Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Goldreich, Daniel, Harold T. Kyriazi, and Daniel J. Simons. Functional Independence of Layer IV Barrels in Rodent Somatosensory Cortex. J. Neurophysiol. 82: 1311-1316, 1999. Layer IV of rodent primary somatosensory cortex is characterized by an array of whisker-related groups of neurons, known as "barrels." Neurons within each barrel respond best to a particular whisker on the contralateral face, and, on deflection of adjacent whiskers, display relatively weak excitation followed by strong inhibition. A prominent hypothesis for the processing of vibrissal information within layer IV is that the multiwhisker receptive fields of barrel neurons reflect interconnections among neighboring barrels. An alternative view is that the receptive field properties of barrel neurons are derived from operations performed on multiwhisker, thalamic inputs by local circuitry within each barrel, independently of neighboring barrels. Here we report that adjacent whisker-evoked excitation and inhibition within a barrel are unaffected by ablation of the corresponding adjacent barrel. In supragranular neurons, on the other hand, excitatory responses to the ablated barrel's associated whisker are substantially reduced. We conclude that the layer IV barrels function as an array of independent parallel processors, each of which individually transforms thalamic afferent input for subsequent processing by horizontally interconnected circuits in other layers.
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INTRODUCTION |
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Sensory areas of the cerebral cortex are
characterized by collections of interconnected neurons having similar
receptive fields. The extent to which these local circuits interact
remains poorly defined, even at the earliest stages of cortical
processing. For example, some models of simple-cell orientation
selectivity in cat visual cortex assume antagonistic interactions
between separate minicolumns serving the same retinal location but
activated by stimuli having orthogonal orientations (Crook et
al. 1997; Sillito et al. 1980
). Other models
base orientation selectivity on convergent thalamic input solely
(Ferster 1987
; Hubel and Wiesel 1962
;
Reid and Alonso 1995
) or in conjunction with locally
mediated, iso-orientation excitation and/or inhibition (Ferster
1988
; Troyer et al. 1998
). Layer IV of rodent
somatosensory cortex contains anatomically identifiable collections of
neurons, called barrels, that represent distinctly different peripheral
locations, i.e., facial whiskers (Woolsey and Van der Loos
1970
). Although differing from orientation minicolumns in this
and other respects, the degree to which barrels interact with each
other remains similarly controversial. Some investigators have
suggested a prominent role for horizontal connections in creating
receptive fields encompassing multiple neighboring vibrissae
(Armstrong-James et al. 1991
; Fox 1994
),
whereas others have proposed that interactions among neighboring
whiskers reflect local, intrabarrel processing of multiwhisker thalamic
inputs (Simons and Carvell 1989
). In both visual and
somatosensory cortices, horizontal connections are thought to
contribute substantially to receptive field properties in nongranular layers.
To what extent do neighboring local circuits function independently of
one another? Because of its anatomic organization, the somatosensory
cortex of rodents is well suited for addressing this issue. A barrel
consists of several thousand synaptically interconnected neurons, each
of which receives the bulk of its afferent input from neurons in an
homologous "barreloid" within the ventral posteromedial (VPM)
thalamic nucleus (Land and Simons 1985). Neurons within
the barrel and throughout its associated column are maximally excited
by a principal whisker (PW) but, depending on laminar location, they
respond also to neighboring whiskers to varying degrees
(Armstrong-James and Fox 1987
; Simons 1978
). Deflection of two or more whiskers in rapid sequence
reveals the presence of surround inhibitory effects that are
considerably stronger in cortical than thalamic neurons
(Brumberg et al. 1996
; Simons and Carvell
1989
).
Previously, we proposed that inhibitory interactions among neighboring
whiskers in the layer IV barrel reflect direct engagement of local
circuitry by thalamic inputs (Simons and Carvell 1989). We hypothesized that inputs to a barrel from nonprincipal whiskers arise directly from thalamic afferents, because neurons within thalamic
barreloids, although driven most strongly by the PW, also respond
robustly to neighboring whiskers (Nicolelis et al. 1993
;
Simons and Carvell 1989
). The absence of a direct
barrel-to-barrel pathway (Akhtar and Land 1989
;
Bernardo et al. 1990b
; Hoeflinger et al.
1995
) further supports the idea that barrels function
independently of each other. Accordingly, destruction of a cortical
barrel should have little, if any, effect on the influence of its
corresponding whisker in neighboring barrels (see Fig.
1). Here we demonstrate that adjacent
whisker-evoked excitation and inhibition within a barrel are virtually
unaffected by ablation of the adjacent barrel.
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METHODS |
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This study was conducted using adult female Sprague-Dawley rats
(202-315 g; Hilltop). Surgery and anesthetic procedures were similar
to those previously described (Brumberg et al. 1996). Briefly, animals were anesthetized with halothane (~1.5% in a 50-50
mixture of N2 and O2) and
tracheotomized. Venous and arterial catheters were inserted for later
drug delivery and blood pressure monitoring. The animal was placed in a
stereotaxic frame, and a craniectomy was made in the skull overlying
part of the whisker representation area of the right primary
somatosensory cortex. A small steel post was attached to the skull with
dental acrylic to hold the animal's head during the experiment. An
acrylic dam was placed around the craniectomy and was kept filled with saline.
The underlying cortex was roughly mapped by multiunit recordings made
through the dura using tungsten microelectrodes (Frederick Haer,
Brunswick, ME; medium point, 3-5 M at 1 kHz), combined with manual
stimulation of the whiskers on the contralateral face. The dura was
then removed over the cortical region of interest (~0.5 × 1.0 mm). The animal was taken off halothane, sedated with fentanyl
(Sublimaze, Jannsen Pharmaceuticals; 5-10 µg · kg
1 · h
1),
immobilized by pancuronium bromide (~1.6 mg · kg
1 · h
1), and
artificially respired with a humidified 50-50 mixture of N2 and O2. Arterial blood
pressure, heart rate, and tracheal airway pressure were monitored by a
program written in LabVIEW 4.0 (National Instruments) running on a
Power Macintosh 7100/66. Pupillary reflexes and electroencephalogram
also were monitored. Ophthalmic ointment was placed over the eyes to
prevent corneal drying.
A video camera attached via a beam-splitter to a surgical microscope
was used to photograph the brain surface, using green light
illumination to enhance blood vessel contrast. A detailed map of the
targeted region of the barrelfield was then made using fine-tipped,
glass/carbon fiber microelectrodes (see Kyriazi et al.
1996), with special attention being paid to the barrel chosen for ablation. To ablate a single barrel, an unused, high-impedance tungsten microelectrode (Frederick Haer: medium tip, 0.010-in. shank
diameter, 10-12 M
at 1 kHz) was inserted normal to the pial surface
overlying the estimated barrel center. To minimize dimpling of the
brain surface and to achieve reproducible penetration depths, the
electrode was advanced initially to a depth of 1,500 µm and then
withdrawn to 1,050 µm. Because preliminary experiments indicated that
electrolytic lesions made with these electrodes produced a conical
abscess that spread superficially, DC (30 µA for 30 s, electrode
negative) was passed initially at a depth of 1,050 µm followed by a
second application at 700 µm, which we routinely find to correspond
to the layer III/IV boundary. Immediately thereafter spontaneous unit
activity could be recorded deep to the lesion but not at middle or
superficial cortical depths.
Subsequently, we examined the receptive field properties of units in a
barrel/column (test barrel) immediately adjacent to the ablated barrel
(see Fig. 1). We intentionally selected units that gave vigorous
excitatory responses to the PW, because we assumed at the outset that
such units were unlikely to be in close proximity to damaged tissue
(but see RESULTS). Also, we assumed that
suppression of such responses by adjacent whisker (AW) stimulation would be a robust indicator of intact inhibitory mechanisms within the
test barrel. Unit recordings were obtained using double-barreled glass
micropipettes, one barrel of which contained 3 M NaCl and the other
10% wt/vol horseradish peroxidase (HRP) for marking selected recording
sites (Simons and Land 1987).
Electromechanical stimulators were used to deflect the test barrel's
PW and two AWs (see Simons and Carvell 1989), one
corresponding to the lesion-ablated barrel (AWL) and the
other to an intact (normal) barrel (AWN) on another side of
the test barrel (see Figs. 2 and 4). The
excitatory influence of each AW was quantified as the average number of
spikes/stimulus taken over eight angles of deflection. Each deflection
angle was repeated 10-20 times. Spike counts were measured during the
5-25 ms following stimulus onset. To quantify inhibitory AW effects,
the AW was deflected in each of eight directions followed 20 ms later
by PW deflection at its maximally effective angle. A condition-test
ratio (CTR) was calculated as the ratio of the average response to the
PW when deflected after the AW to the response to PW deflection alone. Data from the responses evoked by the two AWs were compared using two-tailed paired-sample t-tests. For all trials in
which at least one spike occurred during the stimulus onset window, the
time of the first spike was measured at 0.1-ms resolution, and the mean
and modal latencies across all trials and deflection angles were
determined. For modal latencies, spikes were placed in 0.5 ms bins, and
the bin with the greatest number of spikes was taken as the mode. No
modal latencies were returned for units in which no bin contained more
than one spike. All data are expressed as means ± SD unless
indicated otherwise.
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In two control experiments, the nerves innervating a whisker follicle were reversibly inactivated by infusion of 5 µl of 4% lidocaine (Xylocaine, Astra USA). Under halothane anesthesia, a 30-gauge needle was inserted 2-3 mm into the hair follicle on the caudal side of the whisker (where the afferent fibers enter the capsule). PE-10 tubing connected the needle to a Hamilton syringe, and the entire assembly was positioned such that the needle was suspended in the approximate plane of the whisker. This minimized mechanical effects of the needle's presence on the mystacial pad and permitted attachment of a stimulator to the whisker.
At the conclusion of each experiment, animals were administered a lethal dose of pentobarbital sodium (Nembutal, Abbott Laboratories) and perfused transcardially with phosphate-buffered saline followed by a solution of 2% paraformaldehyde and 1.5% glutaraldehyde. Brains were sectioned on a freezing microtome in the tangential plane, and alternate sections were stained for HRP or cytochrome oxidase and counterstained with thionin. Lesions consisted of a large abscess fringed by a region of cellular disruption in which Nissl-stained neuronal cell bodies were clearly absent. We used the latter as a conservative estimate of the extent of direct physical damage.
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RESULTS |
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Data are reported from 12 experiments in which we estimate from serial tangential sections that, on average, 90% of a barrel was destroyed. Figure 2 shows photomicrographs of barrel fields from four experiments that illustrate the range of destruction as well as the locations of the test barrels. The distribution of lesion sizes, which ranged from 70-100% destruction, for all experiments is plotted in Fig. 3. In the vertical dimension, damage extended downward, having a blunt conical shape whose apex extended well into layer V. From upper layer V through layers IV and lower layer III, the lesion was shaped cylindrically. More superficially, the lesion also tapered conically; the largest lesions extended almost to the pial surface. Lesions rarely extended into neighboring barrels but typically involved the septum between the lesion and the test barrel.
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Dendrites of barrel neurons in rats have an average radius of 100 µm,
and neurons near the barrel side can have dendrites that extend into
the septum and even into the neighboring barrel (Simons and
Woolsey 1984). We therefore classified the recorded units into
two groups: those in the cytochrome oxidase-rich barrel center at least
100 µm distant from the side of the barrel nearest the lesion
(hereafter denoted as "Barrel") and those within this 100 µm zone
("Near-lesion"). The latter included some cells located in the
barrel side and possibly in the immediately adjacent septum. The
relative proportions of units studied in experiments involving different lesion sizes were approximately equal (see Fig. 3). Because
all lesions were extensive and because the number of units studied in
each experiment was relatively small, we pooled data across
experiments. All but 4 of 30 supragranular units were located above the
barrel center; those 4 Near-lesion units were not included in the analyses.
Peristimulus-time histograms of recordings taken from the E2 barrel of an E1 barrel-ablated animal are shown in Fig. 4. The onset and offset of PW (E2) deflection elicit prominent excitatory responses. AWN (E3) evokes a clear, but weaker, excitatory response and a pronounced suppression of the response evoked by subsequent PW deflection. Most notably, the AWL (E1) also evokes virtually identical excitatory and inhibitory effects, despite the near-total ablation of the E1 barrel. On average, a Barrel unit's AW-evoked excitatory response was ~20% that of its PW. Pooled Barrel results demonstrate that neither AWL-evoked excitation nor AWL-evoked inhibition were diminished by destruction of the AW's associated barrel (Fig. 5). Furthermore, there were no significant differences between the AWL and AWN latencies, either mean (15.4 ± 1.9 ms, 15.9 ± 1.4 ms, mean ± SD, n = 27), or modal (14.2 ± 3.6 ms, 15.4 ± 3.3 ms, n = 21). The PW latencies (mean:12.9 ± 1.7 ms, n = 43, modal: 11.6 ± 2.2 ms, n = 35) were significantly shorter than those of either AW (all P values <0.001).
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Near-lesion units displayed statistically significantly less AWL-evoked excitation and inhibition compared with those evoked by AWN. In addition, excitatory AWL responses were 42% smaller than those at locations more distant from the lesion, and these effects were greater with larger lesions (R2 = 0.47, P < 0.001). PW-evoked excitatory responses in Near-lesion units also were slightly (~17%) but not significantly smaller than those of Barrel units. Interestingly, AWN-evoked inhibition was stronger in Near-lesion than in Barrel units, and this, too, was correlated with lesion size (R2 = 47, P = 0.007).
We also recorded from neurons in layers II/III superficial to the center of the test barrel (Fig. 5). Condition-test ratios evoked by AWL and AWN were not significantly different from each other. There was, however, a 54% reduction in the size of the AWL-evoked excitatory response (P = 0.03, paired t-test); no correlation with lesion size was observed.
One possible explanation for the normal levels of
AWL-evoked excitation and inhibition observed in
the Barrel recordings is that AWL effects were
mediated by adjacent barrel remnants that may have survived the lesion
and continued to communicate with the test barrel by means of direct,
straight-line connections across the interbarrel septum (see Fox
1994). We therefore performed one experiment in which 14 additional, smaller lesions (10 µa, 10 s) were made in 7 penetrations, at 1,050 and 700 µm depths, in a line along the septal
region between the ablated adjacent barrel and the test barrel. This
procedure resulted in extensive damage to both the ablated barrel and
the intervening septum and eliminated any possibility of direct,
straight-line barrel-to-barrel communication. Nevertheless,
AWL-evoked excitation and inhibition remained at
normal levels (Barrel, spikes/stimulus: AWL = 0.65 ± 0.28, AWN = 0.69 ± 0.30;
condition-test ratio: AWL=0.50 ± 0.15, AWN = 0.56 ± 0.15; n = 5).
Another possible explanation for the ineffectiveness of the lesion is
that slight, unintended movements of the test barrel's PW that occur
on AW deflection directly activate the test barrel. This may be of
particular concern when the PW remains held by a stationary stimulator
during AW deflection (Simons 1985). To address this
issue, we performed two experiments in which the peripheral nerves
innervating the PW were reversibly inactivated by injection of
lidocaine into the follicle. Immediately after injection, units in the
test barrel were completely unresponsive to the PW and partially
responsive to the AW. Within 45 min after injection, AW-evoked
excitation had recovered to near-normal levels, whereas responses to
the (anesthetized) PW were absent entirely or reduced to below AW
levels for an additional 15-60 min. We therefore consider it unlikely
that mechanical transmission across the mystacial pad accounts for the
bulk of the AWL, or AWN,
response in the test barrel.
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DISCUSSION |
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The major finding of this study is the remarkable preservation in
the center of the test barrel of AW-evoked excitatory and inhibitory
effects despite near-total ablation of the AW's barrel. We consider
layer VI neurons deep to the lesion an unlikely source of the normal
levels of AWL-evoked effects, because at the
minimum their apical dendrites were severely damaged by the lesion,
which extended well into layer V. Results support our hypothesis that thalamic afferents normally provide direct excitatory AW input to a
barrel (Fig. 1B). These thalamic inputs may originate from multiwhisker neurons in the homologous thalamic barreloid
(Brumberg et al. 1996; Simons and Carvell
1989
) and/or from neurons in adjacent, nonhomologous barreloids
(Land et al. 1995
; Land and Simons 1985
). Further, AW-evoked inhibition is, on average, weaker in barreloid than
barrel neurons. The observation of normal levels of
AWL-evoked inhibition in the test barrel is
consistent with the idea that thalamic activation of barrel circuitry
on AW stimulation evokes surround inhibition by a feed-forward,
intrabarrel mechanism, enhancing the response tuning of barrel neurons
(Brumberg et al. 1996
; Kyriazi and Simons
1993
; Simons and Carvell 1989
).
We attribute the abnormalities observed in Near-lesion cells to altered
synaptic circuitry resulting from direct damage to thalamic afferents
and/or dendritic processes of test barrel neurons. Lesion by-products,
e.g., elevated levels of extracellular potassium or glutamate, may also
have contributed to Near-lesion abnormalities. If excitatory
by-products disproportionately affect inhibitory barrel neurons, which
are normally highly excitable, this could account for the somewhat
paradoxical finding that AWN-evoked inhibition was greater in Near-lesion than in Barrel units. In either case, the
fact that receptive field abnormalities are observed in some neurons
(Near-lesion) but not in other, more distant ones (Barrel) residing in
the same barrel suggests that a barrel may contain several
relatively independent subnetworks (see Chmielowska et al.
1989; McCasland et al. 1992
). The nature and
sizes of such possible circuits, and the degree to which they may or
may not interact, remain to be determined.
In contrast to results in layer IV, adjacent barrel lesion led to a
clear reduction in AWL responses recorded in
layers superficial to the test barrel. We suggest that AW input
normally reaches supragranular layers via several pathways (see
Bernardo et al. 1990a,b
; Gottlieb and Keller
1997
): 1) an intracolumnar, vertical pathway
originating within the test barrel itself, 2) a pathway originating in the adjacent barrel, which includes an additional horizontal, intercolumnar component within the supragranular layers, and 3) recurrent collaterals from infragranular neurons deep
to the adjacent barrel. The lesions eliminated the second and possibly the third of these routes.
The present findings in layer IV differ markedly from those of two
previous studies, which used a similar experimental paradigm but in
urethan-anesthetized animals (Armstrong-James et al.
1991; Fox 1994
). In those studies, barrel lesion
reduced excitatory AWL responses in proportion to
the extent of the lesion, and modal latencies increased from 15.2 to
24.3 ms. Inhibitory interactions were not assessed. The authors
concluded that direct barrel-to-barrel connections normally mediate AW
responses (see Fig. 1A). In the study of Armstrong-James et
al., the mean barrel destruction was 58% compared with 90% in the
present study. Moreover, it appears that the present lesions extended
deeper and more superficially (judging from Fig. 4 in
Armstrong-James et al. 1991
). Although Armstrong-James
and colleagues did not categorize the barrel units with respect to
their proximity to the lesion, as done here, it is clear from their
METHODS section that most of their data were obtained >100
µm from the side of the barrel closest to the lesion. Thus
differences in results cannot be explained by differences in the
location of the recorded units or by differences in lesion size.
The most likely explanation for the discrepant findings is that
AW-evoked excitatory responses are qualitatively different in the two
experimental preparations. In terms of AW response latency and
magnitude, relative to PW responses, the present data are comparable to
values obtained previously in awake, undrugged rats (Simons et
al. 1992). As discussed in that study, urethan anesthesia
increases the magnitude and duration of AW-evoked responses, possibly
through involvement of N-methyl-D-aspartate
(NMDA) receptors (see Armstrong-James et al. 1993
).
After exposure of tangential barrel field slices to bicuculline
methiodide, NMDA-dependent paroxysmal discharges can propagate across
the barrel field (Fleidervish et al. 1998
). Thus it
appears that long latency, long-duration, barrel-dependent AW responses
are expressed under conditions where NMDA-dependent synaptic
transmission may be more prominent.
Taken together with results of previous modeling studies
(Kyriazi and Simons 1993; Pinto et al.
1996
), the present findings demonstrate that local, intrabarrel
circuitry is sufficient to account for the integration, both excitatory
and inhibitory, of multiwhisker information within individual layer IV
barrels. Although there are almost certainly some means for barrels to
influence each other, directly or indirectly, interactions are likely
to be modulatory, perhaps contributing to the overall excitability of
the barrel field during different behavioral states (see
McCasland et al. 1997
for a discussion). Whatever role
such interactions might play, available anatomic and physiological
evidence indicates that barrels function as an array of independent,
parallel processors of afferent information. Accordingly, barrel
circuitry transforms multiple-whisker inputs into predominantly
single-whisker outputs, which are then distributed to other layers of
the cortical column, where larger and more complexly organized
receptive fields are synthesized via intercolumnar connections.
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ACKNOWLEDGMENTS |
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We are especially grateful to R. Bruno for extensive participation in preliminary studies and to J. Brumberg for help with some of the experiments.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-19950.
Present address of D. Goldreich: Dept. of Occupational Therapy, Duquesne University, Pittsburgh, PA 15282.
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FOOTNOTES |
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Address reprint requests to D. J. Simons.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 February 1999; accepted in final form 13 May 1999.
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REFERENCES |
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