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The Journal of Neurophysiology Vol. 82 No. 2 August 1999, pp. 963-977
Copyright ©1999 by the American Physiological Society
1Department of Neurobiology,
Katz, Donald B.,
S. A. Simon,
Aaron Moody, and
Miguel A. L. Nicolelis.
Simultaneous Reorganization in Thalamocortical Ensembles Evolves
Over Several Hours After Perioral Capsaicin Injections.
J. Neurophysiol. 82: 963-977, 1999.
Reorganization of the somatosensory system was quantified by
simultaneously recording from single-unit neural ensembles in the
whisker regions of the ventral posterior medial (VPM) nucleus of the
thalamus and the primary somatosensory (SI) cortex in anesthetized rats
before, during, and after injecting capsaicin under the skin of the
lip. Capsaicin, a compound that excites and then inactivates a subset
of peripheral C and A The last 15 years of research on the somatosensory
system has revealed that low-threshold mechanoreceptor receptive fields (RFs), once thought to be static, reorganize after a variety of peripheral manipulations, including amputation (Elbert et al. 1997 Recent analyses of electrophysiological data obtained from rats has
suggested that this reorganization may arise from changes in the
feedback and feedforward interactions throughout the trigeminal somatosensory system that tend to maintain a dynamic equilibrium between excitatory and inhibitory afferents (Krupa et al.
1997 There is substantial evidence that the C- and A In this study, we further investigated the involvement of the
nociceptive pathway in the process of somatosensory reorganization in
anesthetized rats by simultaneously recording the extracellular activity of populations of single neurons located in the SI
"barrel" cortex and the ventral posterior medial (VPM) nucleus of
the thalamus, before and after injecting small amounts of capsaicin
under the skin of the lip. Chronic multisite, multielectrode techniques and computer control of stimulus delivery enabled us to quantify how,
after capsaicin injections, the spatial and temporal relationships among these simultaneously recorded neural ensembles change. We found
that capsaicin injections caused spontaneous levels of activity to
simultaneously increase in VPM and SI. More importantly, RFs in VPM and
SI rapidly changed and continued to reorganize over the course of
6 h. The extended time course of the VPM/SI changes, compared with
the much shorter time course previously reported for capsaicin-related
reorganization in second-order nociceptive relays (Chiang et al.
1997 Subjects
Eight female Long Evans rats (230-280 g at the time of surgery)
served as the subjects in this study. Animals were maintained on a 12 h/12 h light/dark schedule, with experiments carried out in the light
portion of the cycle. Animals in home cages had ad libitum access to
normal rat chow and water.
Implantation of microwires
A complete description of these techniques can be found
elsewhere (Nicolelis et al. 1997 Simultaneous multisite single-unit recording
Details of this technique have been described elsewhere
(Nicolelis and Chapin 1994 Experimental protocol
After 5-7 days of postoperative recovery, subjects underwent
one or two experimental sessions separated by 2-4 days (a separation of 2 days was required to ensure that animals recovered fully from
anesthesia following the 1st session; no differences in spontaneous firing rates or RF sizes were observed between sessions). In each session, the rats were reanesthetized and moved to the experimental chamber, where thalamic and cortical single units were discriminated. The placement of the recording electrodes, combined with the
consistently low spontaneous firing rates that were observed and the
3:1 signal-to-noise (S/N) ratio set as a thresholding criterion,
allowed us to identify the discriminated units as pyramidal cells in
the SI cortex and thalamocortical projection neurons in VPM
(Simons and Carvell 1989 After the neurons were discriminated, single neuron RFs were measured
by positioning a mechanical stimulator directly beneath a single
vibrissa, ~5 mm from the follicle. Low-amplitude whisker deflections
of 100-ms duration were delivered at a frequency of 2 Hz. Preliminary
observations showed that stimulation rates between 0.5 and 2.5 Hz
produce similar poststimulus time histograms (PSTHs) and no
statistically significant adaptation over the course of stimulus
presentations (D. Katz, D. Krupa, and M. Nicolelis, unpublished observations). Each whisker was deflected 300 times (i.e., for 2.5 min), after which the stimulator was repositioned under another whisker
and the procedure was repeated. Up to 20 of the large facial vibrissae
were stimulated in each protocol, which typically was completed in
1.25-1.5 h.
Subcutaneous injection of capsaicin
After this initial RF mapping, 25-30 µl of a 10% capsaicin
dispersion in 30% ethyl alcohol with 0.5% Tween-80 (added to improve solubility) or, alternatively, 25-30 µl of the vehicle alone, was
injected subcutaneously to the perioral region through a 30-gauge needle. Injections were made into the upper lip, ~6 mm away from the
whisker pad, nearest to whiskers E2 and E3 (see Fig. 3D). RF
mapping was then repeated one to three times, using parameters identical to those used in the original protocol.
Data sets also included 30-min periods of spontaneous activity,
recorded before each full mapping protocol. Capsaicin or vehicle injection took place halfway through the recording made between the
first and second RF mappings. Small (0.05 ml) intraperitoneal pentobarbitol boosters were given if breathing became light and fast or
if the rat responded to tail pinch.
Data analyses
POSTSTIMULUS TIME HISTOGRAMS (PSTHS).
The spiking responses of each unit were first summed into PSTHs for all
trials in which a given single whisker was stimulated. This was
repeated for all the stimulated whiskers. A one-way Kolmogorov-Smirnov test was used to determine, for each PSTH, whether the stimulus-locked firing rate rose to a level significantly above spontaneous activity (difference between baseline and response, P < 0.05).
If a significant change occurred, the neuron was categorized as
responsive to that whisker, and that whisker was considered a part of
that neuron's RF. For purposes of visualization, population
poststimulus time histograms (PPSTHs) were used to depict the
simultaneous sensory responses of ensembles of simultaneously recorded
neurons in a single graph (Nicolelis et al. 1999 MULTIDIMENSIONAL SCALING.
To gain insight into population level processes that might be driving
activity in the system, linear multivariate techniques were used to
examine the trial-by-trial activity across the recorded ensembles of
neurons during individual whisker stimulation. One such technique,
multidimensional scaling (MDS), offers a straightforward, quantitative
way to measure how neurons may cluster into "functional groupings"
(Borg and Groenen 1997
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
fibers, triggered increases in spontaneous
firing of thalamocortical neurons (10-15 min after injection), as well
as rapid reorganization of the whisker representations in both the VPM
and SI. During the first hour after capsaicin injection, 57% of the
139 recorded neurons either gained or lost at least one whisker
response in their receptive fields (RFs). Capsaicin-related changes
continued to emerge for 
6 h after the injection: Fifty percent of the
single-neuron RFs changed between 1-2 and 5-6 h after capsaicin
injection. Most (79%) of these late changes represented neural
responses that had remained unchanged in the first postcapsaicin
mapping; just under 20% of these late changes appeared in neurons that
had previously shown no plasticity of response. The majority of the
changes (55% immediately after injection, 66% 6 h later)
involved "unmasking" of new tactile responses. RF change rates
were comparable in SI and VPM (57-49%). Population analysis indicated
that the reorganization was associated with a lessening of the
"spatial coupling" between cortical neurons
a significant
reduction in firing covariance that could be related to distances
between neurons. This general loss of spatial coupling, in conjunction
with increases in spontaneous firing, may create a situation that is
favorable for the induction of synaptic plasticity. Our results
indicate that the selective inactivation of a peripheral nociceptor
subpopulation can induce rapid and long-evolving (6 h) shifts in the
balance of inhibition and excitation in the somatosensory system. The
time course of these processes suggest that thalamic and cortical
plasticity is not a linear reflection of spinal and brainstem changes
that occur following the application of capsaicin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
), nerve cut (Garraghty and Kaas 1991;
Merzenich et al. 1983; Rasmussen 1996),
injection of local anesthetics such as lidocaine (Nicolelis et
al. 1993b) or injection of irritants (mustard oil, formalin,
bradykinin, or most relevantly for our purposes capsaicin; see
Baumann et al. 1991; Cook et al. 1987;
Fitzgerald and Woolf 1982; Hoheisel et al.
1993; Kwan et al. 1996; Lin et al.
1998; McMahon et al. 1993; Nussbaumer and
Wall 1985; Raboisson et al. 1995; Simone
et al. 1989, 1991; Wall 1987; Yu et al.
1993).
; Nicolelis 1996, 1997; Nicolelis et
al. 1998). It has been suggested that this
excitatory/inhibitory balance sets up a situation whereby a fraction of
the neural connections that could transmit information from a peripheral (skin) region to a CNS neuron are "latent," suppressed by patterns of inhibition (Jacobs and Donoghue
1991; Schroeder et al. 1995). In many cases
peripheral manipulations induce a different fraction of the RF to
become active (Byrne and Calford 1991). Subcutaneous
lidocaine injections well outside the recorded RFs of single neurons,
for instance, have been shown to cause both unmasking and masking of
sensory responses throughout multiple relays of the somatosensory
system almost immediately (Faggin et al. 1997;
Nicolelis et al. 1993a).
-fiber nociceptors
may play an important role in modulating RFs of somatosensory system
neurons (Baron and Maier 1995; McMahon et al.
1993; Simone et al. 1991). For example, methods
used to produce RF changes frequently alter nociceptor activity.
Notable among these methods is the administration of capsaicin, the
pungent ingredient in hot pepper, which specifically targets C-fiber
nociceptors and A-thermoreceptors, first exciting and then silencing
them (Baumann et al. 1991; Caterina et al.
1997; Green 1991; Serra et al.
1998; Szolcsanyi et al. 1988). Capsaicin induces
RF changes when it is applied directly to the peripheral nerve
(Fitzgerald and Woolf 1982; Mannion et al.
1996; Nussbaumer and Wall 1985; Wall
1987), injected subcutaneously (Calford and Tweedale
1991; Pettit and Schwark 1996; Rasmussen
et al. 1993; Simone et al. 1989), or applied systemically in neonates (Cervero and Plenderleith 1985;
Chiang et al. 1997; Kwan et al. 1996;
Wu and Gonzalez 1995). RF changes induced by these means
have been detected at the cortical (Calford and Tweedale
1991; Nussbaumer and Wall 1985; Toldi et
al. 1992), thalamic (Rasmussen et al. 1993),
brain stem (Chiang et al. 1997; Kwan et al.
1996), and spinal cord (Cook et al. 1987;
Ma and Woolf 1996; Pettit and Schwark
1996; Simone et al. 1989) levels of the CNS. In
some cases, these changes have been associated with changes in tactile
sensibility and to decreased pain thresholds (Green and Flammer
1988; McBurney et al. 1997; Simone et al.
1991; Treede et al. 1992).
; Cook et al. 1987; Kwan et al.
1996; Ma and Woolf 1996; Pettit and
Schwark 1996; Simone et al. 1991), suggests that
the processes responsible for the observed reorganization are at least
partially intrinsic to the thalamocortical loop of the somatosensory
system. In addition, our ensemble analyses revealed that manipulation
of the capsaicin-sensitive nociceptive pathway decreases the local
interaction between neighboring cortical neurons without changing their
temporal relationships.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). Briefly, animals were
anesthetized using a 5% halothane/air mix, quickly followed by an
intraperitoneal injection of pentobarbital (50 mg/kg). Stable levels of
anesthesia (i.e., no tail pinch or eye blink reflexes) were maintained
with small (0.05 ml) additional pentobarbital injections. The
anesthetized animal was placed on a standard stereotaxic frame, after
which the scalp was excised. Small craniotomies were made for four to six ground screws and two microelectrode assemblies (NBLabs, Denison, TX). Each microelectrode assembly included 16 50-µm Teflon-coated stainless steel microwires (see Fig. 1 in Nicolelis et al.
1997). After resection of the dura over cortex and thalamus,
microwire arrays (SI cortex) and bundles (VPM thalamus) were lowered
slowly into layer V of SI cortex and the barrelloid region of the VPM, guided by stereotaxic measurements, constant examination of the electrophysiological signals, and monitoring for responses to whisker
stimulation. Thalamic bundles were lowered in the vertical stereotaxic
plane, and cortical arrays were lowered normal to the exposed cortical
surface. Once in position, the electrodes were cemented to the skull
with dental acrylic, the scalp was sutured or stapled around the
implant, and antibiotic ointment was applied liberally to the wound.
Postoperative analgesic (buprenex) was administered as needed.
; Nicolelis et al.
1997). Briefly, spontaneous and evoked neural activity from all
implanted microwires was simultaneously digitized at 40 kHz (Plexon,
Dallas, TX). Single and multiple units were discriminated from each
digitized record. Clearly identifiable spikes (minimum 3:1 S/N ratio)
were discriminated, using a combination of an amplitude threshold and
two time/amplitude windows (Nicolelis et al. 1997). Time
stamped records of action potentials were saved to a desktop Pentium
computer as were the sampled waveform segments themselves and vibrissa
deflection times. Off-line examination of waveforms, discrimination,
and interspike interval statistics (<5% of the recorded spikes
occurring within 1 ms of another such spike and a mode of >2-3 ms)
allowed definitive classification of single units. Only data from
single units that were discriminable for entire sessions are reported here.
). Four of the eight animal
subjects had viable placements in both SI and VPM. Two had
discriminable units in SI only, and two had discriminable units in VPM only.
). The
gain and loss of significant responses across time was tabulated,
allowing us to characterize the reorganization that followed capsaicin
and vehicle injections for several hours.
; Davison 1985;
Erickson et al. 1993; Johnson and Wichern
1992) by setting the neurons into a space of predetermined
dimensionality in which the proximity of any two neurons estimates
their similarity in temporal response property. The use of concatenated
single trials as the input matrices (see following text) caused MDS to
calculate these spaces in terms of temporal firing relationships
between the neurons on a trial-by-trial basis. Correlations of the
pairwise distances between the neurons in separate MDS solutions
offered estimates of how functional groups changed with different
experimental manipulations, and according to stimulus identity.
) and encoded for a
commercially available statistical package (S-plus, MathSoft) by Brian
Ripley, relaxes assumptions about the nature of the distances, instead
making use only of the rank order of pairwise dissimilarities.
SEMIVARIANCE.
Spatial statistics imported from physical geography (Burrough
and McDonnell 1998; Cressie 1993;
Kitanidis 1997) were employed to provide estimates of
how the measured cortical population activity could have resulted from
orderly transmission of information across the "cortical field."
For the purposes of this analysis, it was first necessary to
approximate the locations of the discriminated cortical neurons on a
2 × 8 grid representing the tips of the rigid arrays implanted
normal to the cortical surface.
Histology
After the experimental sessions, subjects were deeply anesthetized with pentobarbitol and perfused through the heart with saline followed by 5% formalin in saline. Seven seconds of DC current (7 µA) were passed through selected microwires in preparation for staining. The brain was then removed and immersed in a 30%/10% sucrose formalin solution, and was refrigerated until saturated. Sections (80 µm) cut through the implanted areas were stained with Prussian blue for ferrous deposits blasted off of the electrode tips and counterstained with cresyl violet for cell bodies.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Neural data
Eight animals received chronic VPM and SI implants. Of these, four
provided single neuron records in both thalamus and cortex, two
provided mainly cortical single units, and two provided mainly thalamic
single units. A total of 139 single neurons were recorded60 in VPM
and 79 in SI. The average number of neurons recorded from these
implants was thus 13.2 for SI and 10.0 for VPM.
Effect of capsaicin injections on single-unit SI and VPM whisker responses
CHANGES IN SPONTANEOUS FIRING.
Increases in the spontaneous firing rates of cortical and thalamic
neurons were observed after subcutaneous capsaicin injections (Fig.
1). These increases appeared within 15 min of injection in both VPM and SI and continued for up to 15 min
(whereupon the RF mappings began). The increases partially consisted of
bursts of high activity (Fig. 1)similar capsaicin-induced bursting
has been reported previously in recordings from neurons in the
spinothalamic tract (see Fig. 4 in Simone et al. 1991).
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RF REORGANIZATION.
Extremely small (25-30 µl) injections of a 10% capsaicin solution
caused rapid, spatially distributed, and lasting RF changes in VPM and
SI neurons. That is, neurons developed or lost responses to at least
one whisker at the very first measurement and maintained these changes
for 
6 h. An example showing mainly excitatory changes can be seen in
Fig. 3, which contains
a series of PPSTHs (and some of the constituent PSTHs) taken from
animal 97-103. This figure shows the responses of all
simultaneously recorded single-units to deflection of whisker D5 before
and 0.5 and 6.5 h after capsaicin treatment. New responses
appeared in both cortex and thalamus (see the extracted PSTHs as well)
soon after the injection, and most of these new RFs were still apparent
6 h later. Analysis of all eight animals revealed that
subcutaneous capsaicin injections consistently caused long-lasting
changes in thalamocortical responses.
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6 h later, the percentage of neurons with at least one RF that
had changed since the previous postcapsaicin RF mapping was 50% (66%
of which were unmasked responses), a percentage mainly (79%) made up
of responses that had not changed immediately after capsaicin
injection. A subset of these new responses (19%) involved neurons that
previously had shown no capsaicin-related responses to any whisker.
Control injections caused some response changes themselves, but the
alterations wrought by vehicle injections were far less extensive than
those caused by capsaicin injections. Overall, 24% of recorded neurons
developed new RFs soon after vehicle injections (69% of these were
unmasked responses). An additional control was performed on one
subject, involving a dry needle stick without injection. A negligible
rate of RF change (12%) was observed after this procedure, suggesting
that the changes after vehicle injections were not merely the result of
damage from puncture. Still, for every neuron that developed or lost a
whisker response after vehicle injection, more than two did so after
capsaicin injection.
For one subject, vehicle-related changes were followed 6 h
postinjection. Only 22% of the recorded RFs changed in between the
earlier and later postvehicle mappings. It is unclear whether this
percentage reflects long-term effects of the vehicle injection or
"spontaneous" changes occurring across recordings of several hours.
It is clear, however, that a much higher percentage of neurons changed
after capsaicin injection, both soon after and long after the injections.
The percentages of neurons for which RFs changed in response to
capsaicin and vehicle injection were compared via a simple one-way
ANOVA for mapping time (1 h postcapsaicin, 6 h postcapsaicin, and
1 h postvehicle). This analysis revealed that significantly more
neurons gained or lost responses after capsaicin injection than vehicle
injection (F(2,28) = 3.46, P < 0.05). Overall,
these results demonstrate that capsaicin reorganizes the somatosensory system, that this reorganization is much larger than that caused by
control injections, and that this reorganization continues for 6 h.
Similar percentages of neurons changed in thalamus or cortex, both 1 and 6 h after capsaicin injection (Fig.
5). In cortex, 59% of the neurons
changed immediately after capsaicin injection, 54% 6 h after; in
thalamus, the 54% of the neurons developed new RFs rapidly, and 44%
developed new RFs in later mappings. A 2 × 2 mixed ANOVA for
recording site and time postcapsaicin revealed no differences in the
percentage of SI and VPM neurons showing reorganization (all
P values > 0.25). Similarly, the number of whiskers in
the unmasked responses did not differ between cortex and thalamus. On
average, SI neurons began responding to 0.68 ± 0.10 (mean ± SE) new whiskers soon after injection, while VPM neurons began
responding to 0.52 ± 0.14 new whiskers. At the later mapping,
similar RF unmasking was found (0.61 ± 0.11 in SI, 0.45 ± 0.17 in VPM); none of these differences between SI and VPM was significant.
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). As can be
seen in this example, which is representative of much of the neuronal
sample, the spatial pattern of peak response changed as a function of
poststimulus time, and the overall spatiotemporal pattern changed after
capsaicin injection. For this cortical neuron, shifts in the spatial
location of the short-latency RF center predominated, but longer
latency changes could be observed as well. Although the RF changes were noticed within 1 h after the injection, RFs also changed in the hours after the initial change. Once again, this demonstrated that the
subcutaneous capsaicin injection caused immediate reorganization, and
that this reorganization continued to evolve in both SI and VPM for 6
h.
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Ensemble analyses of changes after capsaicin injections
CHARACTERIZATION OF TEMPORAL RESPONSE PROPERTIES OF ENTIRE
ENSEMBLES VIA LINEAR MULTIVARIATE ANALYSIS: METRIC MDS.
The recent advent of multiple electrode simultaneous recording
technology has allowed researchers to obtain information concerning the
temporal relationships between the firing patterns of two or more
neurons (see, for instance, Deadwyler et al. 1996;
Nicolelis et al. 1995; Seidemann et al.
1996; Vaadia et al. 1995). An initial characterization of these relationships, at zero time lag, can be made
with linear multivariate techniques such as MDS. MDS arranges a space
of predetermined dimensionality, in which the relationships between a
set of variables can be estimated as Euclidean distances. We used this
technique to measure the temporal structure of the ensembles'
responses to particular whiskers at each RF mapping time and then
compared the changes between maps (MDS solutions) that could be related
to the effect of capsaicin.
CHARACTERIZATION OF SPATIAL RESPONSE PROPERTIES OF CORTICAL
ENSEMBLES: TIME-SEMIVARIOGRAMS.
The regular, grid-like arrangement of our multielectrode arrays in the
SI, implanted normal to the surface of the brain, made it possible to
specify, at least approximately, distances between simultaneously
recorded cortical neurons. These approximate distances were used, in
conjunction with the analysis of RFs from SI neurons, as input to a
geostatistical analysis (Burrough and McDonnell 1998;
Cressie 1993; Kitanidis 1997). This
analysis specifically allowed us to isolate one mechanism responsible
for any particular population firing patternspatial spread of
activity. We used this method to examine whether or not capsaicin
injections affect the spatial spread and coupling of SI neuronal
sensory responses. Specifically we used geostatistical analysis to test
whether variance attributable to the relative proximity between pairs
of neurons in layer V contributed to the patterns of ensemble activity
observed before and after capsaicin injection.
0, 250, 500, 750, and 1,000 µm (the
1000-µm lag is unreliable, however, as it approaches half of the
distance across the entire array). Only those four subjects are
included in the analysis of spatial variance.
This analysis demonstrated that firing patterns from SI of anesthetized
rats were spatially interpretable. Figure
7A presents the basic
time-semivariogram analysis for one animal (97-103). The
x axis is spatial lag (distance between pairs of neurons) from 0 to 1,000 µm; the y axis represents continuous
sequences of 10-ms time bins, with time bin 1 beginning at stimulus
onset. Variance progresses from low (black to dark red) to high (bright red to white) and has been interpolated into contours connecting equivariable points on the graphs.
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at time bins 1 and 4-10, no variance appears for neurons
separated by any of the five lags (these are the black regions). This
is true because most of these neurons were not firing at these
instances. Spatial structure emerged only 10-20 ms after stimulus
onset and disappeared soon thereafter. During these epochs, neurons
separated by 500 µm continued to behave similarly to each other
(deep red region), but the variability rapidly increased between lags
of 500 and ~850 µm (that is, the colors progress from deep red to
pink to white as the graph is scanned from left to right). Beyond this
separation, the variance appears to asymptote. Neurons found closer
together tended to behave similarly to each other, whereas neurons that
were further apart tended to behave less similarly. These results
suggest that ensembles of single neurons recorded from the cortical
sheet do behave, during stimulus response, analogously to points in a
geographical data set and that the ensemble itself can be profitably
thought of as a landscape. Simply put, spatial proximity seems to be an important mechanism whereby SI neurons in a population relate to each
other or covary.
The general shape of this spatiotemporal patternfleeting emergence of
an interpretable spatial structure in the population response to
whisker stimulationwas reliable across subject and across repeated RF
mappings. Figure 7, B and C, shows individually normalized time-semivariograms for the same neural ensemble seen in
Fig. 7A recorded ~1 h after and ~6 h after capsaicin
injection. It is important to note that each panel is normalized
individually, to emphasize the fact that the overall pattern replicated
1 and 6 h after subcutaneous capsaicin injections. Overall after
subcutaneous capsaicin injections, the similarity in response between
two neurons decreased as the distance between them increased. For this
individual subject, it appears that, after subcutaneous capsaicin
injections, the increases in spatial variability during epochs 2 and 3 begin at a smaller spatial separation; as neuron pairs become separated by >500 µm, the color becomes lighter (and thus the spatial variance becomes larger).
Figure 8, which shows the across-subject
average peak semivariances at each spatial lag, demonstrates that
capsaicin injections reliably increased the spatial variance at all
lagsthat is, the capsaicin decreased the spatial coupling between
neurons between the first and second RF mappings, effectively sliding
the semivariance curve higher on the y axis. A two-way
repeated measures ANOVA for spatial lag and mapping time revealed a
marginally significant main effect for time, despite the inclusion of
only four subjects in the analysis (F(2,6) = 4.28, P = 0.06). With such a small sample, this represents a
substantial effect (see Cohen 1992) and demonstrates the
robustness of the increase of spatial variance after capsaicin injection. The difference between the first postcapsaicin mapping and
the mapping made several hours later was not significant, but rather
the spatial variance appeared to stabilize at a new, less coupled level
within an hour of the injections. The interaction between mapping time
and spatial lag was significant (F(8,24) = 2.26, P = 0.05) as well.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we quantified the temporal evolution of
changes in firing rate and RF reorganization across simultaneously recorded populations of neurons in the rat somatosensory
thalamocortical loop caused by subcutaneous perioral injections of
capsaicin. We found that capsaicin injections outside the whisker pad
simultaneously increased the spontaneous firing of neurons in SI barrel
cortex and VPM thalamus, with the primary increase occurring between 11 and 15 min after injections; rapidly changed the RFs in both VPM and SI
neurons as well as the distributed patterns of neural activity caused
by stimulation of individual whiskers, in both structures at rates
above those after control injections; caused RFs to continue changing
for 6 h, in a manner that seemed largely identical in the two
structures; and decreased the spatial coupling among ensembles of SI
cortical neurons, causing neurons to respond more independently to
whisker stimulation.
Overall, this study provides evidence of the primary nociceptive pathway's influence on somatosensory system function and suggests that this influence can far outlast the short-term changes typically observed in the spinal and brainstem nociceptive nuclei themselves.
Changes in spontaneous activity after capsaicin injection
The first finding of this study was that subcutaneous capsaicin
injections significantly increased the basal levels of activity in VPM
and SI neurons (Fig. 1). Similar changes in firing rate have been noted
to occur in many preparations and thus represent a fundamental aspect
of the capsaicin response and of capsaicin-induced CNS plasticity
(Carstens et al. 1998; Simone et al. 1989,
1991). Although it is difficult to compare across differences
in capsaicin concentration, the changes in VPM and SI appeared to occur
later than those previously observed in the spinal cord and brain stem. VPM/SI increases developed abruptly between 10 and 15 min after the
injections (Fig. 2), whereas in the dorsal horn, spontaneous changes
develop almost immediately after injection (Simone et al. 1989,
1991), and in the trigeminal subnucleus caudalis, they develop
in 5-10 min (Carstens et al. 1998). Therefore these
data suggest that increases in tonic thalamocortical firing do not temporally track brain stem activity. Rather it appears that
capsaicin-related changes cause a rather sudden state change that
occurs later in the brain stem than in spinal cord and later in the
thalamocortical system than in the brain stem. This suggests that
firing rate increases in the VPM and SI do not simply reflect
alterations observed in the brain stem.
RF changes after capsaicin injections
We observed that subcutaneous capsaicin injections triggered
changes in the RFs of VPM and SI neurons, above and beyond those caused
by vehicle injection, that occurred either rapidly or in the following
6 h (Figs. 3-6). Similar changes have been observed previously in
the dorsal horn (Cook et al. 1987; Fitzgerald and Woolf 1982; Pettit and Schwark 1996;
Simone et al. 1991), principal trigeminal nucleus
(Chiang et al. 1997; Kwan et al. 1996),
ventrolateral thalamus (Rasmussen et al. 1993), and SI
cortex (Calford and Tweedale 1991).
Our results add to the above studies in two important regards: first, we were able to detect more subtle RF changes than had been observed previously, and also observed that these changes occurred in VPM and SI practically simultaneously. Second, we were able to chart the evolution of these changes during 6 h and to observe that unmasking and masking of sensory responses can happen in both VPM and SI throughout this period.
COMPLEXITY OF CHANGES.
Using computer-controlled stimulus delivery to individual whiskers, we
observed distributed, complex RF changes (Figs. 3, 4, 6, and 8)
consisting of both unmasked responses (which accounted for 55-66% of
the observed RF changes) and masked responses (Fig. 4). That is, both
enlargements and contractions of the RFs were observed. Although the
magnitude of the unmasked portion of the RFs was smaller than that
observed after subcutaneous injections of lidocaine made under similar
conditions (Faggin et al. 1997; Nicolelis et al.
1993), both compounds produced similar patterns of change. This
suggests that a subset of the mechanisms involved in the
reorganizations activated by these two pharmacologically different
compounds may be similar. Given that RF unmasking also occurs after
injections of mustard oil (a C-fiber excitant) (see Hoheisel et
al. 1993; Yu et al. 1993), our results support
the hypothesis that any lasting change in peripheral afferents can trigger sensory reorganization throughout the CNS.
; Nicolelis and
Chapin 1994) but appeared to change the fraction of the total population of synapses that could be activated by stimulation of
particular whiskers. This finding is consistent with the suggestion that capsaicin injections altered a balance between excitatory and
inhibitory afferents within the somatosensory relays contributing to
the definition of spatiotemporal RFs rather than simply increasing the
effectiveness of previously inhibited synapses.
RFs in the somatosensory thalamocortical loop frequently have been
assumed to be purely a function of stimulation of A
fibers in the
whisker follicles. The present data, however, represent definitive
evidence of the nociceptive system's influence on somatosensory responses.
EVOLUTION OF CAPSAICIN-RELATED CHANGES.
Overall, our RF quantifications revealed a continuous time course (6
h) for changes in the somatosensory thalamus and cortex that is
different from those reported in the brain stem and spinal cord. The
percentage of neurons undergoing reorganization was approximately as
high 6 h after injection (50%) as 1 h after injection (57%;
Figs. 3-6). Previously, capsaicin-related changes in the dorsal horn
and nucleus gracilis have been reported to peak or stabilize in ~1 h
(Cliffer et al. 1992; Pettit and Schwark
1996; Simone et al. 1991). The difference in
time course between thalamocortical and spinal reorganization make it
clear that changes in the thalamocortical system are not simply a
reflection of changes at second-order nuclei.
; Dickenson and Sullivan 1987; Vaccarino and
Chorney 1994; Vin-Christian et al. 1992).
Cortical feedback also has been shown to affect somatosensory thalamic
responses (Ergenzinger et al. 1998; Krupa et al.
1997).
A recent paper (Ma and Woolf 1996) demonstrated that
after subcutaneous capsaicin injections, repetitive tactile stimulation can cause response plasticity in the dorsal horn. It is conceivable that the protracted RF changes observed here could have resulted, not
from the direct manipulation of C and A fibers, but from the use of
repeated stimulation of individual whiskers. Although we cannot
conclusively dismiss this possibility, we consider it an unlikely
explanation for our findings. Although our protocol called for
individual whiskers to be stimulated 300 times during a 2.5-min period,
no particular whisker was so stimulated more than once every 1.75 h (entire stimulation protocol plus 30 min of spontaneous activity
recording). The infrequency of stimulation probably offered relatively
little opportunity for the development of the sort of changes discussed
by Ma and Woolf. We favor, instead, the interpretation that the
evolving RFs observed in VPM and SI represent the intrinsic response to
a long-lasting manipulation of peripheral nociceptors and that the
reciprocal connections between the VPM and SI are capable of amplifying
and sustaining the reorganization process long after lower subcortical
structures have ceased to show their effects.
The extended time course of the observed changes further suggests the
possibility that changes in C- and A-fiber activity could strongly
contribute both to immediate changes in the pattern of excitation and
inhibition in the somatosensory system after perturbations and to
longer-term changes that may be dependent on modulations of synaptic
strength. Increases in activity in the thalamocortical loop, such as
those that we've observed in this study, could potentially drive the
eventual stabilization of new RF maps. Thus it is possible that the
evolving changes observed here, caused by perturbation of C- and
A-fiber afferents, could represent a bridge between immediate
reorganization (Faggin et al. 1997) and longer-term
plasticity (Merzenich et al. 1983).
Reductions in spatial coupling between cortical neurons after capsaicin injection
In this study, we took advantage of classic geostatistical
analysis to examine the spatial characterization of capsaicin-induced plasticity at the level of neuronal populations (see also
Freeman and Baird 1987). These analyses revealed spatial
structure in the ensemble responses, quantified in time variograms of
activity across trials (Figs. 7 and 8). The initial response to whisker stimulation had a reliable spatial component, reflecting the tendency of nearby neurons to fire in a more related fashion than distant neurons. After capsaicin injection, nearby neurons' responses became
less coupled (i.e., less covariant) to each other (Fig. 8), and thus
the spatial structure of neural ensemble responses, while still
present, was no longer as strong a force in shaping neural firing.
This finding supports the suggestion that the activity of C-fiber
nociceptors can provide tonic inhibition of neurons located in the
somatosensory system, and that the desensitization of C fibers via
capsaicin injection removes this tonic inhibition (Calford and
Tweedale 1991). One difficulty for this theory, already noted by Calford and Tweedale, lies in the fact that C-fiber afferents affected by subcutaneous capsaicin injection typically are reported to
lack spontaneous activity (Szolcsanyi et al. 1988).
Silent afferents, particularly when relatively distant from the RFs of the recorded neurons, hardly can be expected to provide tonic inhibition. It remains unclear, however, whether capsaicin inactivates only silent afferents. Nociceptors with very low spontaneous firing rates may, under certain conditions, be mistaken for silent afferents. Such low rates of spontaneous firing may be enough to provide tonic
inhibition, and the loss of seldom-firing afferents may be enough to
disinhibit the system. It is also possible that the disinhibition is
mediated by the loss of capsaicin-inactivated A fibers, known to be
spontaneously active (Szolcsanyi 1990).
The precise peripheral source of the reduction of spatial couplingand
the attendant RF changesobserved in our data remains unclear. Neither
vehicle injections nor electrical stimulation (Calford and
Tweedale 1991) cause reorganization that approaches the
spatiotemporal extent of that seen after subcutaneous capsaicin injection, suggesting that the brief excitation of nociceptors does not
cause sizeable central plasticity. For several reasons, excitation of
afferents in skin regions surrounding the injection site is also an
unlikely candidate to explain this finding. First, similar
reorganization occurs after lidocaine injections, a compound that
primarily desensitizes afferents (Faggin et al. 1997;
Nicolelis et al. 1993b; Pettit and Schwark
1993). Furthermore researchers have failed to find substantial
excitatory effects in primary afferents near capsaicin injection sites
(Baumann et al. 1991).
Thus, the most likely explanation is that small subcutaneous injections of capsaicin changed the overall excitatory/inhibitory balance in the thalamocortical system and that this effect led to a reduction in the "`functional coupling" between nearby neurons. Regardless of the mechanisms underlying this effect, the algorithm/method used here provided us with a useful way to quantify plastic changes at the level of neural populations.
Change in somatosensory function after capsaicin injection
The reorganization observed in VPM and SI after capsaicin
injection is likely to lead to perceptual changes. When capsaicin is
injected under the skin, subjects experience brief pain followed by
analgesia at and around the injection site (see for instance Baumann et al. 1991; Fitzgerald and Woolf
1982; Iadarola et al. 1998; Serra et al.
1998; Simone et al. 1991; Treede et al.
1992). The subjects also experience hyperalgesia and allodynia,
"tenderness" of the skin marked by lowered mechanical and
thermoreceptive pain thresholds and changes in tactile perception, in a
large area beyond the injection site. Simone et al.
(1991) reported that the extent of hyperalgesia reported by
human subjects was related to increased firing of primate spinothalamic
tract neurons. Our recordings suggest that plasticity at the level of
the thalamus and cortex may be part of the same system response and
that a rat receiving subcutaneous capsaicin injections in its lip would experience hyperalgesia, allodynia, or alterations in somatosensory processing after the stimulation of whiskers (Giamberardino and Vecchiet 1995; Kauppila et al. 1998;
Treede et al. 1992). This interpretation implies that
information from low-threshold mechanoreceptor stimulation may be
processed differently after capsaicin injections, perhaps due to
plasticity induced in the trigeminal nuclei, thalamus, and cortex.
These results further suggest that the somatosensory and nociceptive
circuits are perhaps best thought of not as completely separate systems
(Berkley and Hubscher 1995) but rather as a single dynamic entity, processing a continuous spectrum of stimulus modalities dependent on the condition of the peripheral afferents.
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ACKNOWLEDGMENTS |
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The authors thank A. Ghazanfar for a careful and critical reading of the manuscript.
This work was funded by National Institutes of Health Grants DC-01065 and DE-11121-01, the Philip Morris Company, and the Whitehall Foundation.
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FOOTNOTES |
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Address reprint requests to: D. B. Katz, Dept. of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710.
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 11 February 1999; accepted in final form 8 April 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) and VPM (
). Error bars are SE; *,
difference, at the P < 0.05 level, between the two
postcapsaicin RF mappings and the postvehicle mapping. No other
differences were significant; see text for details.
