1Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, Ohio 43699; and 2Department of Psychobiology, University of California, Irvine, California 92717
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ABSTRACT |
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Stojic, Andrey S., Richard D. Lane, Herbert P. Killackey, and Robert W. Rhoades. Suppression of Hindlimb Inputs to S-I Forelimb-Stump Representation of Rats With Neonatal Forelimb Removal: GABA Receptor Blockade and Single-Cell Responses. J. Neurophysiol. 83: 3377-3387, 2000. Neonatal forelimb removal in rats results in the development of inappropriate hindlimb inputs in the forelimb-stump representation of primary somatosensory cortex (S-I) that are revealed when GABAA and GABAB receptor activity are blocked. Experiments carried out to date have not made clear what information is being suppressed at the level of individual neurons. In this study, three potential ways in which GABA-mediated inhibition could suppress hindlimb expression in the S-I stump representation were evaluated: silencing S-I neurons with dual stump and hindlimb receptive fields, silencing neurons with receptive fields restricted to the hindlimb alone, and/or selective silencing of hindlimb inputs to neurons that normally express a stump receptive field only. These possibilities were tested using single-unit recording techniques to evaluate the receptive fields of S-I forelimb-stump neurons before, during, and after blockade of GABA receptors with bicuculline methiodide (for GABAA) and saclofen (for GABAB). Recordings were also made from normal rats for comparison. Of 92 neurons recorded from the S-I stump representation of neonatally amputated rats, only 2.2% had receptive fields that included the hindlimb prior to GABA receptor blockade. During GABA receptor blockade, 54.3% of these cells became responsive to the hindlimb, and in all but two cases, these same neurons also expressed a stump receptive field. Most of these cells (82.0%) expressed only stump receptive fields prior to GABA receptor blockade. In 71 neurons recorded from normal rats, only 5 became responsive to the hindlimb during GABA receptor blockade. GABA receptor blockade of cortical neurons, in both normal and neonatally amputated rats, resulted in significant enlargements of receptive fields as well as the emergence of receptive fields for neurons that were normally unresponsive. GABA receptor blockade also resulted in increases in both the spontaneous activity and response magnitudes of these neurons. These data support the conclusion that GABA mechanisms generally act to specifically suppress hindlimb inputs to S-I forelimb-stump neurons that normally express a receptive field on the forelimb stump only.
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INTRODUCTION |
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Neonatal forelimb removal in rats results in
reorganization at multiple levels in the somatosensory neuraxis.
Sciatic nerve afferents invade the cuneate nucleus (CN), resulting in
the development of CN neurons (40% of all CN neurons recorded) with
receptive fields that include both the remaining forelimb stump and the hindlimb (Lane et al. 1995). Similarly in the ventral
posterolateral nucleus (VPL), there is evidence of a small, but
significant, number of neurons in the forelimb-stump representation
with receptive fields including both the stump and hindlimb
(Stojic et al. 1998
). Finally, in the forelimb-stump
representation of primary somatosensory cortex (S-I) of neonatally
amputated rats, ~5% of recording sites express a hindlimb receptive
field in addition to that on the stump (Lane et al.
1995
). Thus while expression of inappropriate hindlimb inputs
is evident at all levels of the normal forelimb sensory neuraxis of
neonatally amputated rats, there is a significant reduction in the
expression of these inputs in VPL versus CN and in S-I versus both CN
and VPL (Stojic et al. 1998
). This reduction in hindlimb
expression, at least in S-I, involves suppression of these inputs via
mechanisms that require the activation of either
GABAA or GABAB receptors.
Lane et al. (1997)
showed that application of the GABA
receptor antagonists bicuculline methiodide (BMI, for
GABAA) and phaclofen (for
GABAB) revealed numerous multiple unit recording
sites that were responsive to cutaneous hindlimb stimulation in the S-I
forelimb-stump area of neonatally amputated adult rats.
While the study of Lane et al. (1997) implicated GABA
involvement in suppressing hindlimb signals to the S-I forelimb-stump representation, their results did not make clear what was being suppressed at the level of individual cells. GABAergic mechanisms might
suppress hindlimb information in the S-I forelimb-stump representation
by silencing S-I neurons with dual stump and hindlimb receptive fields,
silencing neurons with receptive fields restricted to the hindlimb
alone, and/or selectively inhibiting hindlimb inputs to neurons that
normally express stump receptive fields only. In this study, we sought
to differentiate these possibilities by using single-unit recording
techniques to evaluate the receptive field(s) of neurons within the S-I
forelimb-stump field before, during, and, in most cases, after
pharmacologic blockade of both GABAA and
GABAB receptors. Additionally, the effects of
GABA antagonists on the responses of neurons within the S-I forelimb
field of normal rats were evaluated for comparison with results from
the neonatally manipulated rats.
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METHODS |
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All protocols described here were developed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Medical College of Ohio.
Neonatal forelimb removal
Neonatal forelimb removals were carried out using methods
previously described (Lane et al. 1995). In brief,
postnatal day 0 rats (>12 h old) were anesthetized by hypothermia, and
the left forelimb was amputated just distal to the shoulder using
iridectomy scissors. The skin was closed with cyanoacrylate adhesive
following cauterization of the brachial artery and application of 0.7%
bupivacaine. The pups were then rewarmed and returned to their mothers.
These animals were used for recording experiments when they reached >60 days of age.
Single-unit recordings from S-I
Recordings in rats that sustained neonatal forelimb removal were
made from the cerebral cortex contralateral to the amputation. Recordings from normal animals were made from the right cerebral cortex. The rats were initially anesthetized with a combination of
ketamine (60 mg/kg) and xylazine (15 mg/kg) administered
intraperitoneally and prepared for recordings as described previously
(Lane et al. 1995, 1997
). The trachea was
cannulated, the left brachial plexus and sciatic nerves were exposed,
and the animals were placed in a stereotaxic headholder and ventilated
mechanically. A bipolar stimulating electrode was placed on the
brachial plexus just proximal to the origin of the median, ulnar, and
radial nerves, and another was placed on the sciatic nerve ~15 mm
distal to the sciatic notch. Rats were paralyzed with gallamine
triethiodide (30 mg, delivered intraperitoneally), and anesthesia was
maintained during the recording session by administration of urethan
(200 mg, delivered intraperitoneally) as needed. A midline incision was
made in the scalp and posterior neck. The cisterna magna was opened and
a large craniotomy was performed over the right cerebral cortex. The
dura and arachnoid were removed, and the surface of the cortex was
photographed at ×44 to record the placement of microelectrode
penetrations. The cortical surface was kept moist by applying culture
medium (Neural basal medium, Gibco) warmed to 37°C.
Single units were recorded using either glass micropipettes
(impedance = 10-40 M) filled with 1 M KCl or parylene-coated tungsten microelectrodes (impedance = 1.5-2.5 M
). Electrodes were inserted into the area of cortex corresponding to the S-I forelimb-stump field and advanced in several-micrometer increments using a microdrive. Neurons were recorded from all cortical layers (typical depth range was 200-1,400 µm).
Protocol for testing neuronal response and data analysis
Spontaneous activity of most neurons was measured over two 20-s
epochs. Cutaneous receptive fields were mapped with tactile stimuli
delivered across the entire body surface using a blunt probe and
plotted on body surface drawings. For quantitative analysis, receptive
fields on the forelimb (for normal rats), stump (for neonatally
amputated rats), and/or hindlimb were stimulated with a pen-motor
stimulator coupled to a waveform generator. A von Frey hair (0.2-mm
hair diam, 0.7 g of force) was attached to the pen motor by
a wooden shaft. The size of the von Frey hair was determined to be
sufficient to produce depression of the skin surface and evoke a
response in all units tested in this study. The tip of the hair was
positioned over the approximate center of the receptive field(s), at a
starting distance of 1.5 mm from the skin surface. The amplitude of
stimulus was 2 mm at a velocity of 0.4 m/s. An interstimulus interval
of 4 s was employed for all cells. A series of 62 responses to
stimulation was collected during a control period, followed by the
application of the GABA antagonists and collection of an additional
series of 62 responses, beginning 10 min after drug application. In
some cases, after the effects of the GABA antagonists were given
sufficient time to wear off, a final series of 62 responses were
collected for the recovery phase of the experiment.
Application of bicuculline methiodide and saclofen in S-I
After initially characterizing a neuron as described above, a 30-µl solution that contained equal parts of 50 µM bicuculline methiode (BMI) and 50 µM saclofen (SAC) (both supplied by Research Biochemicals International) was applied to the cortex. This amount of BMI and SAC applied to the cortical surface was sufficient to block the effects of GABA for a minimum of 30 min. This was based on the observation that BMI produces a characteristic "bursting" of neuronal activity. Bursting activity was evident ~10 min after initial application of the drugs and continued for 30 min following onset. The effects of GABA receptor blockade on receptive fields were evaluated during this bursting period.
After completing data collection during GABA receptor blockade, fresh
culture medium was applied to the surface of the cortex to wash off the
residual antagonist solution, and an interval of 10 additional
minutes was used to permit any GABA antagonist effects to dissipate as
determined by recorded spontaneous activity. After this interval,
post-GABA receptor blockade data were collected, if possible.
Identification of neuron location within cortex
The positions of neurons recorded within the S-I forelimb-stump representation were identified by marking the location with an electrolytic lesion made with a tungsten electrode. In cases where recordings were made with micropipettes, electrolytic lesions were made by replacing the micropipettes with a tungsten electrode that was lowered to the depth at which the neuron was recorded. At the conclusion of the recording session, animals were killed with carbon dioxide and perfused transcardially, initially with heparinized phosphate buffered saline, followed by a 4% paraformaldehyde solution. The brain was then removed and cut into 50-µm-thick coronal sections on a freezing microtome and stained with cresyl violet.
Analysis of receptive field size
Areas of receptive fields plotted on body surface drawings were
determined using a computerized planimeter. The following comparisons
were made: receptive field sizes prior to and during GABA receptor
blockade for supragranular, granular, and infragranular neurons as well
as for cells recorded from all layers were analyzed for normal and
neonatally amputated rats using paired t-tests with the
significance set to P 0.003 (Bonferroni correction); receptive field sizes in different cortical layers for normal versus
neonatally amputated rats were analyzed both prior to and during GABA
receptor blockade by ANOVA with a Newman-Keuls post hoc test
(significance set to P
0.05); and receptive field
sizes for supragranular, granular, infragranular neurons, as well as for cells recorded from all layers together, between normal and neonatally amputated rats were compared using unpaired
t-tests (significance set to P
0.003, Bonferroni correction) both prior to and during GABA blockade.
Categorical data related to the expression of particular receptive
field types (forelimb, forelimb-stump, hindlimb, etc.) were analyzed
using
2 tests with a significance set to
P
0.05.
Analysis of response magnitudes
The responses to mechanical stimulation were summed in
poststimulus time histograms (PSTHs) and counted. The portion of the PSTH used for counting was selected subjectively from the peak area
(2 times background activity) obtained during the control period and
the same interval was then used to count the PSTHs obtained during
application of GABA antagonists and recovery. The change in response
strength of a given cell during GABA antagonist application was
determined by the following formula: {100 × [1
(number of
spikes in the experimental PSTH)]}/number of spikes in the control
PSTH. Differences between control and GABA-blocked responses were
analyzed by a paired t-test (P
0.01).
This procedure is similar to that used by Chiaia et al.
(1997)
.
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RESULTS |
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A total of 71 neurons (18 supragranular, 30 granular, and 23 infragranular) from 20 normal rats and 92 neurons (26 supragranular, 41 granular, and 28 infragranular) from 20 neonatally manipulated rats were isolated and characterized before, during, and in most cases, after pharmacologic blockade of GABA receptors. Confirmation of neurons as supragranular, granular, and infragranular was made by identifying electrolytic lesions in cresyl-violet-stained coronal sections of S-I following each recording session (Fig. 1).
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Expression of inappropriate hindlimb inputs by S-I forelimb-stump neurons during GABA receptor blockade
Figure 2 shows the results of GABA receptor blockade on the expression of hindlimb inputs in S-I forelimb-stump neurons from both normal (Fig. 2A), and neonatally amputated rats (Fig. 2B). Prior to GABA receptor blockade, 86.0% of neurons (61 of 71) from normal rats had receptive fields that were restricted to the forelimb. Only one neuron expressed a hindlimb receptive field before GABA receptor blockade (1.4%), and no cells had dual forelimb and hindlimb receptive fields. The remaining nine (12.7%) neurons were unresponsive to cutaneous stimulation. The effects of GABA receptor blockade were as follows. Of the 61 neurons that had a forelimb only receptive field prior to GABA receptor blockade, 90.2% (55) maintained a forelimb only receptive field. The remaining six neurons changed: three neurons developed dual forelimb and hindlimb receptive fields (4.9%), and the other three expressed a dual forelimb and whisker pad receptive field (4.9%, Fig. 2A). The one neuron that expressed a hindlimb only receptive field prior to GABA receptor blockade demonstrated no change in its receptive field during GABA receptor blockade. Finally, of the nine neurons with no receptive fields under control conditions, GABA receptor blockade revealed cutaneous receptive fields in five neurons, including one neuron with a hindlimb receptive field. The overall increase in hindlimb expression seen during GABA blockade was not statistically significant. Fifty-one of the neurons from normal rats were tested after the effects of GABA receptor blockade were allowed to dissipate, and in each case, the receptive field returned to that observed under initial control conditions.
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Examination of the receptive fields of neurons from the S-I forelimb-stump representation of rats that sustained neonatal forelimb removal revealed several differences when compared with normal rats (Fig. 2B). Prior to GABA receptor blockade, 73.9% of neurons (68 of 92) had stump only receptive fields. Twenty-two cells (23.9%) recorded from neonatally amputated rats were unresponsive to cutaneous stimulation. Finally, two neurons (2.2%) had dual stump and hindlimb receptive fields prior to GABA receptor blockade.
GABA receptor blockade in the neonatally manipulated rats resulted in a significant increase in the expression of hindlimb inputs. Fifty of the 92 units (54.3%) expressed a hindlimb input during GABA receptor blockade (P < 0.0001 when compared with control conditions for these neurons and compared with normal neurons during GABA receptor blockade). In all but two cases, S-I forelimb-stump neurons in the neonatally amputated rats that expressed a hindlimb receptive field also expressed a stump receptive field. Hindlimb receptive fields were found predominantly for S-I forelimb-stump neurons that, prior to GABA receptor blockade, expressed only a forelimb-stump receptive field (e.g., Fig. 3). Of the 68 neurons that expressed a stump only receptive field prior to GABA receptor blockade, 60.3% (41) expressed an additional hindlimb receptive field during GABA receptor blockade (P < 0.0001). Finally, in a small number of neurons (5) that were unresponsive to cutaneous stimulation, GABA receptor blockade revealed dual stump and hindlimb receptive fields (Fig. 4B). In addition to revealing latent hindlimb receptive fields on S-I forelimb-stump neurons of neonatally amputated rats, GABA receptor blockade also demonstrated neurons with receptive fields on the whisker pad (2), and lower jaw (1). Seventy-four neurons were tested after effects of GABA receptor blockade had dissipated, and in each case, receptive fields returned to those observed under control conditions.
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Effects of GABA receptor blockade on receptive field size
Blockade of GABA inhibition produced rapid and significant changes in the shapes and sizes of neuronal receptive fields. Examples of such changes are shown in Figs. 4 and 5 and quantitative assessments are summarized in Fig. 6. In normal rats, prior to GABA blockade, most S-I forelimb neurons had receptive fields that were continuous and restricted to the forelimb (Fig. 4). The size of the receptive fields varied according to the cortical layers from which the neurons were recorded. Granular neurons (Figs. 4B and 6A) had small receptive fields restricted to single forepaw digits, the ventral paw pads, or dorsal paw. Receptive fields of supragranular cells were approximately two times the size of those for granular neurons and often included two digits, and the ventral paw pads or dorsal forepaw (Figs. 4A and 6A). Finally, infragranular neurons had the largest receptive fields (P < 0.01 compared with granular and supragranular neurons) and included multiple digits, the forepaw pads and forearm (Figs. 4C and 6A).
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The majority of S-I forelimb-stump neurons from neonatally amputated rats had receptive fields that were continuous and restricted to the remaining forelimb-stump prior to GABA receptor blockade (Fig. 5). The distribution of receptive field sizes across cortical layers was similar to that seen in normals: granular neurons (Figs. 5B and 6B) had the smallest receptive fields, while supragranular (Fig. 5A) and infragranular neurons (Fig. 5C) had significantly larger receptive fields (P < 0.05 for both comparisons, Fig. 6B). The sizes of neuronal receptive fields overall, and in each cortical layer except for the infragranular layers, were significantly larger in the neonatally amputated rats compared with normal rats (Fig. 6 and Table 1).
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When the effects of GABA were blocked with BMI and SAC, the receptive fields of S-I forelimb-stump neurons from both normal and neonatally amputated animals significantly increased in size (Figs. 4-6, and Table 1). As under control conditions, the receptive fields of neurons from neonatally amputated rats were significantly larger than those for normals during GABA receptor blockade (Fig. 6 and Table 1). For neurons from neonatally amputated rats, in addition to the expansion of the original pre-GABA block receptive fields on the forelimb-stump, GABA receptor blockade resulted in the expression of many discontinuous receptive fields on other regions of the body, especially the hindlimb (see preceding text for more details). Neurons with dual stump and hindlimb receptive fields were found in all cortical layers. In all the cases where receptive field size was quantitatively evaluated, receptive fields returned to their original shapes and sizes after the effects of GABA receptor blockade were allowed to dissipate.
Effects of GABA receptor blockade on functional properties of S-I forelimb-stump neurons
In addition to evaluating the effects of GABA receptor blockade on the receptive fields of S-I forelimb-stump cortical neurons, we examined how GABAergic inhibition affected spontaneous activity and evoked responses.
Figure 7 shows the effects of GABA receptor blockade on the spontaneous activity of S-I forelimb-stump neurons from normal rats and rats that sustained neonatal forelimb removal. Prior to GABA receptor blockade, the spontaneous activity levels of neurons in the normal and neonatally manipulated rats were similar (1.33 ± 0.05 vs. 1.44 ± 0.05 spikes/s, respectively; P > 0.05, unpaired t-test). GABA receptor blockade resulted in significant increases in the spontaneous discharge rates of neurons in both groups. The spontaneous activity rate for normal S-I forelimb neurons increased to 2.80 ± 0.09 spikes/s (P < 0.05, paired t-test), and that for S-I forelimb-stump neurons from neonatally manipulated rats significantly increased to 3.70 ± 0.14 spikes/s (P < 0.01, paired t-test).
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Blockade of GABA inhibition also resulted in increases in the response magnitudes of neurons to controlled tactile stimulation of their receptive fields. Figure 8 shows the change in the response of a granular neuron from a normal rat to stimulation of its receptive field on the second forepaw digit. Prior to GABA receptor blockade (Fig. 8A) the neuron showed a small, but consistent response to stimulation of its receptive field (Fig. 8D, light gray shading). GABA receptor blockade resulted in a significant increase in this response (Fig. 8B, top and middle PSTH). After the effects of GABA receptor blockade had worn off, the response of the neuron, as well as the spontaneous activity (Fig. 8C) returned to control levels (Fig. 8A). GABA receptor blockade produced similar results in S-I forelimb-stump neurons from neonatally amputated rats (Figs. 9 and 10).
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Figure 9 shows the overall effect of GABA receptor blockade on the responses of neurons from normal and neonatally amputated rats to controlled stimulation of forelimb and stump receptive fields, respectively. In normal rats, the mean response of neurons (n = 20) prior to GABA receptor blockade was 1.43 ± 0.16 spikes/stimulation. GABA receptor blockade produced a significant increase in the response of these neurons (2.43 ± 0.19, P < 0.0001, paired t-test). GABA receptor blockade also produced a significant increase in the response of neurons (n = 13) from neonatally manipulated rats (1.01 ± 0.26 control conditions vs. 2.62 ± 0.33 during GABA receptor block, P < 0.01, paired t-test). In six of the neurons from normal rats and six from neonatally amputated rats (data not shown), we were able to characterize the response of neurons after dissipation of GABA receptor blockade: in all cases, the responses and spontaneous activity returned to levels that were similar to those observed prior to GABA receptor blockade.
In addition to demonstrating the effects of GABA receptor blockade on
the response magnitudes of S-I forelimb-stump neurons, PSTH data
illustrated the emergence of hindlimb responses in six neurons from
neonatally manipulated rats. Figure 10 shows the PSTH data from a
granular neuron of a neonatally amputated rat. Prior to GABA receptor
blockade, this neuron had a receptive field on the forelimb stump (Fig.
10A). GABA receptor blockade resulted in a significant
enlargement of the stump receptive field (Fig. 10A) as well
as an increase in the response of the neuron to controlled stimulation
of the receptive field (Fig. 10B). Additionally, GABA receptor blockade revealed a latent hindlimb receptive field (Fig. 10C). Figure 10D shows the PSTH data collected
during controlled stimulation of the hindlimb receptive field. Since
the hindlimb receptive field was not evident under control conditions,
it was not possible to obtain response data prior to GABA receptor
blockade, but data were collected for the hindlimb response after the
effects of GABA receptor blockade had dissipated. As illustrated in
Fig. 10D, right, the hindlimb response
disappeared when the effects of GABA receptor blockade are allowed to
dissipate. Although stump and hindlimb responses to controlled
mechanical stimulation differed in their temporal patternsthe stump
responses being more temporally focused versus more broadly focused
hindlimb responses (compare Fig. 10, B and
D)
the magnitude of the two responses under GABA receptor
blockade were not significantly different (2.5 ± 0.13 vs.
2.04 ± 0.13, respectively, n = 6 neurons,
P > 0.05, unpaired t-test).
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DISCUSSION |
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The results of the present study support the following conclusions. First, the suppression of inappropriate hindlimb inputs in the S-I forelimb-stump area of rats that sustained neonatal forelimb removal often involves the inhibition of latent hindlimb inputs to neurons that normally express a stump receptive field only. Second, GABA inhibitory mechanisms are involved in normal refinement of neuronal receptive fields, and blockade of GABA receptors results in significant enlargements of receptive fields and the expression of inappropriate inputs from other areas of the body surface. In some cases, GABA receptor blockade results in the expression of receptive fields by cells that are normally unresponsive to cutaneous stimulation. Finally, cortical GABA circuits act to modulate the functional characteristics of S-I forelimb-stump neurons, decreasing the rate of spontaneous activity of S-I neurons and attenuating the response of cortical neurons to controlled cutaneous stimulation.
Technical limitations
Since application of BMI and SAC to the entire cortical surface
inhibits GABA inhibitory mechanisms over a very broad area, the present
results do not permit determination of the precise location where GABA
might act to suppress inappropriate hindlimb inputs in neonatally
amputated rats. It is reasonable to suggest that suppression of
hindlimb inputs ascending from the reorganized VPL nucleus
(Stojic et al. 1998) involves inhibition of the S-I cortical neurons receiving these inputs. However, evidence that a
polysynaptic intracortical pathway between the S-I hindlimb and
forelimb-stump fields is a necessary substrate for most dual receptive
fields expressed during GABA receptor blockade in neonatally amputated
rats (Lane et al. 1999
) raises the possibility that suppression of hindlimb inputs might occur some distance from the
cortical neurons that express dual receptive fields.
In attempting to totally remove the influences of GABA inputs to the
neurons isolated in this study, we applied BMI and SAC simultaneously
to block GABA activity at both the GABAA and
GABAB receptors, respectively. This approach did
not permit evaluation of the differential effects that blocking
GABAA or GABAB receptors separately might have on the receptive field and on responses of S-I
forelimb-stump neurons. While results from a previous study suggests
equal involvement of both the GABAA and
GABAB receptors in suppressing hindlimb inputs to
the S-I forelimb-stump field (Lane et al. 1997), studies
in the posterior barrel subfield of rats have attributed changes in
receptive fields and response properties of neurons almost entirely to
actions of GABA at GABAA receptors
(Kyriazi et al. 1996a
,b
). Thus the individual
contributions of GABAA and
GABAB receptors to the overall effect of GABA
modulation of sensory processing in the S-I forelimb-stump
representation remain to be determined.
GABA-mediated suppression of hindlimb inputs in the S-I forelimb-stump field of neonatally amputated rats
In the INTRODUCTION, we suggested three, nonmutually exclusive ways by which blockade of GABA inhibition might lead to the suppression of hindlimb responses in the S-I forelimb-stump representation of neonatally amputated rats: totally silencing cortical cells with dual stump and hindlimb receptive fields, silencing neurons with receptive fields restricted to the hindlimb alone, or suppressing hindlimb inputs to neurons that express a stump receptive field only. The results of this study support the conclusion that GABA mechanisms most often act to suppress hindlimb inputs to S-I forelimb-stump neurons that normally express a receptive field on the stump only. However, it must be noted that there was evidence that GABA-mediated inhibition, to a much smaller extent, also suppressed hindlimb input information to the S-I forelimb-stump representation through the other two mechanisms described in the preceding text.
Previous results have demonstrated that expression of hindlimb inputs
within the forelimb somatosensory pathway of neonatally amputated rats
is significantly reduced in the thalamus and S-I cortex (Stojic
et al. 1998). While 40% of neurons in the CN of neonatally
amputated rats express dual stump and hindlimb receptive fields
(Lane et al. 1995
), only 19% of neurons within the
forelimb-stump representation of the VPL nucleus have such receptive
fields (Stojic et al. 1998
). The results of the present
study show that the expression of these dual receptive fields is
further reduced within S-I: only 2.2% of S-I forelimb-stump neurons
from neonatally amputated rats were found to express dual stump and
hindlimb receptive fields under normal conditions.
Changes in sensory experience, such as those that result from
deafferentation, have generally been found to result in changes in the
functional organization of sensory cortex (e.g., Calford and
Tweedale 1991; Diamond et al. 1993
; Fox
1994
; Kaas 1994
; Kelahan and Doestch
1984
; Kelahan et al. 1981
; Mezernich et
al. 1983
; Rasmusson 1982
; Sanes et al.
1988
; Turnbull and Rasmusson 1990
; Waite and Taylor 1978
; Wall and Egger
1971
), regardless of whether these changes might be considered
beneficial (Pascual-Leone 1993
; Recanzone et al.
1992a
,b
) or deleterious [e.g., such as expression of inputs
from one area of the body into topographically inappropriate areas of
sensory cortex (Aglioti et al. 1994
; Borsook et
al. 1998
; Halligan et al. 1993
; Pons et
al. 1991
)]. Conversely, experiments where
deafferentation has resulted in little cortical reorganization have
been suggested as evidence of an inability of a particular level of the
sensory neuraxis to reorganize (e.g., Waite 1984
). The
present results, and those of our previous experiments, suggest an
alternative explanation for the apparent lack of reorganization: functional suppression of abnormal inputs resulting in maintenance of a
reasonably organized topographic representation of the body surface.
It is worth noting that the present results and those of our previous
studies differ from those of Rasmusson et al. (1985), who reported that limb amputation in raccoons resulted in expression of
hindlimb inputs to the cortical stump representation in the absence of
GABA receptor blockade. The reason(s) for the difference between our
results and those of Rasmusson et al. (1985)
is not clear.
The mechanisms underlying the GABA-mediated suppression of hindlimb
inputs to the S-I stump field of neonatally amputated rats could
involve either nonspecific or selective inhibitory actions. Receptive
field transformation from the ventral posterior nucleus to the
posterior barrel subfield of rat, as well as maintenance of orientation
selectivity in visual cortex, is thought to result from cortical GABA
inhibition acting through nonspecific hyperpolarization of cortical
neurons (Kyriazi et al. 1996a; Nelson et al.
1994
; Somers et al. 1995
). It is possible that
nonspecific inhibition could underlie the suppression of hindlimb input
in the stump representation of S-I. However, it is also possible that
these hindlimb inputs are selectively suppressed. Anatomical data has demonstrated that the majority of GABA synapses in rat S-I are located
on dendritic spines and shafts (Micheva and Beaulieu
1996
). This suggests that GABA neurons might be capable of
selectively inhibiting afferent inputs as they synapse along the
dendritic arbors, independent of generalized inhibitory effects that
might be mediated by axosomatic GABAergic synapses (Micheva and
Beaulieu 1997
; White 1989
). Independent effects
of GABAA-mediated inhibition on the cell bodies
and dendrites of neurons in piriform cortex lend support to such an
interpretation (Kapur et al. 1997
). It is also worth
noting that studies of nonspecific inhibition have focused on the
transformation of thalamic inputs to sensory cortex and not how
GABAergic inhibition acts to refine or suppress afferent inputs that
are conveyed via intracortical circuits. Intracortical inputs appear to
convey the vast majority of hindlimb input to the stump representation
of neonatally amputated rats (Lane et al. 1999
). It has
been demonstrated in piriform cortex that
GABAB-mediated inhibition can selectively
suppress intrinsic inputs within layer 1b but have no effects on
extrinsic afferent inputs from the olfactory bulb that synapse in layer
1a (Tang and Hasselmo 1994
). It may be that sensory
cortex modulates intrinsic and extrinsic inputs via distinct inhibitory mechanisms.
The data in this study, while very limited, provide some support for
the possibility that the suppression of hindlimb inputs involves
selective GABA inhibition. This interpretation is based on comparisons
of the response magnitudes of S-I forelimb stump neurons with dual
receptive fields to stimulation of the stump and the emergent hindlimb
receptive field during GABA receptor blockade. There was no significant
difference in the magnitude of these responses when GABA receptors were
blocked, suggesting that the suppressed hindlimb inputs are not weak,
incapable of driving cortical stump neurons to threshold because of
membrane hyperpolarization produced by nonspecific inhibition. Rather
these data suggest that hindlimb inputs can provide strong excitatory drive to cortical stump neurons, almost equal to that of the stump afferents. However, we cannot exclude the possibility of nonspecific inhibition playing some role in suppressing these hindlimb inputs because GABA receptor blockade also resulted in a significant increase
in the spontaneous activity of S-I cortical neurons, which is
consistent with the nonspecific inhibitory model presented by
Kyriazi et al. (1996a).
Role of GABA in modulating neuronal receptive fields and response properties in the S-I forelimb-stump representation of rats
The present results indicate that GABA-mediated inhibition in S-I
refines neuronal receptive fields in the forelimb and forelimb-stump representations by making them smaller and suppressing inappropriate inputs from other body regions. Under control conditions, in both normal and neonatally amputated rats, the majority of S-I
forelimb-stump neurons had receptive fields restricted to the forelimb
or stump, respectively. The size of the receptive fields of these
neurons varied according to the cortical layers from which they were
recorded: granular neurons had the smallest receptive fields, while the receptive fields of supragranular and infragranular neurons were larger. This pattern is consistent with previous results (Chapin 1986).
The enlargement of S-I forelimb-stump neuronal receptive fields that
resulted from GABA receptor blockade is consistent with previous
reports that have shown that pharmacologic blockade of cortical GABA
inputs results in receptive field enlargements in other areas of
somatosensory cortex (Batuev et al. 1982; Dykes and Lamour 1988
; Dykes et al. 1984
; Hicks
and Dykes 1983
; Kaneko and Hicks 1990
;
Kyriazi et al. 1996a
,b
; see Dykes 1997
for review), visual cortex (Ramoa et al. 1998
), and
motor cortex (Jacobs and Donoghue 1991
). It is
interesting to note that even in rats that have sustained neonatal
forelimb removal, GABA mechanisms appear to function in a similar
fashion as that seen in normal rats.
We also found that GABA receptor blockade increases both the
spontaneous activity and responses of neurons to controlled tactile stimulation. Increases in response magnitude following application of
GABA antagonists have been reported in earlier studies in visual cortex
(Rose and Blackmore 1974; Silito 1975
,
1977
; Wolf et al. 1986
), cat somatosensory
cortex (Dykes et al. 1984
; Hicks and Dykes 1983
), and rat barrel cortex (Kyriazi et al.
1996a
,b
). Increases in the spontaneous activity of cortical
neurons as a result of GABA blockade have been reported in rat barrel
cortex by Kyriazi et al. (1996a)
; however,
Alloway and Burton (1991)
suggest that changes in the
receptive fields of cortical neurons in primate occurs without an
increase in spontaneous discharge rate.
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ACKNOWLEDGMENTS |
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Special thanks go to Drs. Nicolas L. Chiaia and Richard Mooney for excellent technical assistance and to R. Wynn and M. E. Pommeranz for excellent work on the histology.
This work was supported by National Institutes of Health Grants NS-28888 and DE-07734.
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FOOTNOTES |
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Address reprint requests to R. D. Lane.
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 21 December 1999; accepted in final form 2 March 2000.
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REFERENCES |
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