded with
sustained discharges to tone bursts.
In view of the above results, our expectation was that the
axonal projections from an LSO cell would innervate DNLL
neurons in both the anterior and posterior divisions because we could see no reason for segregating projections
from cell groups in each LSO. We were, therefore, surprised
to find that different LSO cells project to the anterior and
posterior regions of the DNLL. Indeed, the strict segregation of INLL neurons as well as the segregation of commissural neurons from the opposite DNLL that project to the
two DNLL regions were equally surprising and represent
one of the principal findings of this study. The prominence
of the segregated projections suggests that they must be
functionally relevant, although exactly what the functional
consequences of such a segregated projection system might
be is unclear to us.
Functional impact of the two DNLL divisions
on their targets in the inferior colliculus
Although the pattern of efferent projections will be
described in a future report, both divisions of the DNLL
target the inferior colliculus bilataterally, and the projections from each division target similar or even overlapping
regions in the two colliculi. As mentioned previously, the
GABAergic influences of the DNLL play a substantial role
in shaping the properties of many EI cells in the ICc (Li and
Kelly, 1992; Vater et al., 199213; Faingold et al., 1993; Park
and Pollak, 1993; Klug et al., 1995). These DNLL influences are thought to be mainly, although not exclusively, on
the contralateral ICc. In view of the differences in the ways
neurons in the two DNLL divisions respond to both tone
bursts and SAM signals, the inhibitory impact of projections from the anterior division must be considerably
different from those of the projections from the posterior
division, and this holds for the projections from both the
ipsi- and contralateral DNLLs. It would follow, therefore,
that the influences of the DNLL on the ICc are more varied
and complex than was previously thought. Specifically, the
results suggest that each DNLL division may differentially
shape not only the binaural properties of their targets in
the inferior colliculus, but also their coding of the time
variations in complex signals.
ACKNOWLEDGMENTS
We thank Albert Berrebi for his advice on the use of
dextran tracers used in this study and Mr. Carl Resler for
designing and implementing the electronics for data acquisition. We also thank Eric Bauer, Michael Burger, Achim
Hug, Carl Resler, Albert Berrebi, Brett Schofield, and John
Zook for their critical comments on the manuscript. This
work is supported by National Institutes of Health grant
DC 20068.
591
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