1Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7; and 2Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada
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ABSTRACT |
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Tremere, Liisa, T. Philip Hicks, and Douglas D. Rasmusson. Role of Inhibition in Cortical Reorganization of the Adult Raccoon Revealed by Microiontophoretic Blockade of GABAA Receptors. J. Neurophysiol. 86: 94-103, 2001. Cortical reorganization was induced by amputation of the 4th digit in 11 adult raccoons. Animals were studied at various intervals, ranging from 2 to 37 wk, after amputation. Recordings were made from a total of 129 neurons in the deafferented cortical region using multibarrel micropipettes. Several types of receptive fields were described in reorganized cortex: restricted fields were similar in size to the normal receptive fields in nonamputated animals; multi-regional fields included sensitive regions on both adjacent digits and/or the underlying palm and were either continuous over the entire field or consisted of split fields. The proportion of neurons with restricted fields increased with time after amputation and was greater than previously found in subcortical regions. A GABAA receptor antagonist (bicuculline methiodide), glutamate, and GABA were administered iontophoretically to these neurons while determining their receptive fields and thresholds. Bicuculline administration resulted in expansion of the receptive field in 60% of the 93 neurons with cutaneous fields. In most cases (33 neurons) this consisted of a simple expansion around the borders of the predrug receptive field, and the average expansion (426%) was not different from that seen in nonamputated animals. In some neurons (n = 4), bicuculline produced an expansion from one digit onto the adjacent palm or another digit, an effect never seen in control animals. Bicuculline also changed the split fields of seven neurons into continuous fields by exposing a responsive region between the split fields. Finally, bicuculline changed the internal receptive field organization of 10 neurons by revealing subfields with reduced thresholds. In contrast to the situation in nonamputated animals, iontophoretic administration of glutamate also produced receptive field expansion in some neurons (n = 6), but the size and/or shape of the change was different from that produced by bicuculline, indicating that the effects of bicuculline were not due to an overall facilitation of neuronal activity. These results are consistent with the hypotheses that an important component of long-term cortical reorganization is the gradual reduction in effective receptive field size and that intracortical inhibitory networks are partially responsible for these changes.
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INTRODUCTION |
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The term "cortical
reorganization" refers to the changes in the sensory responsiveness
of cortical neurons that occur following removal of their dominant
sensory input. Such changes occur in adult, as well as developing,
mammals and consequently have important implications for humans with
peripheral nerve damage. The mechanisms responsible for cortical
reorganization are still unknown, but clearly involve changes in
synaptic strength as well as the formation of new connections. Many of
the studies on cortical reorganization have been carried out in the
raccoon (Kelahan and Doetsch 1984; Kelahan et al.
1981
; Rasmusson 1982
), a species that offers the advantage of extremely large (~25 mm2)
representational areas of each digit that can be reliably identified on
the basis of the unique "triradiate sulcus" (Welker and
Seidenstein 1959
). In addition, the segregation of digit inputs
to each representational area enables one to produce extensive
deafferentation of a digit region by means of digit amputation.
Deafferentation in both the visual and somatosensory systems has been
shown to produce changes in markers for neurotransmitters such as
-aminobutryic acid (GABA) (Jones 1993
) and glutamate (Carder and Hendry 1994
). The decrease in the efficacy
of GABAergic synapses is particularly interesting because it could
alter synaptic efficacy by increasing the excitability of cortical
neurons and by lowering a "modification threshold" that would
permit strengthening of other synapses (Bear et al.
1987
; Bienenstock et al. 1982
).
One important role of GABAergic transmission in normal somatosensory
cortex is to restrict the size of receptive fields (RFs) of many
neurons (Alloway and Burton 1991; Alloway et al.
1989
; Batuev et al. 1982
; Dykes et al.
1984
; Hicks and Dykes 1983
). This action was
first demonstrated using the microiontophoretic administration of a
GABAA receptor antagonist such as bicuculline. Two implications of this finding are that cortical neurons normally receive excitatory inputs from a greater spatial range than is indicated by their RFs and that some of these inputs are being selectively blocked by inhibitory synapses. The RF seen during the
microiontophoretic administration of bicuculline therefore provides an
indication of the total spatial convergence onto these cortical neurons.
To determine the role of GABAA inhibition in
somatosensory processing during reorganization, we first studied
bicuculline effects in control raccoons (Tremere et al.
2001). The results indicated that when expansion of RFs occurs
it is limited to the same part of the forepaw that the original RF was
on; i.e., when the RF is originally on a digit, RF expansion is
restricted to that digit. Following amputation of a digit in this
species, neurons in the deafferented cortical region gradually acquire
new RFs on one or both adjacent digits (Kelahan and Doetsch
1984
; Rasmusson 1982
). Reorganization therefore
cannot be explained by unmasking or removing inhibition of inputs from
these adjacent digits.
On the other hand, the down-regulation in GABA activity that occurs after peripheral denervation raises the possibility that alteration of cortical inhibition plays a significant role during the reorganization process that occurs over several months. Consequently, the present study examined antagonism of GABAA receptors within the clearly defined deafferented digit region of the raccoon cortex during the course of reorganization. The use of microiontophoretic application of antagonists is also useful in distinguishing cortical from subcortical mechanisms of reorganization since any drug effects will be restricted to the cortex. In this paper we show that the organization of both excitatory and inhibitory RFs is different in reorganized cortex than it is in the normal cortex and that the progressive changes in RF structure that follow deafferentation are partially regulated by GABAA receptors.
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METHODS |
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Recordings were made from 11 adult raccoons, ranging in weight from 6.1 to 11.3 kg. The animals were trapped in the wild by a licensed trapper and were maintained in communal housing with ad lib food and water. All procedures for handling and experimentation were approved by the University Ethics Committee and were in accordance of the guidelines set down by the Canadian Council of Animal Care.
The fourth digit of the right forepaw was amputated in all but one
animal. The left forepaw was used in the latter case because this
animal had previous damage to the right forepaw. Our previous experiments have not found any right-left differences in raccoon cortex
or in response to denervation. The surgical procedure was the same as
described previously (Rasmusson 1996a). Briefly, the digit was removed at the metacarpo-phalangeal joint under sterile surgical conditions with the animal anesthetized with halothane. The
nerves were ligated as far proximal as possible before being cut, and
the dorsal hairy skin was sutured to the glabrous skin of the palm.
Immediately after surgery, cesazolin sodium (Keszol, Lilly; 15 mg/kg
im) was injected to prevent infection, and buprenorphin (Buprenex,
Reckitt-Coleman; 0.3 mg/kg im) was injected to reduce postoperative pain.
Recordings were made from primary somatosensory cortex contralateral to
the amputation at 2-, 8-, 11-, 12-, 15-, 18-, 19-, 24- (n = 2), 34-, or 37-wk intervals after the
amputation. The construction of the micropipette assemblies and general
recording procedures are described in detail in Tremere et al.
(2001). The animal was initially anesthetized with halothane
and maintained with
-chloralose throughout the recording session and
was given 2 ml of a corticosteroid (Solu-Delta-Cortef, Upjohn) to
reduce cerebral edema. Body temperature was maintained near 37°C by a feedback-controlled heating pad (Harvard). The animal's head was stabilized in a Kopf stereotaxic frame before opening the skull and
dura. The brain was covered with warmed Elliott's solution (Abbott,
Montreal) throughout the experiment.
Recordings were made using one barrel of a 5- or 7-barrel pipette (A-M
Systems, Carlsborg, WA) containing a 7-µm-diam carbon fiber. The
pipettes were pulled in a vertical Narishige microelectrode puller, and
the carbon fiber was electrochemically etched to yield a final
impedance of 0.5-4 M at 1 kHz. The other barrels contained bicuculline methiodide (BMI, Sigma, 5 mM in 0.9% saline, pH 3.3), L-glutamate (Sigma, 0.5 M, pH 8.0), GABA (Sigma, 0.5 M, pH
3.5), and 0.9% saline, for current balancing. The impedance of these barrels was 10-15 M
. Administration of drugs was controlled using a
Neurophore apparatus (Medical Systems, Greenvale, NY), and a retaining
current of 9-20 nA was applied to each barrel.
When the activity of a single neuron was isolated, the boundaries of
its RF were determined using von Frey hair monofilaments (Stoelting,
Wood Dale, IL). When measuring RF area, the RF was defined using the
lowest suprathreshold monofilament and was marked directly on the skin
with a felt tip pen. The length and width of this field were measured
with calipers and recorded. The RF was also sketched on drawings of the
raccoon forepaw. The activity of each cell was recorded for at least 5 min prior to any drug administration. Since the amount of drug ejected
depends on many variables such as tip diameter, capacitance, transport
number, as well as ejection current (Hicks 1983), the
ejection current was determined empirically for each cell with the
major concern being the avoidance of excessive stimulation with either
glutamate or BMI. The RF was mapped repeatedly before, during, and
after drug administration using the same von Frey monofilament, and the
maximal length and width of the RF were measured in the predrug and
drug states.
The deafferented region of cortex that normally represents the fourth
digit was identified from the gyral patterns surrounding the triradiate
sulcus and confirmed from recordings in the intact third digit, fifth
digit, and palm representations. Data obtained from neurons in the
third and fifth digit cortex were consistent with our observations in
intact, control animals (Tremere et al. 2001) and will
not be dealt with here.
The data obtained in amputated animals were compared with those from
our previous publication (Tremere et al. 2001) that used the same techniques in nonamputated animals. Comparisons of frequency data were made using the
2 test or the
nonparametric Kolmogorov-Smirnov test (Siegel and Castellan
1988
). Other comparisons were made using an unpaired Student's
t-test. All statistical analyses were carried out using the
Statview 5.0 program (SAS Institute, Cary, NC).
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RESULTS |
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Electrode penetrations were made at 150 sites in the deafferented 4th digit representation, and a total of 129 cells were studied in this region. It appeared to be slightly more difficult to find active cells in deafferented than in control animals. The average number of cells isolated per penetration was 0.86 in the deafferented cortex compared with 1.71 in the 4th digit representation of nondeafferented animals, but this difference was not statistically significant (t = 1.82, P = 0.08). The number of cells obtained per penetration was particularly low (<0.5) in the two animals that were studied at 12 and 15 wk postamputation, whereas in all of the remaining animals the values were between 0.75 and 2.0. The majority of the neurons (n = 93, 73%) were rapidly adapting (RA) with RFs on the glabrous skin. A small number responded to stimulation of hairy skin (n = 6) or squeezing a joint or claw (n = 7). The remaining 23 neurons (18%) did not respond to stimulation of any part of the forepaw.
The depth below the cortical surface at which the neuron was
encountered ranged from 40 to 1,400 µm. The greatest number of sampled neurons were around 500 µm (Fig.
1). While the range of depths was
slightly larger than in our sample of neurons in nonamputated animals
(Tremere et al. 2001), there was no statistically
significant difference between the two groups in terms of depth
profiles (Kolmogorov-Smirnov test, P = 0.68).
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Novel RF types in reorganized cortex
In nonamputated raccoons, virtually all of the neurons in the digit representational areas of S1 cortex respond exclusively to stimulation of the glabrous surface of the digit, and the RFs are restricted to a small portion of the digit. Similarly restricted RFs were found in the amputated animals, although the restricted fields often lacked the sharp boundary that is characteristic of the control animals, making it more difficult to quantify RF area. These restricted RFs in reorganized cortex were most often on either the third or fifth digit, but were sometimes on the palm near the scar at the site of the amputation.
In addition to these restricted RFs, several other types of RF were
often encountered in reorganized cortex (Fig.
2). Previous descriptions have called
these RFs "heterotopic" or simply "large" (Kelahan and
Doetsch 1984; Turnbull and Rasmusson 1991
), but, to emphasize the unusual spatial organization of the RFs characteristic of reorganized cortex, we will use the term multi-regional (MR), as the
RFs included parts of two or three regions of the forepaw, namely the
two adjacent digits and/or the palm. Multi-regional RFs could be
further grouped according to whether the RF consisted of spatially
distinct RFs on two or more regions (multi-regional-split, MR-S) or was
continuous over these regions (multi-regional-continuous, MR-C).
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The largest proportion of RFs on glabrous skin fell into the restricted category (49 of 93, 53%). The RF sizes of these restricted fields were not significantly different from the size of RFs on the distal digit in control animals (12.9 vs. 15.4 mm2, respectively; t = 1.0, P = 0.32). Forty-four neurons had multi-regional RFs, 22 neurons (24%) in each of the MR-S and MR-C categories. In the MR-C group, the most common combination of regions was basically a U-shaped RF with responsive regions on both digits 3 and 5 and the distal part of palmar pads C and D proximal to these digits (Fig. 2C). The threshold was usually not uniform throughout these MR-C fields, but there were lower threshold subfields that were spatially joined by higher threshold regions. The low-threshold subfield was usually on the distal pad of a digit or near the wound. In the case of the MR-S group, the majority of cells had subfields on the distal pads of digits 3 and 5.
The frequency of occurrence of these different types of RF changed with
time after the amputation. This is illustrated in Fig.
3, in which animals tested at similar
intervals after the amputation are grouped to give larger sample sizes
at each interval. The change in the proportion of neurons in each of
these four categories over time is statistically significant
(2 = 35.9, P < 0.001). The
most dramatic changes are the increase in restricted RFs, from 5% at
the earliest intervals to over 50% at later intervals, and the
decrease in nonresponsive cells, from 40% to almost zero. At the
earliest intervals almost all of the responsive neurons have
multi-regional RFs, but this proportion drops to 35-40% at longer
intervals. These data support two hypotheses suggested in earlier
reports. First, many RFs in deafferented cortex differ from normal RFs
in being unusually large, often extending beyond the confines of a
single digit. Second, there is a progressive decrease in RF size with
increased time after amputation.
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Comparison with reorganization in thalamus and cuneate
The proportion of neurons falling into these three categories of
RF was compared with previous results in the reorganized thalamus
(Rasmusson 1996a,b
) and cuneate nucleus
(Rasmusson and Northgrave 1997
). These data are
presented in Fig. 4, which illustrates that restricted RFs predominate in the cortex, whereas multi-regional fields are most common in subcortical regions. The differences between
the three levels are statistically significant
(
2 = 30.3, P < 0.001).
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Effects of microiontophoretic administration of drugs
NEURONAL EXCITABILITY.
BMI was administered to 113 cells in deafferented cortex. The range of
ejection currents for BMI was 10-85 nA, and the maximal time of
application was 6 min. In our experience (Tremere et al. 2001), equilibrium is reached within 5 min, and responses do
not show any additional change with longer administration. At these levels, BMI produced a clear increase in spontaneous activity in 18 cells and decreased the threshold for mechanical activation in 37, with
both effects occurring in 9 neurons. Glutamate was tested on 77 cells
in the deafferented cortex. Glutamate resulted in a decreased threshold
in 38 cells and an increase in spontaneous activity in 33 cells, with
12 neurons showing both effects. GABA was administered to 31 cortical
neurons in reorganizing cortex. Fourteen cells showed an increased
threshold to mechanical stimulation, and 14 showed a decrease in
spontaneous activity, with both effects seen at 4 cells. While these
observations give only a general impression about the effectiveness of
each drug in altering neuronal responsiveness, the absence of a change
does not imply that the neuron does not possess the relevant receptors.
Nevertheless it is important to note that these proportions were not
statistically different from those observed in nonamputated animals
(glutamate,
2 = 0; BMI,
2 = 0.12; GABA,
2 = 0.09), indicating that deafferentation did not result in a large change
in responsiveness to these drugs.
BMI EFFECTS ON RF ORGANIZATION. BMI was tested on a total of 113 neurons in deafferented cortex. Fifteen of these neurons did not respond to peripheral stimulation before BMI administration; in none of these was a RF unmasked by BMI. BMI also did not produce any appreciable change in the RF of any of the neurons that responded only to stimulation of deep tissue or of hairy skin. Of the 93 neurons with purely cutaneous RFs, BMI produced changes in RF size or structure in 56 (60%). Three types of changes were produced by BMI. The first type of change was a simple expansion of the predrug RF with preservation of the original RF shape; the second was an expansion that drastically altered the RF shape and necessitated a redefinition of the RF category for that cell; the third was the appearance of lower threshold subfields within the original RF without an increase in total RF area.
Simple expansion of the RF was seen in 33 of 93 neurons with cutaneous RFs (35%). Expansion was more common among the neurons with restricted RFs (21 of 49, 45%) than in the MR-S or MR-C groups. This expansion was similar to that seen in control animals (e.g., Fig. 5A). The amount of expansion was determined by measuring the RF area before and after BMI in a subset of 15 neurons that had RFs with discrete boundaries. The average expansion in these neurons was not significantly different from that seen in nonamputated animals, regardless of whether this was measured as absolute change (28.3 mm2 vs. 40.8 mm2, t = 143, P = 0.15) or relative change (426% of the original RF size vs. 286% in nonamputated; t = 1.16, P = 0.25).
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EFFECTS OF GLUTAMATE ON RFS. In contrast to the situation in nonamputated animals, glutamate did change the RF properties of a small number of neurons (n = 6); four of these had a restricted RF (Fig. 8, A-D), one a MR-C field (Fig. 8E) and one a MR-S field (Fig. 8F). In each of these neurons, BMI also produced expansion. In four neurons, glutamate and BMI had similar effects with BMI producing greater expansion than glutamate. In the other two neurons the effects of the two drugs were quite different. One of these (Fig. 8B) had a restricted field on the distal 5th digit that expanded to cover the entire digit when BMI was administered. Glutamate, on the other hand, did not expand the field on the 5th digit, but revealed a second field on the distal 3rd digit. The other exception had a MR-S field (Fig. 8F). Glutamate produced proximal expansion of both subfields, whereas BMI affected only one of the subfields, producing an expansion in the distal direction on the 3rd digit.
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DISCUSSION |
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Peripheral denervation, as in the case of digit amputation, results in a reorganization within the CNS so that regions that have lost their dominant input gradually come to respond to new sensory inputs. Here we have demonstrated that local inhibitory connections, acting via GABAA receptors, play a role in the changes that occur during reorganization. While this complex process is often discussed in terms of simple alternative mechanisms, such as unmasking versus sprouting, or cortical versus subcortical loci, it likely involves multiple mechanisms occurring at several sites in the somatosensory pathways. The present study provides information about three important issues related to reorganization. One is the time course of reorganization and the progression of changes in RF structure; the second is the role of cortical inhibitory mechanisms in reorganization; and the third is the relative contribution of cortical and subcortical structures to the changes that are seen in the cortex.
Time course
It is useful to divide the process of reorganization into several,
probably overlapping stages (Cusick et al. 1990;
Turnbull and Rasmusson 1991
). During the first phase,
immediately after nerve injury or amputation, there is an unmasking of
secondary inputs to the region that may be either actively suppressed
by the dominant input or simply not obvious when the dominant input is
present. Although the intent of studying the immediate effects of
denervation is to observe the starting point of long-term plasticity, it is important to recognize that rapid changes in synaptic strength may be occurring during the course of these recording experiments that
last for many hours (Mioche and Singer 1989
). In the
raccoon digit amputation model, this phase is characterized by the
cells being largely nonresponsive or showing inhibition to stimulation of an off-focus digit with or without rebound excitation at the end of
stimulation (Rasmusson and Turnbull 1983
;
Turnbull and Rasmusson 1990
). In other models, nerve
section may reveal minor connections that reflect either peripheral
overlap of the sectioned and surviving nerves or overlap in the
projection pathways. For example, median nerve section in the monkey
reveals RFs on the dorsal surface of the hand in some parts of the
affected cortex (Schroeder et al. 1997
).
During the second phase of reorganization, the neurons in denervated
cortex begin responding to stimulation of new regions of skin. In the
raccoon this occurs over the course of several weeks and involves the
disappearance of the strong inhibitory responses, an increase in
spontaneous activity (Rasmusson et al. 1992) and the
appearance of large RFs with poorly defined borders (Kelahan and
Doetsch 1984
; Rasmusson 1982
). In both the
visual and somatosensory system, it has been shown that denervation
results in down-regulation of GABAergic markers (cf. next section).
These changes are consistent with the model of plasticity that involves the ability to vary a "modification threshold" on the basis of overall cell activity (Bear et al. 1987
;
Bienenstock et al. 1982
). For new synapses to be
strengthened, the modification threshold would have to be lowered so
that weak inputs would be sufficient to activate the postsynaptic
neuron and consequently initiate plasticity.
Finally, once the periphery is capable of firing the deafferented
neurons, the pattern and intensity of tactile input may selectively
enhance some synapses and weaken others (Jenkins et al.
1990). The strengthening of some of the synaptic inputs and the
reestablishment of inhibitory shaping could cause the large, diffuse
RFs to become restricted. Several months after amputation in the
raccoon, the new RFs are much smaller and are similar in size to those
in nonamputated animals, although now on an adjacent digit
(Kelahan and Doetsch 1984
; Rasmusson
1982
; Turnbull and Rasmusson 1991
). This final
stage in the progression has also been described as a
"consolidation" of reorganization (Churchill et al.
1998
), which emphasizes the similarities of these later changes
to those underlying learning and memory.
The incidence of restricted and multi-regional RFs seen in the present
study (Fig. 3) is consistent with this interpretation of the time
course of reorganization. The proportion of neurons with restricted RFs
increases dramatically between 8 and 11 wk. This is not, however, a
direct test of the hypothesis that a given neuron first develops a
multi-regional field that gradually becomes restricted to a small part
of this large field. Conclusive evidence would require long-term
recordings from the same cell throughout the reorganization process.
While this technique has been applied in the study of both
somatosensory and visual plasticity for periods of several hours
(Katz et al. 1999; Mioche and Singer
1989
), it is not practical to follow the same cell for many
days or weeks. More convincing support for a continual progression from
large, diffuse fields to small RFs comes from the finding of the
present study that blocking GABAA receptors on
neurons with restricted RFs can reveal multi-regional fields and on
neurons with split fields can unmask a responsive region between the
subfields. These observations are also consistent with the idea that
the spatial properties of RFs are determined by dynamic,
self-organizing processes (Merzenich 1987
).
Role of GABAergic inhibition in reorganization
The second major finding of this paper concerns the relationship
between inhibition and the development of new RFs during reorganization
(cf. reviews, Jones 1993, 2000
).
Following the discovery that visual deprivation (by enucleation)
produces biochemical GABAergic changes in visual cortex
(Hendry and Jones 1986
), similar effects were
shown in the somatosensory cortex. The density of immunoreactive
labeling of the synthesizing enzyme, glutamate decarboxylase (GAD), is
decreased by whisker trimming in rats and mice (Akhtar and Land
1991
; Welker et al. 1989b
) and by digit amputation in the rat (Warren et al. 1989
). GABA
immunoreactivity is reduced in squirrel monkey cortex after median and
ulnar nerve transection (Garraghty et al. 1991
), and
there is a downregulation of GABAA receptors
after whisker trimming in rodents (Fuchs and Salazar
1998
; Skangiel-Kramska et al. 1994
). If the
effectiveness of GABAergic synapses is decreased, it would be expected
that there would be an increase in spontaneous activity; this has been demonstrated electrophysiologically in rat barrel cortex after whisker
trimming (Simons and Land 1987
) and in raccoon cortex 1 wk after digit amputation (Rasmusson et al. 1992
). Both
GAD immunoreactivity and GABAA receptor binding
have been shown to return to normal levels within several months after
whisker destruction in mice (Skangiel-Kramska et al.
1994
; Welker et al. 1989b
) and GABAergic markers
in the visual system recover after restoration of visual input
(Hendry and Jones 1988
). Similarly, training or excessive sensory stimulation produces an increase in GABAergic measures (Siucinska et al. 1999
; Tremere et al.
2001
; Welker et al. 1989a
). These results
indicate that the level of afferent activity is regulating GABA expression.
Normally, GABAergic synapses in somatosensory cortex function to
restrict RF size, as demonstrated by the fact that
GABAA receptor blockade produces RF expansion in
control animals (Dykes et al. 1984; Hicks and
Dykes 1983
; Kyriazi et al. 1996
). The present study shows that, in cortex that is undergoing reorganization, GABAA receptor blockade can produce different
types of RF expansion. One is similar to that seen in control animals,
with a two- to threefold increase in RF area occurring around the
original RF. In contrast to the control situation, however, some
neurons in deafferented cortex showed an expansion onto another digit
or the palm. In other words, in reorganized cortex, RF expansion did
not always respect the boundaries between digits or between a digit and
the palm. This is evidence of significant convergence, within the
cortex, of information from a much larger region than seen before
amputation. In addition, a qualitatively different type of expansion
was observed in which a new field could be revealed that was spatially
separated from the original RF. Together these observations indicate
that inputs from different parts of the RF, for example from different
digits, are sufficiently separated on the dendritic tree that
inhibitory synapses are able to suppress some but not all of the inputs.
The expansion of RFs in the presence of BMI and the conclusion from the
previous section that the final stage of reorganization involves the
shrinkage of RFs support the hypothesis that GABAergic mechanisms are
largely responsible for this progressive shrinkage of RF size. The
expansion effects of BMI also appeared to be much more robust at longer
intervals after amputation, consistent with the idea that the
functionality of the GABAergic synapses is recovering as reorganization
proceeds. It is also worth noting that, early in the reorganization
process, the RFs are ill-defined and lack clear boundaries, whereas at
later intervals the boundaries are distinct as in normal cortex. A
well-recognized function of lateral or surround inhibition is to
enhance response differences at the edge of stimuli (Mountcastle
and Powell 1959), and the recovery of sharp borders may be
another reflection of the recovery of GABAergic mechanisms.
The importance of GABAergic inhibition in reorganization has also been
illustrated in the rat following neonatal forelimb removal (Lane
et al. 1997, 1999
; Stojic et al.
2000
). Since this model involves deafferentation in the
neonatal animal, developmental mechanisms are possibly involved in
addition to those available only in the adult. Nevertheless, these
studies also found that GABAergic synapses restrict the size of new
cortical RFs after reorganization. In addition these studies showed
that this restriction is selective, such that inputs from the hindlimb
were preferentially inhibited, while inputs from the stump were maintained.
In the raccoon a similar selective remodeling would be expected to
favor RFs on parts of the adjacent digits that are now exposed to
greater stimulation by the absence of the digit and by a predominance
of distal rather than proximal RFs. The present data set is too small
to confirm these predictions statistically. However, the issue of
selectivity of inhibition can also be addressed by comparing the
effects of BMI with the effects of glutamate on the same neurons. If
GABA receptor blockade has simply a nonselective effect, similar
changes would be expected with BMI (via removal of inhibition) and
glutamate (via excitation). In contrast to control animals
(Tremere et al. 2001), glutamate did produce RF expansion in a small number of neurons in reorganized cortex (Fig. 8).
Thus these neurons must receive subthreshold excitatory inputs that
were only able to reach threshold when the neuron was depolarized by
glutamate. However, most cells that were influenced by BMI were not
affected by glutamate, and, in those neurons that were altered by both,
the BMI-induced expansion was larger and/or spatially different from
the glutamate-induced expansion. This again suggests that inputs from
different parts of a neuron's RF must be sufficiently separated on the
dendritic tree that inhibitory synapses can suppress some of the inputs
but not others.
An important question is whether the functional pruning of inputs is
random for a particular neuron or whether some large-scale control
mechanism regulates the overall organization of the deafferented cortex. A consistent finding in raccoon experiments (Kelahan and Doetsch 1984; Rasmusson 1982
; Turnbull
and Rasmusson 1991
) has been that while the deafferented cortex
is not randomly organized, it is far less organized than the original
state, and there is less somatotopy than in primate models of cortical
reorganization (Merzenich et al. 1983
,
1984
).
Subcortical versus cortical contributions to reorganization
The results of the present study also relate to the relative
importance of subcortical and cortical plasticity. While the majority
of studies on reorganization have looked only at the cortex, many
studies have also found changes in the thalamus, dorsal column nuclei,
and spinal cord (cf. recent review, Jones 2000).
Obviously any changes that occur in these subcortical regions must
contribute to the responses of cortical neurons. Digit amputation in
the raccoon results in long-term reorganization in both the ventrobasal
thalamus (Rasmusson 1996a
,b
) and the cuneate nucleus (Rasmusson and Northgrave 1997
) that is similar to that
seen in the cortex: the deafferented regions contain neurons that now respond to adjacent digits and the palm. Since the same denervation and
RF mapping techniques were used in these studies on subcortical and
cortical reoganization, it is likely that some of the cortical changes
are due to plasticity at these lower relay nuclei. Furthermore, the
short latencies of novel responses in the cuneate and thalamic nuclei
make it physically impossible for them to be occurring as a result of a
relay through the reorganized neocortex.
The impression gained from this series of studies is that small,
restricted RFs are less common in deafferented subcortical regions than
in the cortex. A comparison of data from the cortex (present study)
with these subcortical regions in terms of restricted, MR-S and MR-C
RFs (Fig. 4) confirms this hypothesis. Most notable is the decrease in
MR-S fields as one ascends from cuneate to thalamus to cortex with a
corresponding increase in restricted RFs. While these data are taken
from different animals and the times after amputation are different in
each study, the cortical sample includes both short and long intervals,
whereas the subcortical data are all from animals studied at least 2 mo
after amputation. If only the long intervals were included in the
cortical sample, the differences from the subcortical samples would be
even greater. A similar difference between subcortical and cortical
levels was seen in the neonatal forelimb amputation model (Lane
et al. 1995; Stojic et al. 1998
); these results
were interpreted as a suppression within the cortex of some of the
subcortical changes.
The microiontophoretic technique used here to block selectively a subpopulation of GABA receptors can contribute to this issue because the drug administered in this way can only affect local, in this case cortical, synapses. The observation that BMI administration within the cortex results in RF expansion is strong evidence that some of the thalamocortical inputs are indeed being suppressed within the cortex but does not, of course, tell us whether local or extrinsic GABAergic neurons are responsible. Similar studies at the subcortical relays would be useful in determining the relative importance of each level in shaping RF properties.
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ACKNOWLEDGMENTS |
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This work was supported by the Canadian Institute of Health Research.
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FOOTNOTES |
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Address for reprint requests: D. D. Rasmusson (E-mail: rasmus{at}is.dal.ca).
Received 7 December 2000; accepted in final form 14 March 2001.
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REFERENCES |
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