Download Central projections of auditory receptor neurons of crickets

THE JOURNAL OF COMPARATIVE NEUROLOGY 493:439 – 447 (2005)
Central Projections of Auditory
Receptor Neurons of Crickets
KAZUO IMAIZUMI AND GERALD S. POLLACK*
Department of Biology, McGill University, Montreal, Quebec H3A1B1, Canada
ABSTRACT
We describe the central projections of physiologically characterized auditory receptor
neurons of crickets as revealed by confocal microscopy. Receptors tuned to ultrasonic frequencies (similar to those produced by echolocating, insectivorous bats), to a mid-range of
frequencies, and a subset of those tuned to low, cricket-like frequencies have similar projections, terminating medially within the auditory neuropile. Quantitative analysis shows that
despite the general similarity of these projections they are tonotopic, with receptors tuned to
lower frequencies terminating more medially. Another subset of cricket-song-tuned receptors
projects more laterally and posteriorly than the other types. Double-fills of receptors and
identified interneurons show that the three medially projecting receptor types are anatomically well positioned to provide monosynaptic input to interneurons that relay auditory
information to the brain and to interneurons that modify this ascending information. The
more laterally and posteriorly branching receptor type may not interact directly with this
ascending pathway, but is well positioned to provide direct input to an interneuron that
carries auditory information to more posterior ganglia. These results suggest that information about cricket song is segregated into functionally different pathways as early as the level
of receptor neurons. Ultrasound-tuned and mid-frequency tuned receptors have approximately twice as many varicosities, which are sites of transmitter release, per receptor as
either anatomical type of cricket-song-tuned receptor. This may compensate in part for the
numerical underrepresentation of these receptor types. J. Comp. Neurol. 493:439 – 447, 2005.
© 2005 Wiley-Liss, Inc.
Indexing terms: insect; hearing; tonotopy; communication
Hearing in crickets is a model system for studying sensory processing. Crickets use acoustic signals to recognize
and localize both conspecifics and predators (echolocating
bats). A number of recent studies have used electrophysiological, behavioral, and computational approaches to investigate the neural mechanisms underlying signal detection, frequency analysis, temporal processing, sound
localization, and signal recognition (Imaizumi and Pollack, 1999, 2001; Samson and Pollack, 2002; Hennig,
2003; Pollack, 2003; Nabatiyan et al., 2003; Reeve and
Webb, 2003; Hedwig and Poulet, 2004; Marsat and Pollack, 2004, 2005; Tunstall and Pollack, 2005). Relatively
little is known, however, about the anatomical substrates
of neural processing.
Each ear in the cricket contains ⬃65 receptor neurons
(Michel, 1974; Young and Ball, 1974; Imaizumi, 2001). In
Teleogryllus oceanicus these can be classified into three
groups based on selectivity for sound frequency. These are
most sensitive, respectively, to low frequencies near the
dominant component of the species’ communication signals, 4.5 kHz; to ultrasonic frequencies similar to those
© 2005 WILEY-LISS, INC.
emitted by a class of predator, echolocating bats; and to an
intermediate range of frequencies (Imaizumi and Pollack,
1999, 2001). Low-frequency receptors are by far the most
numerous, comprising ⬃75% of the receptor population
(Pollack and Faulkes, 1998; Imaizumi and Pollack, 1999,
2001) and, as we previously reported briefly, comprise two
anatomically distinct subgroups (Imaizumi and Pollack,
Grant sponsor: Natural Sciences and Engineering Research Council of
Canada; Grant number: RGPIN 7151; Grant sponsor: Whitehall Foundation; Grant number: F96-13 (to G.S.P.); Grant sponsor: the Government of
Canada (to K.I.).
Kazuo Imaizumi’s present address is Coleman Memorial Laboratory,
W.M. Keck Foundation Center for Integrative Neuroscience, UCSF, San
Francisco, CA, 94143-0732.
*Correspondence to: Gerald S. Pollack, Dept. of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, QC H3A1B1, Canada. Email: [email protected]
Received 16 February 2005; Revised 9 June 2005; Accepted 12 July 2005
DOI 10.1002/cne.20756
Published online in Wiley InterScience (www.interscience.wiley.com).
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K. IMAIZUMI AND G.S. POLLACK
2001). The remaining receptors are divided nearly equally
between the mid-frequency and ultrasound receptors. The
receptor neurons project to the prothoracic ganglion where
they interact with a number of identified central neurons,
the anatomy of which has been thoroughly described
(Wohlers and Huber, 1985; Atkins and Pollack, 1987a;
Stiedl et al., 1997).
Within the ear, receptor neurons are organized tonotopically (Oldfield et al., 1986). However, the anatomy and
organization of their projections to the central nervous
system have been reported only briefly (Eibl and Huber,
1979; Esch et al., 1980; Imaizumi and Pollack, 2001). In
this article we present a detailed description of the afferent auditory projection, as studied using single-cell intracellular staining and confocal microscopy.
MATERIALS AND METHODS
Teleogryllus oceanicus were raised in the laboratory on
a diet of Purina cat chow and water. Adult virgin females,
aged 14 –23 days after the final molt, were used. Coldanesthetized animals were secured with wax to a platform
ventral side up after removing the wings and mid- and
hindlegs. The forelegs, which bear the ears, were held
flexed against the sides of the pronotum in a position
similar to that adopted during flight. The prothoracic ganglion was exposed by ventral dissection, bathed in modified TES ringer (Strausfeld et al., 1983), and supported on
a metal platform.
Axons of receptor neurons were penetrated in the leg
nerve, near its entrance into the prothoracic ganglion,
with microelectrodes, the tips of which were filled either
with 2– 4% Lucifer Yellow CH (Aldrich, Milwaukee, WI;
resistance, 100 –300 M⍀) or with 2.5–25 mM Alexa 488 or
568 hydrazide (Molecular Probes, Eugene, OR; resistance
150 –1,000 M⍀); electrode shafts were filled with 2 M LiCl.
Interneurons were impaled in their dendritic processes
within the prothoracic ganglion. Neurons were stained by
injecting negative current (1–10 nA). Ganglia containing
dye-filled neurons were fixed in 4% formaldehyde in 0.1 M
phosphate buffer (pH 7.3) for 10 –30 minutes followed by
4% formaldehyde in methanol for 1 hour, then washed
with 100% ethanol for 15 minutes. Ganglia were cleared in
methyl salicylate, mounted on a depression slide, and
viewed with confocal microscopy (Leica CLSM or Zeiss
LSM510). Selected specimens were embedded in Spurr’s
resin and sectioned transversely at 20 ␮m. For embedding, ganglia were washed with fresh methyl salicylate for
10 minutes and then transferred sequentially to a 1:1
mixture of methyl salicylate and 100% ethanol (1 hour),
100% ethanol (1 hour), 1:1 mixture of 100% ethanol and
Spurr (1 hour), and 100% Spurr (overnight), all at room
temperature. Ganglia were then transferred to fresh
Spurr and incubated at 60°C (ⱖ19 hours). Sections were
viewed and photographed with standard or confocal fluorescent microscopy and drawn by tracing the image of a
negative projected with a photographic enlarger.
Receptor neurons were classified physiologically on the
basis of their responses to 30-ms sound pulses, presented
twice per second. Sound frequency ranged from 3– 40 kHz
and intensity ranged from 40 –100 dB SPL. Our experimental protocol was to hold intensity constant while scanning through the range of test frequencies. We repeated
this for as many intensity levels as recording duration
(mean duration ⬃3 minutes) permitted. A neuron’s char-
acteristic frequency (CF) was defined either as the frequency at which threshold was lowest or (when recordings
could not be held long enough for complete determination
of the neuron’s threshold curve) as the frequency that
elicited the largest number of action potentials at the
lowest suprathreshold intensity tested. Stimuli were produced by National Instruments (Austin, TX) digitalanalog boards driven by custom software. See Imaizumi
and Pollack (1999) for further details.
Quantification of anatomy
We quantified a neuron’s projection pattern by mapping
the positions of varicosities on its axons terminals in confocal optical sections. Ultrastructural studies show that
varicosities are rich in synaptic vesicles, and thus are good
markers for presynaptic sites (Hardt and Watson, 1999).
Optical horizontal sections were first made at low power
(6.3⫻ objective) to visualize the entire projection, as well
as the borders of the ganglion. A second set of sections was
then made at higher power (25⫻ water-immersion) at
intervals of 1.5–2 ␮m, overlapping the fields of view when
required to encompass all of a neuron’s varicosities. Arbors (or portions of them) were reconstructed by collapsing
series of sections into a single plane.
Counts of varicosities were made directly on the monitor
screen. An acetate sheet was fixed on the screen and
individual varicosities were marked and counted sectionby-section, using the reconstructed arbors for guidance.
We mapped the positions of varicosities in the horizontal plane on confocal, optical sections using the programs
Scion Image for Windows (http://www.scioncorp.com) or
UTHSCA Image Tool (http://ddsdx.uthscsa.edu). When
necessary, neighboring fields of view were aligned with
reference to landmarks common to both of them. Positions
of varicosities were normalized with respect to the length
and width of the hemiganglion (see Fig. 2). We estimate
that errors introduced by the position of the ganglion
along the curvature of the cavity of the depression slide
were less than 0.6%; therefore, we did not correct for them.
Series of confocal sections were collapsed into single
images by setting the value of each pixel in the final image
to the maximum value for the same x-y coordinates in the
contributing sections, using the software native to the
confocal microscope systems (Leica CLSM or Zeiss
LSM510). Image brightness and contrast were adjusted
using Gimp 2.2, running on a Linux workstation. The
image in Figure 5 was additionally sharpened using a
deconvolution filter.
RESULTS
The results presented are based on intracellular fills of
146 auditory receptors (98 low-frequency, 22 midfrequency, and 26 ultrasound receptors). Forty-eight receptors, for which intracellular penetration could be maintained long enough for excellent staining, were selected
for quantitative analysis (32 low-frequency, 7 midfrequency, and 9 ultrasound receptors). The remaining 98
receptors provided additional supporting evidence.
Two anatomically distinct receptor types
Horizontal and transverse reconstructions of axon terminals are illustrated in Figure 1. Axonal arbors are of
two types. One type terminates near the midline of the
PROJECTIONS OF AUDITORY RECEPTOR NEURONS
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Fig. 1. A: Horizontal reconstructions, from confocal sections,
of the terminal branches of auditory receptor neurons. Four different types of receptor neurons
based on morphological and physiological differences are illustrated. B: Termination sites of different types of receptor neurons in
the horizontal (upper drawings in
each panel) and transverse (lower
drawings) planes. Orientation is
indicated by the arrows: A, anterior; P, posterior; D, dorsal; V,
ventral. The characteristic frequency of each neuron is indicated. Drawings of the transverse
sections include several axon
tracts and neuropil areas. DMT,
dorsal median tract; DIT, dorsal
intermediate tract; DCIII, dorsal
commissure III; VIT, ventral intermediate tract; R5, root of nerve
5; mVAC, median ventral association center; VMT, ventral median
tract. Nomenclature after Pflüger
et al. (1988) and Wohlers and Huber (1985). Scale bars ⫽ 50 ␮m in
A; 200 ␮m in B.
prothoracic ganglion (medial termination; MT), and the
other terminates more laterally, with large, bifurcating
branches (bifurcating: BC). BC types occur only among
low-frequency receptors, whereas MT types occur among
all three frequency classes (Fig. 1A). For convenience, in
this work we use the designations BC and MT to refer only
to the two types of low-frequency receptor.
MT-type, mid-frequency, and ultrasound receptors terminate in the anterior part of the auditory neuropil (medial ventral association center, mVAC). The projections of
these receptors are also similar in transverse sections
(Fig. 1B). The mVAC contains the dendrites of several
identified auditory interneurons (Wohlers and Huber,
1985), and is a site of synaptic contact between receptors
and interneurons (Hardt and Watson, 1984; Watson and
Hardt, 1996; Hirtz and Wiese, 1997). The anterior
branches of BC-type receptors enter the lateral part of the
mVAC. Their posterior branches terminate in a region
posterior to the mVAC.
Many receptor neurons have small branches within the
root of nerve 5, lateral to the mVAC, where several central
neurons (ON1, ON2, AN2, DN1) also have processes
(Wohlers and Huber, 1985; Atkins and Pollack, 1987a;
Hardt and Watson, 1994; Watson and Hardt, 1996). These
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Fig. 2. Coordinate system for quantification of varicosity positions.
A: Length of the anterior–posterior (A-P) axis defined as the distance
from the anteriormost to the posteriormost portion of the midline.
Length of the medial–lateral (M-L) axis defined as the distance from
the midline to the posterior edge of the entry point of nerve 5. B: In 25
(of 48) ganglia, the midline (readily apparent by its autofluorescence)
was shifted laterally, probably due to asymmetric shrinkage of the
ganglion during fixation/dehydration. In these cases the M-L axis was
defined as the distance from a line tangent to the most lateral point of
the midline to the junction between the ganglion and the posterior
edge of nerve 5. The shift shown in B is an extreme case. Scale bar ⫽
200 ␮m in A (applies to A,B).
Fig. 3. Positions of varicosities in
the horizontal plane. Each panel
shows three representative receptors.
A: BC types; B: MT types; C: midfrequency receptors, and D: ultrasound receptors. The CF of each receptor is indicated.
small branches are most prevalent on BC-type, midfrequency, and ultrasound receptors.
Quantification of axonal arbors
The axon terminals of receptor neurons bear many varicosities (see Fig. 1A), and ultrastructural studies have
shown that these are sites of synaptic output (Hardt and
Watson, 1999). We quantified the anatomical projections
of receptor neurons by mapping the positions of their
varicosities in the horizontal plane. Figure 2 illustrates
the coordinate system. The position of each varicosity was
expressed as the percentage along the length of the
anterior–posterior (A-P) and medial–lateral (M-L) axes of
the hemiganglion. Figure 3 shows the positions of varicos-
ities of representatives of each of the four receptor types.
BC types differ strikingly from the other receptors in that
they terminate more laterally. We compared this difference quantitatively for the two anatomical types of lowfrequency receptors. The M-L coordinate of the most medial varicosity of BC types was 18.0 ⫾ 2.3% (mean ⫾ SD);
the corresponding value for MT types was 2.3 ⫾ 2.1% (P ⬍
0.0001, t-test).
Tonotopy
We first examined the possibility of tonotopy of the
afferent projection by summarizing the distribution of varicosities of all receptors, including both MT and BC types,
along the A-P and M-L axes as the means of the coordinate
PROJECTIONS OF AUDITORY RECEPTOR NEURONS
443
Fig. 5. Co-occurrence of varicosities of an MT-type receptor (red)
and an ultrasound receptor (green) in the same, 10.2-␮m-thick slice of
neuropil. The image was produced by collapsing three 3.4-␮m-thick
horizontal optical sections. Scale bar ⫽ 10 ␮m.
Fig. 4. Tonotopic organization of mean positions of varicosities of MT
types, mid-frequency, and ultrasound receptors within the mVAC. This
organization is apparent along the M-L axis (A), but not along the A-P
axis (B). Statistical results are from linear regression analysis.
values of all of their varicosities. When all varicosities are
considered, there is no significant relationship between
CF and their mean positions, along either the A-P or M-L
axis (linear regression, n ⫽ 48, data not shown). However,
of particular interest is the organization within the
mVAC, in which extensive interactions occur between receptors and central neurons (see above). It is difficult to
define the mVAC in our confocal reconstructions because
the internal structure of the ganglion is not visible. However, varicosities are densely packed in a region extending
from 35–50% along the A-P axis and from 0 – 40% along
the M-L axis (Fig. 3). We operationally take these boundaries as delimiting the mVAC. To compute mean termination position within the mVAC we included only varicosities located within these boundaries. The anatomical
differences between BC types and the other three types,
considered together with the anatomy of central neurons
(Wohlers and Huber, 1985; Hardt and Watson, 1994;
Watson and Hardt, 1996), suggest that the target neurons
of BC types may differ from those of the other types. In
particular, BC types are unlikely to make extensive, direct
connections with the three central neurons that have been
most directly implicated in auditory behavior, namely,
ON1, AN1, and AN2 (Atkins et al., 1984; Nolen and Hoy,
1984; Schildberger and Hörner, 1988; see below). To examine whether the axonal projections onto a common set
of potential central targets might be tonotopic, therefore,
we excluded BC types from this analysis. Figure 4A shows
that, for this restricted set of receptor neurons, the projection to the mVAC is indeed tonotopic along the M-L
axis, with receptors that are tuned to higher frequencies
terminating more laterally (n ⫽ 29, r2 ⫽ 0.252, P ⫽ 0.005).
Although statistically significant, the magnitude of the
difference in mean position is only 4% of the M-L width,
corresponding to ⬃20 ␮m. There is no significant relationship along the A-P axis (Fig. 4B; n ⫽ 29, r2 ⫽ 0.054, P ⫽
0.225). Nor is there a significant relationship between CF
and M-L position within any of the three receptor populations (low-frequency receptors, n ⫽ 14, r2 ⫽ 0.06, P ⫽
0.4; mid-frequency receptors, n ⫽ 7, r2 ⫽ 0.239, P ⫽ 0.219;
ultrasound receptors, n ⫽ 8, r2 ⫽ 0.239, P ⫽ 0.219).
Receptor terminals might, in principle, also be distributed systematically in the dorsoventral plane, as occurs in
tettigoniids (Römer, 1983). However, as shown in Figure
1B, receptors of all types terminate at approximately the
same depth within the neuropil. Figure 5 shows projections from an MT-type receptor (red) and an ultrasound
receptor (green) within the same, 10.2-␮m-thick region of
neuropil. Varicosities from both receptors are intermingled. Co-occurrence of varicosities from the two receptors
occurs throughout the entire depth of their projections
(not shown). Thus, although we cannot rule out the possibility of a subtle segregation of receptor terminals along
the dorsoventral axis, their projections overlap considerably in depth.
Overlap between receptor projections and
interneuron dendrites
We examined possible patterns of connectivity between
receptor neurons and central neurons by staining both in
the same individual (Fig. 6). Figure 6A,B shows low-power
views of the interneurons ON1 and DN1, respectively.
ON1’s medial dendrites occur in the same restricted re-
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K. IMAIZUMI AND G.S. POLLACK
Fig. 7. Number of varicosities varies between different types of
receptor neuron. Significant differences between receptor types were
determined by t-tests, adjusted for multiple comparisons using the
sequential Bonferroni technique (Rice, 1989), and are indicated by
bars and asterisks; **P ⬍ 0.01; ***P ⬍ 0.001; ****P ⬍ 0.0001. There
is no significant difference between BC and MT types, nor between
mid-frequency and ultrasound receptors. Sample size was 18, 14, 7,
and 9 for BC-type, MT-type, mid-frequency, and ultrasound receptors,
respectively.
Fig. 6. Anatomical relationships between axon terminals of receptor neurons and dendrites of central neurons. A,B: Low-power views
show the overall structures of the identified interneurons ON1 (A) and
DN1 (B) and their positions in the prothoracic ganglion. White rectangles indicate the approximate regions that are shown in C–F. C,D:
The termination sites of MT types (C) and ultrasound (D) receptors
(green) overlap with the medial dendrites of ON1 (red). E: The terminal branches and varicosities of BC types are lateral and posterior of
ON1’s medial dendrites. The string of varicosities (white arrows) that
appears to overlap with a large process of ON1 is in fact 18 –20 ␮m
more dorsal than the ON1 branch. F: BC types do, however, overlap
with the dendrites of another identified interneuron, DN1. Insets in
C,D,F are enlarged views of selected regions (indicated by white
rectangles), produced by collapsing three adjacent horizontal confocal
slices, for a total section thickness of 2.5 ␮m. Scale bars ⫽ 100 ␮m in
A,B; 20 ␮m in C–F (main image); 10 ␮m for insets C,D,F.
gion as the projections within the mVAC of two other
identified neurons, AN1 and AN2 (Wohlers and Huber,
1985), and thus serve as a marker for the medial dendrites
of these neurons. DN1’s dendrites lie mainly outside of
this region. Figure 6C illustrates the overlap between MT
types and the medial dendritic tuft of ON1. Thus, MT
receptors are anatomically well suited to provide extensive direct input to all three of these interneurons (although probably with carrier-frequency-specific connectivity). Figure 6D shows that the same is true of ultrasound
receptors. By contrast, as a consequence of the more lateral and posterior projections of BC-type receptors, there
is little or no overlap between their terminal branches and
ON1’s medial dendrites (Figs. 3A, 6E). Absence of overlap
applies as well to the short processes arising from the
axons of BC-type receptors, which are posterior to the
lateral process of ON1 (not shown). Thus, BC types probably make few, if any, direct connections with ON1, AN1,
or AN2 (Pollack and Imaizumi, 1999). This observation
raises the question: what are the targets of BC types? DN1
has extensive dendritic processes in the same region as
the branches of BC receptors (Atkins and Pollack, 1987a),
and is thus a potential postsynaptic target of BC types.
Figure 6F indeed confirms that the terminal branches of
BC receptors overlap with DN1’s dendritic processes.
Number of varicosities
The impact of a receptor on central neurons will be
determined in part by the amount of neurotransmitter it
releases. As varicosities on axon terminals are known to
be presynaptic sites (Hardt and Watson, 1999), we
counted varicosities on the terminals of the different types
of receptor neuron as one indicator of their probable central impact. The number of varicosities per receptor neuron varies with CF. Mid-frequency and ultrasound receptors have significantly more varicosities per neuron than
either type of low-frequency receptor, which do not differ
from one another (Fig. 7).
DISCUSSION
Sound frequency-specific anatomical
differences
Systematic relationships between the peripheral positions of the cell bodies of sensory neurons and the locations of their central terminals have been described for
several insect sensory systems, for example, mechanosensory hairs (Mücke and Lakes-Harlan, 1985; Johnson and
Murphey, 1985; Newland, 1991) and chordotonal sensilla
(the sensillum type of auditory receptors) (Nishino, 2000,
2003; Nishino and Field, 2003).
PROJECTIONS OF AUDITORY RECEPTOR NEURONS
445
In auditory systems, the position of a receptor neuron’s
cell body within the ear is related to its frequency sensitivity, as well as to the location of its terminations in the
central nervous system. Tonotopy has been described in
other orthopteran insects. Both in tettigoniids (Römer,
1983; Stölting and Stumpner, 1998) and in acridids (Halex
et al., 1988; Jacobs et al., 1999), receptor neurons that are
tuned to different sound frequencies project systematically to different regions of auditory neuropil. Although
our quantitative analysis did reveal central tonotopy of
cricket auditory receptors (Fig. 4), this was rather minor
compared to other orthopterans. There is considerable
overlap, for example, between the projection areas of MT
types, mid-frequency, and ultrasound receptors.
The difference in degree of tonotopy between crickets
and tettigoniids is particularly notable because the peripheral organization of auditory receptors is similar in
these groups (Oldfield, 1982; Oldfield et al., 1986). In both
cases, receptors are arrayed along the proximodistal axis
of the tibia, with proximal receptors tuned to low sound
frequencies, and distal receptors to high frequencies. Despite anatomical similarity, the receptor arrays differ
physiologically. In tettigoniids, there is a relatively gradual shift in tuning from receptor to receptor (Oldfield,
1982; Römer, 1983; Stölting and Stumpner, 1998),
whereas in crickets the representation of sound frequency
by the receptor array is discontinuous, with receptors
falling into three distinct groups based on frequency tuning (Imaizumi and Pollack, 1999). This physiological organization in crickets coincides with behavioral tests that
demonstrate that crickets perceive sound frequency categorically, rather than as a continuous range of frequencies
(Wyttenbach et al., 1996).
At least one identified interneuron, ON1, receives input
from both low-frequency and ultrasound receptors; this
neuron has a W-shaped tuning curve, with enhanced sensitivity in both of these frequency ranges (Atkins and
Pollack, 1986). Faulkes and Pollack (2001) showed previously that ultrasound input to ON1 is monosynaptic but,
based on several electrophysiological observations, they
proposed that low-frequency input might be mainly
polysynaptic. However, our double fills suggest strongly
that MT types provide direct input to ON1 for processing
of low-frequency sounds (Fig. 6). This is supported by
simultaneous paired intracellular recordings from MT
types and ON1 (G.S. Pollack, G. Marsat, P. Sabourin,
unpublished observation).
Our earlier work demonstrated the numerical imbalance between low-frequency and ultrasound receptors
(Pollack and Faulkes, 1998; Imaizumi and Pollack, 1999,
2001). However, despite the underrepresentation of ultrasound at the receptor-neuron level, ultrasonic stimuli
evoke robust responses in central neurons, including ON1,
in part because unitary, ultrasound-elicited EPSPs, recorded in ON1’s large, dendritic integrating segment, are
relatively large (Pollack, 1994; Faulkes and Pollack,
2001). One possible explanation for this is that the termination sites of ultrasound receptors are electronically
more favorable than those of low-frequency receptors, resulting in less attenuation of ultrasound inputs. The
present findings suggest that this is unlikely; both MT
types and ultrasound receptors terminate in approximately the same region. Indeed, the slight difference in
location, with ultrasound receptors terminating more laterally than low-frequency receptors, would favor in-
creased attenuation of ultrasound-elicited EPSPs. Our
counts of varicosities suggest another factor that might
contribute to the large sizes of ultrasound-induced unitary
EPSPs. Ultrasound receptors (and mid-frequency receptors) have approximately twice as many varicosities per
receptor as low-frequency receptors, suggesting that they
may make more contacts with their target neurons.
Segregation of low-frequency information at
the primary-neuron level
The most striking anatomical distinction among the
physiologically different receptors occurs between BC and
MT types. BC types project more laterally and posteriorly
than MT types. This difference is particularly interesting
in view of the fact that both anatomical types are tuned to
cricket-like sound frequencies.
Projections similar to those of MT and BC types have
been described previously. Esch et al. (1980) described
receptors in the cricket Gryllus bimaculatus that resemble
BC and MT types, and were tuned to the sound frequency
of the species’ song (4 –5 kHz). However, in their illustration (their fig. 7B), both types terminate at similar locations along the M-L axis. It is not possible to determine
from their results whether the BC-like morphology was
restricted to low-frequency-tuned receptor neurons. Mason et al. (1999) also described receptors with MT-type
and BC-type morphology in the haglid Cyphoderris monstrosa, a primitive member of the suborder Ensifera,
which includes crickets. The two types were similar in
minimum threshold and best frequency but differed in
bandwidth; receptors with BC-type morphology responded
relatively poorly to the higher sound frequencies used for
communication.
Double fills of receptor neurons and ON1 suggest that
MT-type and BC-type receptors may interact with different central pathways. MT types are anatomically well
suited to provide direct input to AN1, an ascending, lowfrequency-tuned interneuron that plays an important role
in behavioral responses to cricket song (Wohlers and Huber, 1982, 1985; Schildberger and Hörner, 1988). MT types
can also influence ascending information indirectly
through their connections with ON1, which provides contralateral inhibition to AN1 (Faulkes and Pollack, 2000).
AN1 appears to be the main source of afferent input to
brain circuits that filter stimulus temporal pattern
(Schildberger, 1984), a stimulus feature that is crucial for
signal recognition.
By contrast, BC types make few if any direct connections with ON1 or, by extension, with AN1, and thus may
have little or no influence on ascending auditory information. They are, however, a possible source of input to DN1,
a descending neuron that relays information about cricket
songs to more posterior ganglia (Wohlers and Huber,
1982; Atkins and Pollack, 1987b). Consistent with this, in
T. oceanicus both BC types and DN1 have relatively high
minimum thresholds (BC types: 62.3 ⫾ 7.3 dB SPL,
Imaizumi and Pollack, 2001; DN1: 65–70 dB SPL, Atkins
and Pollack, 1987b) compared to MT types (52.2 ⫾ 8.3 dB
SPL; Imaizumi and Pollack, 2001) and ON1 (44 ⫾ 3.3dB
SPL, Atkins and Pollack, 1986) or AN1 (46.1 ⫾ 4.2 dB
SPL, Marsat and Pollack, 2005).
A possible function of descending auditory information
is to provide input to motor circuitry that accomplishes
phonotactic steering. This circuitry has access to temporally detailed information about sound stimuli (Pollack
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K. IMAIZUMI AND G.S. POLLACK
and Hoy, 1981; Hedwig and Poulet, 2004). Recent experiments (Hedwig and Poulet, 2004) suggest that phonotaxis
might be accomplished by reflex-like (though not direct)
circuitry between auditory afferents and motor neurons,
gated by descending inputs from the brain reflecting recognition of the stimulus temporal pattern (Pollack and
Hoy, 1981; Doherty, 1991; Hedwig and Poulet, 2004). Two
physiological features of the BC types are interesting in
this respect. First, although threshold for phonotaxis is
similar to that of MT types (Doolan and Pollack, 1985;
Schmitz, 1985), behavioral performance improves with
increasing intensity, reaching a plateau at ⬃60 dB SPL,
similar to the intensity at which BC types and DN1 would
come into play (Pollack and Plourde, 1982; Schmitz, 1985).
Second, intensity-response functions of BC types are
steeper than those of MT types (Imaizumi and Pollack,
2001), perhaps facilitating their coding of stimulus amplitude at each ear, which is the dominant cue for sound
direction (Pollack, 2003). Our results raise the intriguing
possibility that the neural substrates for two different
components of acoustic behavior, signal recognition and
phonotactic steering, are segregated from the earliest
stage of processing, with input for the former carried
mainly or exclusively by MT-type receptors, and that for
the latter by BC-type receptors.
The relative numbers of BC and MT types are similar (of
98 low-frequency receptors stained in the present study,
53 were BC types and 45 were MT types). Although much
attention has been paid to how acoustic signals are represented and processed by ON1 and by ascending neurons,
relatively little is known about the descending pathway
(Wohlers and Huber, 1982; Atkins and Pollack, 1987b).
Our finding, that more than a third of the entire receptor
population may interact mainly, if not exclusively, with
descending interneurons, suggests that this component of
auditory circuitry warrants further investigation.
ACKNOWLEDGMENTS
We thank Alan Watson for advice on nomenclature,
Maureen Leung for editorial assistance, and Christoph
Schreiner, Jeffery Winer, and Catherine Carr for comments on an early version of the article.
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