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Cross-modal Circuitry Between Auditory
and Somatosensory Areas of the Cat
Anterior Ectosylvian Sulcal Cortex: A ‘New’
Inhibitory Form of Multisensory
Convergence
Lisa R. Dehner1, Leslie P. Keniston2, H. Ruth Clemo2 and
M. Alex Meredith2
Examples of convergence of visual and auditory, or visual and
somatosensory, inputs onto individual neurons abound throughout
the brain, but substantially fewer incidences of auditory–somatosensory neurons have been reported. The present experiments
sought to examine auditory–somatosensory convergence to assess
whether there is a feature of this type of convergence that might
obscure it from conventional methods of multisensory detection.
Auditory–somatosensory convergence was explored in cat anterior
ectosylvian sulcus (AES) cortex, where higher-order somatosensory
area IV (SIV) and auditory field of the anterior ectosylvian sulcus
(FAES) share a common border. While neuroanatomical tracers
documented a projection from FAES to SIV, physiological studies
failed to reveal the bimodal neurons expected from such cross-modal
connectivity. Stimulation of FAES through indwelling electrodes also
failed to excite any of the SIV neurons examined. However, when
stimulation of auditory FAES was combined with somatosensory
stimulation, a large majority (66%) of SIV neurons showed a significant response attenuation. FAES-induced response suppression was
specific to SIV, could not be elicited by activating other auditory
regions and was blocked by the microiontophoretic application of the
GABAergic antagonist bicuculline methiodide. Based on these data,
a novel, cross-modal circuit is proposed involving projections from
auditory FAES to somatosensory SIV, where local inhibitory interneurons ‘reverse the sign’ of the cross-modal signals to produce
auditory–somatosensory suppression. This form of excitatory–inhibitory multisensory convergence has not been reported before and
suggests that the level of interaction between auditory and somatosensory modalities has been substantially underestimated.
Even though multisensory neurons have been identified within
a host of cortical areas (e.g. Jiang et al., 1994a,b; Benedek et al.,
1996; Duhamel et al., 1998; Xing and Andersen, 2000;
Schroeder et al., 2001), the neural circuitry underlying multisensory convergence is not well understood and broad organizational principles for multisensory processing have yet to be
proposed. The overwhelming number of cortical multisensory
studies has reported the convergence of visual and auditory, or
visual and somatosensory inputs (e.g. Calvert, 2001). In
contrast, comparatively little is known about neuronal
responses to converging auditory and somatosensory information. Except for recent studies of somatosensory inputs to
auditory cortex (Schroeder et al., 2001; Schroeder and Foxe,
2002; Brett-Green et al., 2003) and several attentive or
cognitive studies (Jousmaki and Hari, 1998; Caclin et al., 2002;
Fujiwara et al., 2002; Guest et al., 2002; Hotting et al., 2003),
published accounts of auditory–somatosensory convergence
or integration are comparatively rare. Furthermore, when
neurons receiving auditory and somatosensory inputs have
been reported, they showed the lowest incidence among the
different patterns of multisensory convergence (Meredith and
Stein, 1986; Wallace et al., 1992, 1993; Jiang et al., 1994a,b).
Although multisensory neurons represented ∼55–60% of the
neurons in the deep layers of the cat superior colliculus, the
proportion of auditory–somatosensory neurons was only 4%
(Meredith and Stein, 1986); auditory–somatosensory neurons
in the cat anterior ectosylvian sulcal cortex comprised only
4% as well (Wallace et al., 1992); for the primate superior
colliculus, the ratio was 27% multisensory to 0.9%
auditory–somatosensory (Wallace et al., 1996). Despite this
low level of occurrence, these reports also indicated that
auditory–somatosensory convergence and integration operate
under the same constraints as the other modality combinations. Therefore, further examination of this infrequent
phenomenon will provide insight not only into the incidence
of this particular pattern of sensory convergence, but will also
contribute to our understanding of the basic principles of
multisensory convergence and integration in general.
The anterior ectosylvian sulcus (AES) is one of but a few
cortical locations that hosts adjoining representations of the
auditory and somatosensory modalities and because the probability of connectivity increases with physical proximity
(Young et al., 1995), this area seemed well suited for investigating potential cross-modal connections between these
sensory modalities. The AES, as depicted in Figure 1, is found
at the junction of the frontal, parietal and temporal regions of
the cat cortex and is composed of a horizontal (anterior) limb
and a vertical (posterior) limb. Within the dorsal bank of the
horizontal limb, there is a representation of the body surface
identified as somatosensory area SIV (Clemo and Stein, 1982,
1983, 1984). Receptive field reversals and cytoarchitectonic
differences distinguish SIV from SII on the adjacent anterior
ectosylvian gyrus (Burton et al., 1982; Clemo and Stein, 1983).
To its medial/deep side, SIV transitions into an area of the AES
fundus termed para-SIV, where neurons have somatosensory
receptive fields that are not topographically arranged, expand
to include hemi-body and whole-body representations, and
exhibit multisensory responses (Clemo and Stein, 1983). Posterior to SIV the somatosensory representation transitions into
the auditory field AES (FAES) (Clarey and Irvine, 1986, 1990a,b;
Meredith and Clemo, 1989; Wallace et al., 1992, 1993; Korte
and Rauschecker, 1993; Rauschecker and Korte, 1993; Middlebrooks et al., 1994; Rauschecker, 1995, 1996; Benedek et al.,
1996). The FAES occupies the anterior and posterior banks of
the vertical limb of the AES and is distinguished from both the
anterior auditory field and AI by its lack of tonotopic organiza-
Cerebral Cortex V 14 N 4 © Oxford University Press 2004; all rights reserved
Cerebral Cortex April 2004;14:387–403; DOI: 10.1093/cercor/bhg135
Keywords: association cortex, field AES, GABA, multisensory, SIV, superior
colliculus
Introduction
1Department
of Health Sciences, Physical Therapy Program,
College of Mount St Joseph, Cincinnati, OH 45233, USA and
2Department of Anatomy and Neurobiology, Virginia
Commonwealth University School of Medicine, Richmond,
VA 23298, USA
tion, the presence of neurons broadly tuned for sound frequency (Clarey and Irvine, 1986; Korte and Rauschecker,
1993; Rauschecker and Korte, 1993; Middlebrooks et al., 1994;
Rauschecker, 1996) and the sensitivity to the spatial features of
auditory cues (Korte and Rauschecker, 1993; Rauschecker and
Korte, 1993; Middlebrooks et al., 1994; Nelken et al., 1997). Of
these two regions of the AES, SIV is perhaps the best understood because it contains a topographic map of the body surface, connects heavily to other somatosensory cortices and
exhibits distinct cytoarchitectonic properties. Therefore, the
somatosensory SIV cortex was selected to test the possibility
that it receives cross-modal inputs from the adjacent auditory
FAES and, if so, to assess the functional nature of that connection.
Materials and Methods
All procedures were performed in compliance with the Guide for Care
and Use of Laboratory Animals (NIH publication 86-23) and approved
by the Institutional Animal Care and Use Committee at Virginia
Commonwealth University.
Anatomical Studies
Surgical Preparation
Cats (n = 9) were anesthetized with sodium pentobarbital and their
heads placed in a stereotaxic frame. Under aseptic surgical conditions,
a craniotomy and durectomy were performed to expose the AES
cortex. In two of four cases in which retrograde tracers were injected
into SIV, standard extracellular recording techniques were used to
map the extent of SIV, as described elsewhere (see Clemo and Stein,
1983). To make tracer injections, a modified electrode carrier was
used to support a 5 µl Hamilton syringe and its needle (31 gauge). For
injections into SIV, the carrier was angled 50–60° with a 90° cant
(lateral to medial) and the needle tip was inserted 2.0–2.5 mm into the
dorsal bank of the AES. For injections into FAES, the carrier was
angled 53–60° (from vertical), with 35–40° cant (posterior from
coronal plane) and the needle tip was inserted at a point 0.8–1.5 mm
anterior to the vertical limb of the AES to a depth of 5.25–5.70 mm.
After a 5 min pause, horseradish peroxidase (HRP; 20% in sterile
saline; 125–425 nl total volume) or biotinylated dextran amine (BDA;
10 000 mol. wt, lysine fixable, 10% in 0.1 M phosphate buffer,
345–700,nl) was pressure injected at a rate of 15 nl/min. The needle
was then retracted, the cortex was covered with gel foam, the skin
around the wound sutured closed and standard postoperative care
was provided.
Histological Processing
After a 24 h survival period, the animals injected with HRP were given
a barbiturate overdose and perfused intracardially with heparinized
saline followed by fixative (1.0% paraformaldehyde, 1.25% glutaraldehyde solution). The brain was blocked stereotaxically, removed and
refrigerated overnight in 0.1 M phosphate buffer. Serial, coronal
sections (50 µm thick) were cut using a vibrating microtome. One
series of sections, at 200 µm intervals, was reacted using a standard
diaminobenzidine (DAB) procedure to demonstrate the zone of
maximal uptake around the injection site (LaVail and LaVail, 1974). A
second series of sections, at 100 µm intervals, was processed using
tetramethylbenzidine (TMB; Mesulam, 1978) in conjunction with
nickel–cobalt intensification. Standard procedures were used to
mount, counterstain and coverslip the tissue.
For animals that received BDA injections there was a 7–14 day postinjection survival period before they were given a barbiturate overdose and perfused intracardially with heparinized saline followed by
fixative (4.0% paraformaldehyde, 0.5% glutaraldehyde solution). The
brain was blocked stereotaxically, removed and stored in 0.1 M
phosphate buffer with 25% sucrose (for cryoprotection) at 4°C until
the tissue sank. Coronal sections (50 µm thick) were cut using a
freezing microtome and collected serially. One series of sections, at
200–250 µm intervals, was processed for visualization of BDA using
388 Cortical Cross-modal Circuit • Dehner et al.
the avidin–biotin peroxidase method, according to the protocol of
Veenman et al. (1992) and then intensified using silver nitrate (1.42%
at 56°C) and gold chloride (0.2% at room temperature). The sections
were mounted and coverslipped without counterstain. An additional
series of sections, taken at 200–250 µm intervals, was counterstained
using a standard cresyl violet protocol to assist in cytoarchitectonic
and laminar identification.
Data Analysis
Neuronal labeling was visualized using a light microscope (Nikon
Optiphot-2) and the data plotted using a PC-driven digitizing stage
controlled by Neurolucida software (MicroBrightField Inc.). A calibrated tracing of each section outline with the border between gray
and white matter, the position of the injection site, labeled neurons
and axon terminals was produced. Injection sites were defined as the
large aggregate of densely labeled cell bodies, dendrites and axons at
the terminus of the injection needle track. HRP-labeled neurons were
identified by the presence of dense black beads within the cytoplasm
of the soma and proximal dendrites. BDA-labeled neurons were
sharply black throughout their soma which sometimes spread into the
distal dendrites, although some reactive neurons revealed a lighter,
reddish-brown label. BDA-labeled axon terminals appeared as sharp,
black swellings at the end of thin axon stalks or as symmetrical
varicosities along the course of an axon.
Tissue outlines and injection sites were plotted at 40× magnification. Labeled neurons and boutons were plotted at 200× magnification
and the Neurolucida software kept a count of numbers of identified
neurons and boutons. Labeled cell bodies were plotted in the region
of the posterior AES cortex. The average diameter was determined by
measuring the longest and shortest widths that passed through the
cell center, and dividing by two. A shrinkage factor of 7.5% was then
applied, as determined by the average difference in tissue area measured before and after processing. Labeled boutons were plotted
within the anterior and middle AES cortex, in regions corresponding
to that of SIV and para-SIV.
Once the locations of labeled neurons/boutons were plotted for a
given section, the cortical laminae were traced from an adjacent,
cresyl-violet-stained section and imported back to the digitized plot.
The software then calculated the number of labeled neurons or
boutons in supragranular (above layer IV), granular (layer IV) and
infragranular (below layer IV) locations for a specific area of tissue.
The number of granular, supragranular and infragranular boutons was
compiled for each case and the ratios of boutons in each laminar
region were calculated.
Physiological Studies
Surgical Procedures
Acute (n = 5) as well as chronic preparations (n = 6) were used
because, at the initiation of this portion of the study, it was not known
which anesthetic would best permit recordings from these cortical
areas. Both surgical procedures were essentially the same, but asepsis
was maintained for animals scheduled to recover from the surgery.
Animals were anesthetized (acute = isoflurane, n = 3; pentobarbital,
n = 2) (chronic = sodium pentobarbital, n = 6) and their heads were
placed in a stereotaxic frame. The animals were intubated through the
mouth and artificially ventilated. Expiratory carbon dioxide levels
were monitored and kept to within 4.0–4.5%. A heating pad was used
to maintain temperature, monitored rectally, at 37°C. An intravenous
line was placed in the saphenous vein to administer additional medications, fluids and, if appropriate, supplemental anesthesia.
A craniotomy was performed to expose the cortex around the AES.
A stainless steel recording well (14 mm inner diameter) was placed
over the opening for recording access to SIV in the chronic experiments, as well as to support the animal’s head without obstructing the
eyes, ears or face. Stimulating electrodes (parylene coated tungsten,
<1 MΩ, two rows 1 mm apart, four electrodes in each row at 1 mm
spacing, angled 45–50°) were implanted in FAES to a depth 6.2–7.2
mm from the surface of the middle ectosylvian gyrus (MEG). Additionally, control electrodes were positioned either in other auditory (AI,
nine cases) or somatosensory (SV, two cases) cortices. Implanted electrodes were banked with gel foam and secured in place with dental
Figure 1. Somatosensory (SIV), auditory (FAES) and visual (AEV) representations in the AES cortex. (A) Lateral view of the left cortical hemisphere of the cat; the box indicates
the location of the AES (arrow). The vertical lines labeled ‘C–E’ indicate the location coronal sections through the AES shown in the lower panel. (B) AES magnified and opened to
reveal its banks and fundus, as well as the posterior portion (stippled) that is submerged under the middle ectosylvian gyrus (MEG). The dashed lines indicate the borders between
sensory representations. (C) Coronal section through the anterior portion of the AES, with SIV in the dorsal bank and para-SIV in the fundus. (D) Coronal section through the posterior
portion of the horizontal limb of the AES, with FAES in the dorsal bank and AEV in the ventral bank. (E) Coronal section further posterior than in (D) with the FAES (dorsal) and AEV
(ventral) enclosing a circular remnant of the AES and underlying the MEG.
acrylic. For chronically implanted animals, the scalp was sutured
closed around the implant, routine postoperative analgesic and antibiotic care was provided, and ∼5–7 days elapsed before the initial
recording experiment. The acute preparations were moved immediately into the recording phase.
FAES, each FAES electrode (and control AI or SV electrode) was individually activated (1 anodal pulse, 0.1 ms duration, 500–600 mA
current intensity, 3–5 iterations) in a fashion identical to that used in
our previous studies (Clemo and Stein, 1984; Wallace et al., 1993;
Meredith, 1999).
SIV Recording
For chronically prepared animals, recording experiments were
conducted in a fashion similar to that described in detail in Meredith
and Stein (1986, 1996). Briefly, recording was initiated by anesthetizing the animal (ketamine/acepromazine, n = 3; isoflurane, n = 3)
and securing the implant to a supporting bar. These animals, as well
as the acutely prepared ones, were intubated and ventilated. The
saphenous vein was cannulated for administration of fluids and
supplemental anesthetics, as needed. Each of the implanted stimulating electrodes was connected, through a stimulation isolation unit
(Grass SIU-5), to a Grass stimulator (S-88). For recording, a glass insulated tungsten electrode (tip exposure < 20 µm, impedance < 1.0 MΩ)
was supported by a 16 gauge steel cannula which, in turn, was guided
by the X–Y slide (10 mm A-P/M-L excursion; Kopf Instruments) that
covered the well. In acute preparations, no cannula was used and the
recording electrode was inserted at a 40–50° lateral–medial angle
directly through the craniotomy.
In both preparations, neuronal activity was amplified, displayed on
an oscilloscope and played on an audiomonitor. Neurons were first
identified by their responses to manually presented stimuli: somatosensory (puffs of air through a pipette, brush strokes and taps, manual
pressure and joint movement); auditory (claps, clicks, whistles and
hisses); visual (flashed or moving spots or bars of light from a hand
held ophthalmoscope projected onto the translucent hemisphere and
dark stimuli from a rectangular piece of black cardboard). In addition,
a neuron’s somatosensory receptor type (hair, skin, deep or joint) was
determined and its receptive field was mapped. Next, to assess
whether a neuron received excitatory (orthodromic) input from the
SIV Sensory Tests
To determine whether somatosensory responses of SIV neurons were
influenced by auditory stimuli or auditory FAES activation, somatosensory stimuli were presented within a neuron’s receptive field
alone and then in combination with a natural auditory cue or with
electrical stimulation through one of the FAES electrodes. Somatosensory stimuli were produced by a narrow (3 mm diameter) metal
shaft moved by an electronically driven, modified shaker (Ling 102A)
as described in previous studies (Clemo and Stein, 1984; Meredith and
Stein, 1986, 1996). Free-field auditory cues were electronically generated white noise bursts, 100 ms duration, 54–70 dB SPL on ‘A’ level.
This stimulus was delivered through a speaker, mounted on 18″ diameter hoop [as also described in previous studies (Meredith and Stein,
1986, 1996)] and was positioned to be in spatial register with the
somatosensory receptive field. During combined sensory presentations, the onsets of the stimuli were adjusted to compensate for the
differences in the average latency of auditory and somatosensory
responses in AES cortex [somatosensory evoked responses ∼15 ms
later than auditory (Wallace et al., 1992)]. FAES activation was
effected by electrical stimulation delivered through an indwelling
FAES electrode (trains of pulses at 25–250 Hz, 80–100 ms duration,
0.1 ms/pulse, 37–600 µA anodal current intensity). Separate and
combined modes of stimulation were interleaved to compensate for
possible shifts in baseline activity. When somatosensory and electrical
stimuli were combined, the electrical activation of FAES preceded the
somatosensory stimulus by 85–100 ms to avoid interference between
stimulation artifact and neuronal action potentials. An interstimulus
interval of 10 s was employed to avoid habituation; each test was
Cerebral Cortex April 2004, V 14 N 4 389
repeated 10–14 times. Neuronal activity was digitized (rate > 25 kHz)
using Spike2 (Cambridge Electronic Design) software and stored for
off-line analysis.
Data Analysis
Off-line analysis consisted of using Spike2 software to examine data
files and, within each file, only reliably isolated waveforms were
subjected to further analysis. For a given spike waveform, the file was
collated according to the testing conditions and a perstimulus-time
histogram was constructed for each. From these histograms, the duration of a response was determined and the mean spike number per
response (and standard deviation) was calculated over that period.
Responses to the combined stimuli were statistically compared
(paired t-test, P < 0.05) to that of the most effective single stimulus,
and responses which showed a significant difference were defined as
response interactions (Meredith and Stein, 1983, 1986, 1996; Wallace
et al., 1992). In addition, the magnitude of a response interaction was
determined by the following formula: C – T/T × 100 = %, where C is the
response to the combined stimulus, and T is the response to the tactile
stimulus alone (Meredith and Stein, 1983, 1986, 1996).
Histological Procedures
The depth of each identified neuron within a penetration was noted
and recorded. Several recording penetrations were often performed in
a single experiment and each successful recording penetration was
marked, at its conclusion, with a small electrolytic lesion (0.5–1.0 mA
for 0.5–2.0 s). At the conclusion of an acute experiment, or after a
series of experiments using a chronically-prepared animal, the animal
was given a barbiturate overdose and perfused with physiological
saline followed by 10% formalin. The brain was removed, frozen
sections (50 µm) were cut in the coronal plane through the recording
and stimulation sites, processed using standard histological procedures and counterstained with cresyl violet. A projecting microscope (Bausch & Lomb) was used to trace sections and to reconstruct
stimulating electrode positions, recording penetrations and the
locations of examined neurons from the lesion sites. Gyral/sulcal
patterns, cytoarchitectonic characteristics and observed physiological
properties were used to demarcate the relevant functional cortical
subdivisions. These anatomical data were then tabulated with the
physiological observations (described above).
In Vitro Slice Recordings
These procedures were essentially the same as reported previously
(Meredith and Ramoa, 1998). Two animals were deeply anesthetized
and, immediately prior to euthanasia, their AES cortex was exposed
and surgically removed en bloc. This tissue was immediately
submerged in 5°C modified (sodium free, 10 mM glucose) Ringer’s
solution. Parasagittal sections 400 µm thick through the AES were
taken using a Vibratome. Slices were transferred to an interface-type
recording chamber where extracellular multiunit recordings were
made using glass-insulated tungsten electrodes (∼1 MΩ at 1 KHz, tip
exposure <20 µm). Once a neuron was isolated, its electrical activity
was amplified, routed to an oscilloscope and audiomonitor, and
stored on tape. Next, a site within the same column as the recording
electrode, or a location anterior or posterior was stimulated electrically (concentric bipolar electrode, 250 µm diameter, 0.1 ms duration,
600 µA maximal stimulus intensity) and unit activity was recorded in
response to 50 stimulus iterations (2–3 s interstimulus interval).
Whether a neuronal response was observed or not, the same
stimulation paradigm was repeated after the local administration
(picospritzed) of bicuculline methiodide (BIC; 50 µM), the co-administration (through two separate picospritzing pipettes) of BIC and D -2amino-5-phosphonovaleric acid (d-APV; 40 µM) and then the coadministration of BIC with 6-nitro-7-sulphamoylbeno(f)-quinoxaline2,3-dione (NBQX; 10 µM). Following each experiment, the taped data
was transferred to a computer and the activity of each neuron was
analyzed quantitatively (Spike2 software) for its response to electrical
stimulation of another region of the slice, whether that response was
enhanced by the presence of the inhibitory antagonist BIC, and
whether that enhancement could be diminished by the presence of
the excitatory antagonists, d-APV and NBQX. Data from the popula-
390 Cortical Cross-modal Circuit • Dehner et al.
tion of neurons sampled was tabulated to assess the general pattern of
response to the different treatments.
Microiontophoresis and Recording
Two animals were prepared chronically for recording in the manner
described above (see Physiological Studies: Surgical Procedures). On
the day of the experiment, each animal was anesthetized (ketamine/
acepromazine) and, after the recording well was opened, the dura
was reflected to admit the microiontophoresis pipette/electrode
assembly. The assembly was constructed from a standard glass insulated recording electrode (impedance 0.6–1MΩ), to which was glued
(cyanoacrylic, supported by dental acrylic) a double-barreled pipette
(4″ long, 1.5 mm OD capillary glass with microfilament; A-M Systems)
with tips pulled and trimmed to reveal an internal diameter opening of
∼10 µm. The pipette tips were positioned, using a dissection microscope and a pair of micropositioners, within ∼40–50 µm of the electrode tip. Approximately 30 min prior to use, a fine needle was used
to fill one barrel of the pipette with BIC (10 mM, pH 3.5) while the
other received normal saline. The assembly was supported by a stereotaxic carrier. Each barrel of the pipette was attached, via a deeply
inserted silver wire, to a separate channel of a microiontophoresis
current programmer (WPI model 260). For the barrel containing BIC,
the current programmer generated a negative holding current of
15–20 nA, while a positive current of the same value was delivered
through the saline channel to balance the net current flow. Once a
neuron was identified, its activity was amplified and stored in the
manner described above (see SIV Recording) and the following
battery of tests (same as described in SIV Sensory Tests, above) was
presented. Either the somatosensory probe was used to stimulate the
neuron’s receptive field, or an electrical current (100–600 mA, 0.1 ms
pulse duration; 100 Hz for 100 ms) was passed through one of the
indwelling FAES stimulating electrodes, or the two forms of stimulation were combined in an interleaved fashion (10 repetitions) with
100 ms separating the onsets of the combined stimuli. This stimulation paradigm was presented first prior to the ejection of BIC, then
immediately after its iontophoresis into the tissue (ejection current
was positive 20–50 nA, continuous for 3–5 min, balanced by a negative current of the same magnitude through the saline channel) and
then at regular intervals afterward until the effects of the BIC were no
longer apparent. Neuronal activity was collected using Spike2 and
analyzed offline using custom software (L. Keniston). This analysis
calculated neuronal responses (mean and standard deviation of
spikes/response) to somatosensory (alone) and somatosensory with
FAES stimulation (combined) and then compared responses for preejection, immediate post-BIC ejection and recovery periods to establish (i) if the somatosensory response was significantly suppressed by
the FAES-stimulation, (ii) whether that suppression was blocked by
the presence of BIC and (iii) when, during the recovery period, the
disinhibition effect had cleared. These data for each neuron were then
tabulated and graphed, so that the time course of the BIC effect could
be assessed for the sample. Statistical evaluation of neuronal and
population responses were conducted using the Wilcoxon signed
rank test (P < 0.05).
Results
SIV Injections: Retrograde
Injection of retrograde tracers (HRP, n = 2; BDA, n = 3) into the
dorsal bank of the horizontal (anterior) limb of the AES filled
the somatosensory SIV to varying degrees. Injections were
directed toward the most anterior portion of SIV, where the
representation of the head and forelimb reside. This was done
to maximize the distance between the SIV injection site and
FAES and AEV, thereby avoiding direct contamination of the
posterior AES cortex by the injection site or track. In two
cases, the injection was entirely confined to the dorsal bank of
the AES, but in the other three (larger) injections, tracer also
entered the fundus (para-SIV) and portions of the ventral bank.
As expected from previous reports (Reinoso-Suarez and Roda,
1985; Barbaresi et al., 1989; Mori et al., 1991), neurons retrogradely labeled from these SIV injections were identified
within the ipsilateral somatosensory cortical regions of SI–SIII
and SV.
Injection of HRP (or BDA) into SIV also retrogradely labeled
neurons in the posterior 3–5 mm of the AES cortex, where the
sulcus becomes submerged under AI/AII auditory regions of
the MEG. As depicted in Figure 1B,D,E, the dorsal and medial
portion of this region corresponds to the location of the FAES,
while the more ventral portion represents the AEV. FAES
neurons labeled from SIV were visualized using either HRP or
BDA techniques, as shown in Figure 2A. The somatodendritic
labeling for these and other retrogradely marked neurons was
consistent with a pyramidal-type morphology, the average
diameter of which was 17.6 ± 1.67 µm (from three cats, n = 231,
range = 11–27 µm).
FAES neurons retrogradely labeled from SIV were consistently found along a dorsal–ventral arc along its medial aspect.
A large HRP injection that included para-SIV (Fig. 2B) as well as
a smaller BDA injection that was restricted to portions of SIV
(Fig. 2C) both yielded retrogradely labeled neurons sequestered along the dorso-medial aspects of the FAES. Within this
region of the FAES, neurons projecting to SIV predominantly
resided in lamina III (∼78% supragranular versus 19% infragranular), as depicted in Figure 2D.
Figure 2. Retrograde documentation of FAES projections to SIV. (A) Injections into SIV retrogradely labeled neurons with pyramidal morphology in FAES (400×) using HRP (left) or
BDA (right). (B, C) HRP or BDA injections (respectively) into SIV produced retrogradely labeled neurons in the posterior-medial aspects of the AES cortex, corresponding to FAES. In
each panel, the three coronal sections on the left indicate the anterior, middle and posterior extent of the SIV injection site (shaded). Further to the right, serial coronal sections (at
400–500 µm intervals) through the posterior end of the AES are shown with the location of retrogradely labeled neurons (1 black dot = 1 neuron) plotted on each. On the schematic
of the brain (far right), the vertical lines bracket the area from which the coronal sections were taken; the blackened area approximates the location of the injection. (D) Magnified
views of the FAES with the cortical lamination indicated (dashed lines), illustrating that FAES neurons projecting to SIV originate primarily in cortical lamina III, but with some also
found in lamina V.
Cerebral Cortex April 2004, V 14 N 4 391
FAES Injections: Anterograde
BDA (10 000 mol. wt) is also a strong anterograde tracer and
injections were made in the posterior AES (n = 5) to evaluate
the projection to SIV. These injections were distributed such
that they collectively covered an arc of tissue from the dorsal
regions of the FAES, to its medial/inferior-medial areas and then
most inferiorly into the representation of AEV. The four injections that included FAES (Cases 1–4) produced terminal label
observed in the adjacent auditory fields (e.g. AI, AII, AAF; see
Clarey and Irvine, 1990b), while the most inferior injection (in
AEV; Case 5) did not.
Following BDA injections into auditory FAES cortex, labeled
axons and axon terminals were sought within somatosensory
SIV. Here, networks of labeled axons were frequently observed
and numerous BDA-positive boutons were clearly visible, as is
evident from Figure 3. As demonstrated in Figure 4, each of the
injections that encroached on the medial bank of the FAES
(Cases 2–4; corresponding to where neurons retrogradely
labeled from SIV were concentrated) consistently produced
the densest terminal labeling in SIV. By comparison, injections
placed too high (Case 1) or too low (Case 5) in the posterior
AES cortex yielded reduced or no terminal labeling in SIV.
Terminal labeling from FAES was found throughout the
rostro-caudal and medial–lateral extent of SIV, as also shown in
Figure 4. The distribution of this terminal labeling occurred in
clusters or patches that were interrupted by regions in which
boutons were sparse. Some of the bouton-sparse regions occupied the full thickness of the cortical mantle, as if to segregate
FAES-recipient patches. While the areas containing labeled
boutons included all cortical laminae, the distribution was
not homogeneous. Instead, the regions of heaviest terminal
labeling were consistently found in the layers closest to the pial
surface. This pattern is illustrated in representative sections
from four cases in Figure 5, where FAES-labeled boutons were
plotted according to their laminar location in SIV. The densest
region of terminal label within SIV was consistently observed
in laminae I, II and III, the supragranular layers. Lamina IV
showed little terminal labeling from FAES. In the deepest layers
(infragranular; V and VI), BDA-positive boutons were again
evident. When the number of boutons per layer was tabulated
for all cases according to their supra-, infra- or granular
location, the predominance of the supragranular projection
was apparent: 75% supragranular versus 21% infragranular
locations.
Physiology: SIV Responses
The presence of projections from auditory FAES to somatosensory SIV was unexpected, because the wealth of physiological
Figure 3. Tracer injections into FAES orthogradely labeled axon terminals within SIV. (A–C, D–F) Photomicrographs from two cases of BDA injections into FAES. Both (A) and (D)
are low-power (40×) views of the anterior, dorsal bank of the AES where SIV is located. The square box in each indicates the location of tissue magnified in the subsequent panels.
The mid-power (400×) views, shown in (B) and (E) reveal networks of FAES axons within SIV. (C, F) High-power (1000×) views showing BDA-positive FAES axon terminals (arrows
indicate boutons) in SIV.
392 Cortical Cross-modal Circuit • Dehner et al.
Figure 4. The distribution of orthogradely labeled FAES terminals within SIV. This figure represents the results of five cases in which BDA was injected in or near FAES (coronal
sections on right show the anterior, middle and posterior extent of the injection site) with the resulting pattern of terminal label within SIV (coronal sections labeled A–D; 1 dot =
1 terminal). Terminal labeling within AEV, when present, is not shown. BDA injections that included the medial bank of FAES consistently produced moderate to heavy patterns of
terminal labeling in SIV (Cases 2–4). In contrast, BDA injections superior (Case 1) or inferior (Case 5) to this medial FAES region yielded modest to no terminal labeling in SIV. The
brain schematic (far right) indicates the location of the coronal sections through SIV (lines A–D) as well as that of the injection site (blackened).
studies of SIV have indicated that it is a unimodal, somatosensory area. Clemo and Stein (1983) used natural stimuli from
somatosensory, as well as auditory and visual modalities to
probe responses of SIV neurons, and reported SIV was exclusively excited by somatosensory inputs. Subsequent reports
have substantiated these observations, although some exceptions have noted examples of visual-somatosensory convergence there (Minciacchi et al., 1987; Jiang et al., 1994a,b).
Therefore, electrophysiological recording techniques were
used to examine the functional role of this cross-modal projection in a total of 256 neurons located in and near SIV. Upon
reconstruction of the recording tracks, 144 of the neurons
were identified within SIV as depicted in Figure 6. Of the SIV
neurons examined, nearly all (130/144, 90.3 %) were excited
exclusively by somatosensory stimulation; visual or auditory
stimulation was ineffective in all but six (4.2%) neurons (see
Table 1). Similarly, all neurons encountered in SII (n = 44)
showed unimodal somatosensory responses, but those
observed in para-SIV (n = 56) revealed a variety of sensory
responses whose properties are displayed in Table 1.
All SIV neurons responsive to somatosensory stimuli were
excited by low-threshold, high velocity stimuli (e.g. air puffs)
consistent with activation through hair-type receptors. As illustrated in Figure 7, the different somatosensory receptive fields
of the identified SIV neurons were found on all areas of the
body. The most frequently encountered receptive field location was that on the forelimb/paw (40.3%; n = 102 of 253
receptive fields), while relatively fewer included locations on
the face (21.3%; n = 54/253), trunk (17.8%; n = 45/253) and
hindlimb/paw (19%; n = 48/253). The bias toward forelimb/
Cerebral Cortex April 2004, V 14 N 4 393
Figure 5. Laminar distribution of FAES axon terminals in SIV. For the each of the cases (1–5) illustrated in Figure 4, axon terminals were plotted on a magnified view of SIV (pial
surface at top). Laminar boundaries (dashed lines) were determined using adjacent Nissl-stained sections. The center of the corresponding injection site in FAES is depicted in the
top row. For BDA injections that included the medial bank of FAES (Cases 2–4), terminal label was found primarily within the supragranular and, to a lesser extent, the infragranular
layers of SIV. A similar supragranular preference, but much weaker projection, was evident for the less effective injections located high and lateral in FAES (Case 1), or low in AEV
(Case 5).
Figure 6. The distribution of somatosensory-responsive neurons examined for responses to auditory FAES stimulation. (A) Lateral view of the cat cerebral cortex indicating the
location of the AES and the levels from which the coronal sections, shown in B (dashed vertical lines) and C (solid vertical lines), are taken. (B) Sections through FAES show the
position of the eight, indwelling stimulating electrodes for a single case. Note that the tip of all but one of the electrodes is positioned within the medial part of the FAES. (C) Plot
of the histological location of somatosensory-responsive neurons encountered in SII, SIV, or para-SIV that were tested for orthodromic and subthreshold inputs from auditory FAES.
All of the neurons depicted were examined for but failed to show orthodromic responses to FAES stimulation. The effect of combined somatosensory + FAES stimulation on each
neuron is indicated by the symbol, as defined in the ‘key.’ The asterisks on the first and last sections denote two penetrations at widely different locations in the same animal.
paw receptive fields may have resulted from the enhanced
probability of encountering the magnified representation of
this body region in SIV (Clemo and Stein, 1983, 1984)
394 Cortical Cross-modal Circuit • Dehner et al.
Othodromic Stimulation Tests for Excitation
Because the FAES projection to SIV arises from layer III/V
pyramidal neurons that are typically glutamatergic, the possi-
Table 1
Sensory modality distribution for neurons identified in SIV, SII and para-SIV shown as number (and
percentage) of sample by region
Sensory modality
SIV
SII
para-SIV
4 (7%)
V
1 (0.7%)
0
A
2 (1.4%)
0
S
130 (90.3%)
44 (100%)
3 (5%)
38 (68%)
VA
0
0
0
VS
0
0
4 (7%)
AS
3 (2.1%)
0
2 (4%)
VAS
0
0
0
0
5 (9%)
Unresponsive
8 (5.5%)
Total
144
44
56
Figure 7. Receptive field locations of SIV neurons examined for inputs from FAES.
Each bar indicates the number of neurons whose receptive field included the stipulated
body region. Of the neurons tested for FAES-induced suppression, a majority was
suppressed in each receptive field category. Thus, even though forelimb receptive fields
were most frequently encountered, tested, and suppressed, FAES-inputs appear to
similarly influence all the represented regions of the body. Note that because some
receptive fields encompassed more than one body region, numbers sum to greater
than the number of neurons examined. FL = forelimb; HL = hindlimb; Hemi = hemibody receptive field.
bility that this projection conveyed excitatory auditory inputs
to SIV neurons was further tested using orthodromic stimulation. In 11 animals, a total of 78 stimulating electrodes were
implanted within the caudal AES cortex. Of these, the tips of
58 electrodes were histologically confirmed to be located
within FAES (see Fig. 6B) and 50 revealed exclusively auditoryevoked activity. Only those electrodes which exhibited
auditory-evoked activity were used for subsequent combined
FAES-stimulation and somatosensory tests (see Combined
Somatosensory and FAES Stimulation, below). However, all
implanted electrodes were used for orthodromic stimulation
tests. By activating each of the indwelling FAES electrodes individually (500–600 µA, 0.1 ms duration), a total of 120 SIV
neurons were examined for orthodromic input from FAES.
However, none (0/120) of the SIV neurons were excited by
FAES stimulation. Similarly, none of the SII (0/44) or para-SIV
(0/56) neurons tested was orthodromically activated by FAES
stimulation.
Combined Modality Tests for Subthreshold Excitation
Although the projection from FAES did not excite SIV neurons,
additional tests explored the possibility that inputs from FAES
might carry subthreshold excitatory or suppressive signals to
SIV. These tests used a standard multisensory paradigm (see
Meredith and Stein, 1986, 1996) to collide excitatory responses
evoked by somatosensory stimulation with acoustically-evoked
activity. A total of 12 SIV neurons (and 18 para-SIV neurons)
were presented a somatosensory cue (within the cell’s receptive field), a free-field auditory cue (in spatial alignment with
the tactile stimulus) and the combination of somatosensory
and auditory stimuli (within 15 ms of synchrony) in an interleaved fashion. All of these neurons were responsive to
somatosensory cues, but none were excited by the auditory
stimulus presented alone and none revealed a significant
response increase when the two cues were combined.
However, in the example provided in Figure 8, an SIV neuron
that was unresponsive to an auditory stimulus presented alone
had its somatosensory response significantly suppressed by the
presence of an auditory stimulus. That suppression was not
observed in all of the neurons tested in this manner may result
from the presence of anesthesia and/or the possibility that
Figure 8. Combined-modality stimulation results in response suppression. A neuron located in SIV (coronal section labeled A) produced the responses shown in B and summarized
in C. A vigorous response (mean = 5.0 ± 3.8 spikes/trial) was produced by the rapid deflection of hairs (ramp labeled ‘T’) on the trunk, but a response to an auditory stimulus
(squarewave labeled A; 0° elevation; 60° azimuth; 69 dB SPL, 100 ms duration) could not be distinguished from baseline (mean = 0.7 ± 3.1 spikes/trial). No auditory cues were
effective in producing an excitatory response in this neuron. However, when these same stimuli were combined, the level of activity was reduced (mean = 2.8 ± 2.8 spikes/trial),
representing a significant (indicated by asterisk; P < 0.05; paired t-test) 44% reduction from the response to the tactile stimulus alone. Peristimulus time-histogram bin width =
10 ms; these conventions are the same in subsequent figures.
Cerebral Cortex April 2004, V 14 N 4 395
Figure 9. Somatosensory responses of a majority of SIV neurons were suppressed by concurrent stimulation of auditory FAES. (A) The coronal sections illustrate the SIV recording
and FAES stimulation sites for the neuron whose activity is depicted in B. (B) tactile stimulation alone (ramp labeled ‘T’) elicited a robust response (mean = 4.4 spikes/trial), but
stimulation of FAES (line and pulses labeled ‘S’) did not. However, when these same stimuli were combined, the neuronal response was significantly suppressed (mean = 2.1
spikes/trial), as summarized in the bar histogram at the far right (response suppression =56%; P <0.05, paired t-test). (C) Data for the population of SIV neurons tested in this
same manner, where the y-axis represents the responses to the combined somatosensory + FAES stimulation, while the x-axis represents the response to the somatosensory
stimulus alone. Note that the majority of responses (66%) fall well below the unity line (grey line); the regression line (black line; slope = 0.58) for these suppressed responses
was significantly lower than unity. In contrast, the plot labeled D illustrates the responses to the same stimuli from the population of neurons sampled in SII (open squares) and in
para-SIV (black diamonds), where somatosensory responses were not significantly affected by FAES stimulation (grey unity line nearly equaled the black regression line, slope =
1.01).
acoustically-induced suppression might be relatively weak and,
therefore, might not achieve a statistically significant response
change within a small number of stimulus iterations. However,
it is important to note that the neuron depicted in Figure 8 also
had its somatosensory response suppressed by FAES stimulation, as described below.
Combined Somatosensory and FAES Stimulation
While combined-modality tests suggested that auditory cues
could suppress SIV responses to somatosensory stimuli, electrical activation of the auditory FAES documented the effect. In
this series of tests, somatosensory SIV responses were examined before and in combination with FAES activation through
an indwelling electrode. A representative example is shown in
Figure 9, where excitation of an SIV neuron elicited by a tactile
stimulus was significantly reduced (by 52%; P < 0.05, paired ttest) when combined with activation of auditory FAES. For the
entire sample, combining tactile and FAES stimulation significantly suppressed responses to the tactile stimulus in 66% (51/
77) of the SIV neurons and the level of suppression ranged
from 24–100%, as summarized in Figure 9C and Table 2. FAESsuppressed SIV neurons were distributed throughout the
anterior–posterior and medio-lateral axis of SIV (see Fig. 6,
filled circles) and, accordingly, no particular body region
appeared to be disproportionately affected (see Fig. 7, black
396 Cortical Cross-modal Circuit • Dehner et al.
Table 2
The number (and percentage) of somatosensory-responsive neurons (in SII, SIV, para-SIV) that
were tested with stimulation of auditory FAES or control area (AI)
Stimulation site
FAES
FAES
FAES
AI-control
Recording site
SIV
SII
para-SIV
SIV
Suppression
51 (66%)
0 (0%)
0 (0%)
Facilitation
4 (5%)
0 (0%)
2 (6%)
0 (0%)
No effect
22 (29%)
8 (100%)
34 (94%)
42 (100%)
Total
77 (100%)
8 (100%)
36 (100%)
42 (100%)
0 (0%)
bars). Furthermore, the effect was widely divergent and a
single FAES stimulation site was capable of suppressing activity
at different SIV locations. For example, each of the widely
separated recording penetrations denoted by asterisks in
Figure 6 were taken from a single animal and contained
neurons whose responses were suppressed from the same
FAES stimulation site. Similar examples of divergence were
revealed in the four additional animals in which suppression
was observed in more than one penetration. In contrast, FAESsuppression effects were not observed in neurons from the
adjoining SII and para-SIV representations. As demonstrated in
Figure 9D, responses in both SII and para-SIV to somatosensory
stimulation were not significantly affected by concurrent FAES
stimulation.
Whether the observed response suppression arose from the
FAES, or was a general result of stimulation of adjacent auditory
cortices, was assessed by comparing SIV responses to FAES
stimulation with those evoked from sites in AI (n = 42
neurons). Figure 10A depicts such a comparison, where the
tactile stimulation of an SIV neuron was paired with activation
of FAES (top row) or auditory area AI (bottom row). In this
example, the tactile response was significantly suppressed by
the FAES stimulus, but not by the activation of AI. None of the
SIV neurons whose tactile responses were suppressed by FAES
stimulation were significantly influenced by activation of
control electrodes in AI (see Table 2). Together with previous
estimates of stimulation current spread (<1 mm; Wallace et al.,
1993; Meredith, 1999), these data are consistent with the
notion that the observed suppressive effects specifically
involved FAES activation.
Simultaneous, dual single-unit recordings indicate that some
SIV neurons are preferentially targeted over others by FAES
inputs. Figure 10B illustrates records of two neurons recorded
at the same time and location within SIV. The tactile response
of the neuron in the top row was significantly (P < 0.05, t-test)
suppressed by concurrent FAES stimulation, while that of the
other neuron (bottom row) was unaffected. This same pattern
of FAES suppression/non-suppression was observed in five
other pairs of simultaneously recorded SIV neurons.
The nature of FAES suppression of SIV responses was characterized by systematically changing the parameters of the
FAES stimulation. These tests revealed that the magnitude of
suppression was directly related to the level of stimulation
intensity or frequency. For example, the tactile responses of a
single SIV neuron depicted in Figure 11 were suppressed by
concurrent FAES stimulation and, as the rate or intensity of
FAES stimulation was systematically increased, the level of
response suppression likewise increased. As shown in Figure
11, this relationship was clearly monotonic for both stimulation parameters. In addition, the same relationship between
increased levels of FAES stimulation intensity/frequency and
increasing levels of SIV suppression was observed in all the
Figure 10. FAES-induced suppression of SIV is specific for FAES stimulation ‘A’ and for particular SIV neurons ‘B’. (A) The coronal sections illustrate the location of the SIV neuron,
as well as its FAES and AI stimulation sites, whose activity is depicted in the panels to the right. The top row of panels shows that the response to tactile stimulation (mean = 1.6
spikes/trial) was reduced by 56% (mean = 0.7 spikes/trial) when combined with FAES stimulation. However, the bottom row shows that the response to the same tactile stimulus
(left; mean = 1.4 spikes/trial) was essentially unchanged by concurrent stimulation in AI. The bar histograms to the far right summarize these effects, illustrating that FAES
stimulation significantly (P <0.05, paired t-test) suppressed SIV responses, but AI stimulation did not. (B) As illustrated by simultaneous recordings from two SIV neurons at the
same site, FAES-induced suppression targeted some neurons, but not others. The coronal sections show the FAES stimulation site and the SIV location at which both neurons were
recorded. On the right, the top row of panels show that the tactile responses (mean = 1.0 spikes/trail) of this neuron were reduced (–60%) by concurrent FAES stimulation (mean
= 0.4 spike/trial). In contrast, the bottom panels show that the same tactile and FAES stimulation parameters failed to influence the activity of a simultaneously recorded neuron.
Here, the response to tactile alone (mean = 1.1 spikes/trial) was not significantly different from that evoked by the combined stimulation (mean = 1.0 spikes/trial). These effects
are summarized in the bar histograms (far right).
Cerebral Cortex April 2004, V 14 N 4 397
Figure 11. Systematic changes in FAES stimulation parameters yielded parallel changes in suppression of SIV activity. In A, the coronal sections show the location of an SIV neuron,
as well as the FAES and control (AI) stimulation sites. The panels in B show the responses of the neuron to tactile stimulation alone (left) were suppressed when combined with
FAES (middle) but not AI (right) stimulation. The bar histogram (far right) summarizes this activity. In C the tactile responses (mean = 1.6 spikes/trial) of the same SIV neuron were
tested with increasing levels of FAES stimulation intensity from 37 to 600 µA (37 µA = 2.0 spikes/trial; 75 µA = 1.0 spikes/trial; 150 µA = 0.9 spikes/trial; 300 µA = 0.8 spikes/
trial; 600 µA = 0.2 spikes/trial) and, as FAES stimulation intensity increased, the level of response suppression also increased. This monotonic effect is graphed (far right) for this
neuron (black trace) as well as for five other similarly tested neurons (grey traces). Note that each level of suppression was calculated by comparison to results from tactile
stimulation alone for a given FAES stimulus intensity (not shown). Part D illustrates the same SIV neuron’s responses to tactile stimulation when combined with increasing FAES
stimulus frequency (from 25 to 250 Hz). As the frequency of the FAES stimulation was systematically increased, the level of suppression also gradually increased (25 Hz = 2.8
spikes/trial; 50 Hz = 2.0 spikes/trial; 100 Hz = 0.8 spikes/trial; 150 Hz = 0.3 spikes/trial; 250 Hz = 0.2 spikes/trial). The graph (far right) summarizes this monotonic effect on the
SIV neuron’s responses (black trace) and that of seven other SIV neurons (grey traces). Each level of suppression was calculated by comparison with responses to tactile stimulation
alone for a given FAES stimulation frequency (not shown).
other neurons examined in a similar fashion (n = 5, 7, respectively).
GABAergic Inhibition
Given that FAES stimulation suppressed SIV responses, the
possibility that this effect was mediated through GABAergic
inhibition was initially assessed using in vitro techniques
amenable to pharmacological manipulation. Parasagittal slices
through the longitudinal extent of the AES were used to make
extracellular multiunit recordings before and after application
of BIC, the GABA antagonist. A total of 29 AES neurons were
examined, of which 69% (n = 20/29) were sensitive to BIC
treatment. In these cases, neurons that were either initially
unaffected (n = 7/20) or minimally activated (n = 13/20) by
electrical stimulation became highly excited by this stimulation
in the presence of BIC, indicating that inhibitory GABAergic
398 Cortical Cross-modal Circuit • Dehner et al.
circuits normally suppressed the evocation of excitatory
responses. In addition, in those slices whose plane of section
preserved connections between FAES and SIV, 58% (n = 7/12)
of the SIV neurons that were minimally activated by electrical
stimulation of FAES were strongly activated by that same
stimulus in the presence of BIC. An example is provided in
Figure 12. In this case, the neuron responded to only 5 of 50
FAES shocks. However, when BIC was applied to the preparation, the neuron was strongly activated by the same stimulus
and for a longer time period. Co-application of the excitatory
antagonist d-APV served to attenuate the duration of the BICenhanced response, while co-administration of excitatory
antagonist NBQX completely blocked all synaptic activity.
These same effects of excitatory antagonists were observed in
5 of seven cells tested with d-APV and in all six neurons
presented with NBQX.
Figure 12. Excitation in SIV neurons is held in check by GABAergic inhibition. An in vitro slice preparation of the AES in a parasagittal plane included both dorsal (i.e. SIV) and ventral
banks, as well as its posterior end where FAES is located. Under normal conditions, electrical stimulation (concentric bipolar, 600 µA, 0.1 ms, 50 repetitions) of FAES minimally
excited neurons at the recording site in SIV. However, when bicuculline methiodide (BIC; 50 µM, picospritzed) was introduced, GABAergic inhibition was blocked and the SIV was
strongly activated by the same FAES stimulus. When BIC was co-applied with the excitatory antagonist, d-APV (40 µM), the duration but not the amplitude of the excitatory effect
was truncated. Ultimately, when the excitatory antagonist NBQX (10 µM) was co-applied with BIC, all synaptic activity was blocked.
While the pharmacologic disinhibition of inputs to SIV
neurons in vitro was consistent with inhibition of SIV neurons
by GABAergic circuits controlled by FAES, the general results
of these tests might be expected from most neocortical regions
within which ∼30% of the neurons are GABAergic. To examine
the involvement of GABAergic inhibition directly in FAES
stimulation-induced suppression of SIV responses, two animals
were used to make electrophysiological recordings before and
after the microiontophoretic application of BIC. These
methods were successful in reducing or blocking FAES stimulation-induced suppression of SIV responses. A representative
example is provided in Figure 13, where the response of an SIV
neuron to a tactile stimulus was significantly suppressed by
concurrent stimulation of the FAES (compare black histogram
of FAES suppression superimposed on the grey, tactile-alone
histogram). This suppression, however, was almost completely
blocked by the microiontophoresis of BIC, as evidenced by the
nearly equal responses to the BIC + FAES +tactile combination
(black histogram) overlying the BIC + tactile-alone tests (grey
histogram). Ultimately, when the tissue had recovered from
the effects of the BIC, FAES stimulation induced suppression of
SIV responses returned, as evidenced by the widely separated
responses to the FAES + tactile (black histogram) versus the
tactile-alone (grey histogram) tests. A total of 29 neurons were
examined in this fashion, of which 10 completed the entire
series of tests, as summarized in Figure 13D.
Discussion
These experiments have demonstrated a novel form of multisensory convergence, whereby inputs from one modality act to
suppress, rather than enhance, activity in another. This effect
was specific for auditory–somatosensory convergence
resulting from the FAES projection to SIV: FAES activation did
not suppress responses in adjacent SII and para-SIV regions nor
did stimulation of non-FAES auditory areas have an effect on
SIV activity. However, its effect was broadly distributed within
SIV rather than topographic. Also, cross-modal suppression,
which occurred in 66% of SIV neurons, was directly (not
inversely) related to the efficacy of the FAES stimulation.
Furthermore, the cross-modal effect was mediated through
GABAergic interneurons, since the FAES-induced suppression
was initiated through pyramidal neurons in FAES but blocked
by iontophoretic application of BIC (the GABA antagonist) in
SIV.
Collectively, these observations do not support the likelihood that the FAES projection onto SIV neurons results in the
conventional, well-known form of multisensory convergence.
At present, the neuronal basis for multisensory convergence
and integration has largely been explored in the deep layers of
the superior colliculus (e.g. Meredith and Stein, 1983, 1986,
1996; King and Palmer, 1985) and the principles revealed there
have been upheld by additional examples identified
throughout the neuroaxis and across phylogeny (for a review,
see Stein and Meredith, 1993). In these reports, the multisensory neurons that were encountered were excited by a
stimulus presented in one modality and excited by an independent cue presented in another (i.e. excitatory–excitatory
convergence). Therefore, without combining stimuli for
concurrent presentation, it has been possible to establish the
multisensory nature of many neurons by their independent,
excitatory responses to the different modalities. However, in
the present case (and previous studies as well; see Clemo and
Stein, 1982, 1983, 1984; Jiang et al., 1994a,b; Wallace et al.,
1992; Rauschecker and Korte, 1993), the multisensory nature
of SIV neurons was not apparent when stimuli from different
sensory modalities were presented alone. In the present study,
the multisensory properties of SIV neurons became evident
only during combined tests, where excitation elicited through
one modality was suppressed by the effects of stimuli from
another. Because the nature of the processing controlled by
FAES is GABAergic, this ‘new’ arrangement revealed for SIV
neurons might best be regarded as an excitatory–inhibitory
form of multisensory convergence.
That inhibition plays a role in multisensory processing,
however, is not new. Multisensory response depression occurs
as a result of inhibition of responses to one modality by stimulation of another (Meredith and Stein, 1983, 1986, 1996; King
and Palmer, 1985). Of the reported examples of multisensory
response depression, these effects occurred in neurons that
were excitable by stimuli from more than one modality. Thus,
some excitatory–excitatory inputs apparently carry complex
signals whereby one or both excitatory channels are accompanied by inhibitory receptive field surrounds (Meredith and
Stein, 1996) or by potent post-excitatory inhibitory periods
(Meredith et al., 1987). In this way, a stimulus in one modality
that was out of spatial and/or temporal alignment could
depress activity initiated by another. These mechanisms for
multisensory inhibition do not appear to apply to the present
Cerebral Cortex April 2004, V 14 N 4 399
Figure 13. Iontophoretic application of the GABA antagonist bicuculline methiodide (BIC) blocks (or reduces) FAES-induced suppression of SIV tactile responses. (A) Location of
an SIV neuron tested for the influence of FAES stimulation before, immediately after ejection of BIC through a microiontophoresis/electrode assembly and then after a period of
recovery. The responses of this neuron are depicted in the histograms in B, which first shows the responses evoked by the tactile stimulus alone (mean = 8.0 spikes/trial). Next,
its responses to the same tactile cue are shown (mean = 8.0 spikes/trial; same as in first panel, but histogram is now grey) overlaying the responses to tactile + FAES stimulation
(mean = 3.5 spikes/trial; black histogram), where the response reduction is easily apparent. When BIC was iontophoresed (bar labeled ‘BIC’; 50nA ejection current; 3 min ), not
only was neuronal activity elevated, but also the response to tactile alone (mean = 20.5 spikes/trial; grey histogram), when overlain by the combined tactile + FAES stimulation
(mean = 20.5 spike/trial; black histogram), was virtually identical, indicating that FAES-induced suppression was blocked. After a period for recovery, the difference between tactile
alone (mean = 13.2 spikes/trial; grey histogram) and combined tactile + FAES stimulation (mean = 5.5 spikes/trial; black histogram) returned, indicating that the suppressive
effects of FAES stimulation were again present. The bar histogram to the far right (C) summarizes these results, showing that the tactile responses that were significantly
suppressed by FAES stimulation were disinhibited in the presence of BIC. (D) Summary of the responses of 10 SIV neurons tested in the same fashion and showing the same BICinduced disinhibition of FAES-mediated suppression. The large black squares linked by heavy black lines represent the mean for the sample. *Significant difference between the
tactile response alone and the suppression induced by the tactile and FAES costimulation; **indicates that there was a significant disinhibition due to the presence of BIC (Wilcoxon
signed rank test, P < 0.05).
observations. The involvement of spatial inhibition seems
unlikely here, because all natural stimulation tests were
conducted with stimulus combinations that were presented in
spatial and temporal alignment. On the other hand, the
suppression between FAES and SIV may represent a crossmodal adaptation of GABAergic circuitry which has been welldocumented in modality-specific cortices to underlie the
generation of a variety of response properties. For example,
local GABA-mediated circuits have been demonstrated to
modulate the sharpness of frequency tuning in auditory cortex
(e.g. Fuzessery and Hall, 1996; Richter et al., 1999; Wang et al.,
2000), orientation tuning in visual areas (Sillito, 1975; Sillito et
al., 1985) and spatial inhibition in barrel field/somatosensory
cortex (e.g. Chowdhury and Rasmusson, 2002; Li et al., 2002).
Therefore, it seems reasonable to speculate that the crossmodal GABAergic suppression observed between FAES and SIV
may represent a recruitment of these well-established withinmodality connectivity patterns for multisensory purposes.
Furthermore, this excitatory–inhibitory form of convergence
may underlie a variety of cross-modal inhibitory phenomena,
such as that induced by visual motion on vestibular activity in
parieto-insular cortex (Brandt et al., 1998).
400 Cortical Cross-modal Circuit • Dehner et al.
While the precise organization of the cross-modal excitatory–inhibitory convergence onto SIV neurons is not yet
known, numerous clues exist in the present data that are
consistent with the connectivity schematized in Figure 14.
First, the size, morphology and laminar location of the
FAES–SIV projection neurons are consistent with excitatory
cortico-cortical connections described for numerous other
regions. Next, upon reaching SIV, FAES terminals excite inhibitory interneurons which, in turn, powerfully inhibit principal
SIV neurons perhaps by terminations on their soma, proximal
dendrites or axon hillock [as documented for different varieties
of inhibitory interneurons (see Kawaguchi and Kubota, 1997)].
These arrangements would be consistent with the present
in vivo and in vitro experiments demonstrating GABAergic
involvement in the FAES suppression of excitatory SIV
responses. Furthermore, the results from in vitro slices also
revealed excitatory SIV responses to FAES stimulation which
appear to be held at subthreshold levels by local GABAergic
circuits. Therefore, there is a possibility that principal SIV
neurons also receive excitatory inputs from FAES, but do so on
their distal dendrites in a manner that is easily regulated by
more proximally effected inhibition.
Figure 14. Possible architecture of the cross-modal circuit linking auditory FAES to
somatosensory neurons in SIV. Anatomical evidence shows that pyramidal FAES
neurons in lamina III and, to a lesser extent, lamina V project to the supragranular
(predominantly) and infragranular layers of SIV. Electrophysiological data demonstrated
that FAES activity does not elicit suprathreshold excitement of SIV neurons, but
suppresses their somatosensory responses and this suppression was sensitive to
GABA antagonism. Such cross-modal suppression could be generated by excitatory
FAES projections terminating on SIV inhibitory interneurons, which in turn inhibit or
reduce the activity of other SIV neurons. In addition, data from in vitro slice experiments
show that excitatory inputs to SIV neurons are powerfully held in check by GABAergic
mechanisms, indicating that excitatory inputs from FAES may reach distal dendrites of
SIV neurons.
In addition to illustrating the possible neuronal connectivity
between FAES and SIV, Figure 14 also summarizes their areal
relationships. FAES projection neurons were primarily located
in lamina III, although some were also found in lamina V. FAES
axon terminals in SIV primarily targeted the supragranular
layers, with a smaller proportion of boutons also found in the
infragranular layers. Projections that exhibit these primarily
supragranular-to-supragranular characteristics do not fall neatly
within classical hierarchical connectional schemes (Felleman
and Van Essen, 1991). However, projections considered to be
‘feedback’ in nature preferentially target the supragranular
layers can also have a bilaminar origin, as was observed here.
Therefore, the projection between the FAES and SIV regions
most likely represents a feedback hierarchical relationship.
The functional or behavioral role of SIV is unknown. Only
one study, published as an abstract (Jiang and Guitton, 1995),
has examined SIV in behaving animals and showed that SIV
stimulation evokes goal-directed movements of the head and
body. Given the lack of behavioral data regarding SIV, the
possible function of FAES modulation of SIV is rather speculative. Because FAES neurons encode the spatial components of
auditory cues (Korte and Rauschecker, 1993; Rauschecker and
Korte, 1993; Jiang et al., 1994a,b; Middlebrooks et al., 1994;
Rauschecker, 1996) and SIV contains a map of the body surface
(Clemo and Stein, 1983), connections between the two areas
could serve to provide updates on the spatial alignments
among the two modalities. However, such an effect would
argue for a point-to-point or block-to-block connectional relationship between the two areas, which is not consistent with
the divergent projection pattern observed here. Alternatively,
these connections may play a role in cross-modal selective
attention, whereby attention to one modality (e.g. auditory)
broadly suppresses activity in another (e.g. somatosensory).
Attention to stimuli from one modality is well known to
suppress event-related potentials evoked by another (Hillyard
et al., 1984; Teder-Salejarvi et al., 1999; Hotting et al., 2003)
and the cross-modal connections identified here may provide
the neuronal basis for such an effect. This notion is supported
further by the anatomical divergence of the projection from
FAES to SIV, whereby broad areas of tactile activity in SIV
might be modulated by the auditory attentional state of the
animal. Along this line of reasoning, because both SIV and FAES
are heavily connected with the superior colliculus, it is
possible that shifts in attention, for example, toward an
auditory stimulus, could reduce activity levels (via FAES–SIV
suppression) of cortical somatosensory afferents to the colliculus while elevating those for auditory afferents, thereby
enhancing the likelihood of attending to an auditory stimulus.
These effects would be consistent with a role in selective attention.
These results underscore some of the fundamental differences between excitatory–excitatory and excitatory–inhibitory forms of multisensory convergence. Unlike the wellknown excitatory–excitatory forms of multisensory convergence, the present excitatory–inhibitory multisensory neurons
are virtually undetectable as being multisensory using standard
extracellular recording and search techniques. Afferents from
the inhibitory modality, such as those to SIV neurons from
auditory FAES, become apparent only when directly paired
against effective stimulation in the other modality (or during
high levels spontaneous activity). In the present experiments,
sequential single-modality tests revealed a nearly uniform
unimodal sample (only 2.3% were multisensory), while
combined tests revealed a surprisingly high proportion of
auditory FAES-affected SIV neurons (66% were multisensory).
Ultimately, the revelation of these ‘silently multisensory’
neurons suggests that the incidence of multisensory processing
in the brain, or at least that between auditory and somatosensory modalities, has been vastly underestimated. Consequently, these observations strongly support the assertion,
based on large numbers of behavioral and brain imaging
studies, that ‘cross-modal interactions are the rule and not the
exception . . . and that the cortical pathways previously
thought to be sensory-specific are modulated by signals from
other modalities’ (Shimojo and Shams, 2001).
Notes
We are indebted to Dr A. Ramoa for the use of his equipment to
conduct the in vitro slice experiments and to Dr T. Salt for his expertise in microiontophoresis which he generously shared with us.
Supported by NIH NS39460 and by the Human Frontiers Science
Program RGO174.
Address correspondence to Dr M. Alex Meredith, Department of
Anatomy and Neurobiology, Virginia Commonwealth University
School of Medicine, Richmond, VA 23298-0709 USA. Email:
[email protected].
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