Cortical influences on rapid brainstem plasticity

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Cortical influences on rapid brainstem plasticity Xin Wang, John T. Wall ⁎ Department of Neurosciences, Medical University of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Cortical contributions to brainstem plasticity in the somatosensory system are poorly

Accepted 4 April 2006

understood. Tactile receptive fields (RFs) of brainstem dorsal column nuclei (DCN) neurons

Available online 12 May 2006

rapidly enlarge when peripheral inputs are disrupted by local anesthetic blocks with lidocaine (LID). Cortical inputs appear to influence this plasticity because enlargements

Keywords:

have been shown to be greater when cortical inputs are disrupted. Like disruptions of

Somatosensory cortex

peripheral inputs, disruptions of DCN inhibition by DCN administration of the GABAA

Disinhibition

receptor antagonist bicuculline methiodide (BMI) also cause rapid enlargements of DCN RFs

Dorsal column nuclei

when cortical inputs are intact. These findings leave questions about interactions between

Cuneate nucleus

cortical inputs, DCN inhibition, and DCN RF plasticity. To study potential interactions, the

Gracile nucleus

present experiments evaluated RF sizes of DCN tactilely responsive neurons in anesthetized

Deafferentation

rats following DCN microinjection of BMI when cortical inputs were acutely disrupted or intact. These tests were also supplemented by subsequent LID tests to directly compare post-BMI and post-LID effects on the same RF. BMI caused DCN RF enlargements when cortical inputs were disrupted or intact; however, enlargements after cortical input disruption were greater than when cortical inputs were intact. Following RF enlargement and retraction after BMI, LID often caused a second enlargement of the same RF, across skin that partially matched skin involved in the enlargement after BMI. This occurred when cortical inputs were disrupted or intact. We hypothesize that cortical inputs are not required for BMI and LID to initiate partially matching enlargements in individual DCN tactile RFs, however, cortical inputs constrain magnitudes of these enlargements. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Tactile inputs from the skin are first processed in the brainstem dorsal column nuclei (DCN) before subsequent processing at thalamic and somatosensory cortical levels (Kaas et al., 2002). This ascending system, in turn, is complemented by descending cortical inputs to each subcortical level, including the DCN. DCN neurons commonly have excitatory tactile receptive fields (RFs) whose sizes can be assessed by measuring the skin area that elicits responses to tactile stimuli (Willis and Coggeshall, 2004). RF size presum-

ably reflects activity of inputs to DCN neurons, including ascending peripheral inputs, cortical inputs, and intrinsic DCN inputs from DCN inhibitory interneurons. RF size analyses provide a valuable approach for assessing somatosensory function. These analyses have proved particularly useful for assessing functional plasticity that occurs over periods of minutes or hours because the size of an RF can be accurately compared over short periods before and after treatments (Dostrovsky et al., 1976; Merzenich et al., 1983; Calford and Tweedale, 1991; Pettit and Schwark, 1993; Ergenzinger et al., 1998; Klein et al., 1998; Dostrovsky, 1999;

⁎ Corresponding author. Fax: +1 419 383 3008. E-mail address: [email protected] (J.T. Wall). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.04.015

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Calford, 2002; Chowdhury and Rasmusson, 2002; Kawamata et al., 2005; Wang and Wall, 2005). In the DCN, there is evidence that RF sizes can rapidly change following disruption of either peripheral inputs or intrinsic inputs from DCN inhibitory interneurons. In this regard, if peripheral inputs are disrupted by cutaneous injections of the local anesthetic lidocaine (LID), DCN single- and multi-unit RFs frequently immediately enlarge (Pettit and Schwark, 1993; Panetsos et al., 1995; Wang and Wall, 2005). DCN RFs can also immediately enlarge if inputs from DCN inhibitory interneurons are disrupted by DCN administration of the GABAA receptor antagonist bicuculline methiodide (BMI) (Schwark et al., 1999). The rapidness of RF enlargements seen in LID or BMI studies has led to thinking that these enlargements may be related, i.e., that LID, like BMI, may cause DCN RF enlargements by decreasing DCN inhibition. DCN neurons receive direct descending inputs from cortex (Desbois et al., 1999; Martinez-Lorenzana et al., 2001). Contributions of cortical inputs to the above RF enlargements have received limited study, with all work to date focused on cortical influences on post-LID enlargements. This work suggests that LID continues to cause RF enlargements following acute lesion of cortical inputs (Pettit and Schwark, 1993; Wang and Wall, 2005). In addition, although not required for post-LID enlargements, cortical inputs appear to constrain the magnitudes of these enlargements because post-LID RF enlargements are larger when cortical inputs are lesioned than when they are intact (Wang and Wall, 2005). The above findings leave unanswered questions about cortical influences on RF enlargements after BMI and on potential relationships between enlargements after BMI and LID. To explore these issues, we compared DCN RF size changes following disruption of DCN inhibition with BMI when cortical inputs were removed or intact. We also supplemented BMI tests with subsequent LID tests to further compare post-BMI and post-LID changes in the same RF, when cortical inputs were removed or intact. The objective was to examine cortical influences on individual RF plasticity after these treatments and to study potential relationships of DCN effects of these treatments.

single- and multi-unit RFs had pre-injection RFs that overlapped; following BMI, single-unit RFs expanded into skin areas that were within the starting and/or enlarged areas of the paired multi-unit RF (e.g., Fig. 1F), suggesting that BMI caused RF enlargements of single neurons and clusters of adjacent neurons. Finally, in some cases (8/51), little or no changes in RF size were detected (e.g., Figs. 1E, G). The above results applied to starting RFs on toe and/or more proximal hindpaw and forepaw locations (33 gracile and 18 cuneate RFs). Post-BMI changes in sizes of all RFs were charted in a scatterplot and boxplots. The scatterplot, which charted individual RF data, indicated that percentage changes in post-BMI RF size ranged from little change to maximal enlargement of 244% (mean = 77%; Fig. 1G, DCN BMI). Boxplots showing indices of the distributions of group data from singleand multi-unit RFs indicated that BMI caused similar enlargements in single- and multi-unit RFs (Fig. 2, cortex intact; mean single unit = 78%, mean multi-unit = 76%). In control tests, individual DCN RFs were repeatedly defined before and after DCN microinjections of saline (e.g., Fig. 1H; 4 single- and multi-unit RF pairs + 1 multi-unit RF). RFs underwent little or no changes after saline (Fig. 1G, DCN saline; mean = 3%). Saline tests in 8 of these RFs (4 single- and multi-unit RF pairs) and BMI tests in 12 RFs (6 single- and multi-unit RF pairs) used a blind microinjection procedure in which the investigator defining RFs did not know whether saline or BMI was given. The scatterplots of the individual RF changes from these blind tests indicated the distribution of RF enlargements after BMI was shifted above the distribution of small to no changes after saline (Fig. 1G, compare blind DCN BMI vs. blind DCN saline; mean BMI = 69%, mean saline = 3%). In some cases, upon completing a first BMI test, BMI was microinjected a second time to explore whether an additional BMI microinjection might reveal further RF enlargement (6 single- and multi-unit RF pairs). Further RF enlargements were seen in 33% (4/12) of these tests (e.g., Fig. 1I, arrows after 2nd DCN BMI). Thus, RF enlargements after BMI sometimes reflected a fraction, rather than the full extent, of possible enlargement.

2.1.2.

2.

Results

2.1.

Effects of BMI

2.1.1.

BMI tests with intact cortical inputs

To test RF effects of BMI when the DCN had cortical inputs, cortex was left intact and individual DCN RFs were repeatedly defined before and after a DCN microinjection of BMI (e.g., Fig. 1A; 24 single- and multi-unit RF pairs + 3 single-unit RFs). Following BMI, most RFs (43/51) underwent enlargements into skin areas that were outside the pre-injection RF (e.g., arrows in Figs. 1A–D and see G). These enlarged areas involved, for example, one or more adjacent digits and/or skin areas on paw pads or dorsal paw surfaces. RF enlargements usually emerged within a few minutes after injection (e.g., Fig. 1A, between 3 and 7 min) following which they were maintained or further progressed before significantly or completely retracting over about the subsequent hour. Paired

BMI tests following acute removal of cortical inputs

To test if DCN inhibition can regulate RF size in the absence of cortical inputs, cortical areas that project to the DCN were acutely lesioned, and each RF was tested before and after a DCN microinjection of BMI (e.g., Fig. 3A; 20 single- and multiunit RF pairs). The procedures were as in the above BMI tests when cortex was intact (mean BMI volume, cortex intact = 20.8 nl, cortex lesion = 20.5 nl). Following cortical lesion, BMI initiated enlargements of RFs onto one or more adjacent digits and/or skin areas on paw pads or dorsal paw surfaces that were outside the preinjection RF (e.g., arrows in Figs. 3A–E). RF enlargements usually became apparent within a few minutes (e.g., Fig. 3A, within 0–2 min) following which they were maintained or further progressed before retracting to a significant or complete degree over about an hour. Paired single- and multi-unit RFs had pre-injection RFs that overlapped; following BMI, single-unit RFs expanded into skin areas that were within the starting and/or expanded areas of the paired multi-

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Fig. 1 – Effects of BMI on DCN RFs when cortical inputs were intact. (A) As illustrated by this example, each RF was repeatedly defined at several-minute intervals before and after a single microinjection of BMI (DCN BMI). Continuous time is shown in minutes, with resetting to 0 at microinjection to also indicate post-injection times. Arrows indicate examples of RF enlargement into skin that was outside the pre-injection RF. RF enlargement was followed by later RF retraction. (B–F) Further examples of BMI test results. RFs were tested as in A, but only one pre-injection RF (pre) and the maximal post-injection RF (post) is shown as a summary. Times under ‘post’ RFs indicate the time the illustrated maximal RF was defined after BMI microinjection. In B–D and F, BMI caused RF enlargements (arrows), whereas in E no enlargement was seen. F shows results from a single (top)- and multi (bottom)-unit RF pair. (G) Scatterplots of percentage changes in sizes of RFs in all BMI and saline tests. ‘0’ indicates no change in RF size after microinjection. Above ‘0’ values indicate magnitudes of post-injection RF enlargements. Each single- or multi-unit RF is charted as an individual point, and tests that used blind procedures are distinguished. (H) Example of a control test in which DCN microinjection of saline did not cause RF enlargement. (I) In some RFs, after completing a first BMI test, a second BMI microinjection was given which revealed further RF enlargement (e.g., arrows) not seen after the first test. ‘Blind’ in A, F, G, and H indicates that the RF was tested without knowledge of whether BMI or saline was given. All illustrated RFs are single-unit RFs except the multi-unit RF (‘multi’) in F.

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Fig. 2 – Boxplots showing indices of the distributions of RF enlargements after BMI for single- and multi-unit RFs. The boxes indicate interquartile (25th–75th percentile) ranges, and the brackets indicate total ranges for each distribution. Note that when: (1) cortex was intact, single- and multi-unit RFs showed similar enlargements (left), (2) cortex was lesioned, single- and multi-unit RFs showed similar enlargements (right), and (3) cortex was lesioned, enlargements of single- and multi-unit RFs were shifted upward compared to when cortex was intact (single-unit RFs, z = −2.30, P < 0.021; multi-unit RFs, z = −2.40, P < 0.016).

unit RF (e.g., Fig. 3C); thus, BMI continued to initiate RF enlargements of single neurons and clusters of adjacent neurons. Finally, in some cases (4/40), little or no RF enlargement was detected (e.g., Figs. 3F, G). The above results applied to starting RFs on toe and/or more proximal hindpaw and forepaw locations (20 gracile and 20 cuneate RFs). As in tests with intact cortex, RF measures from all tests after cortical lesions were charted in a scatterplot showing individual RF data and in boxplots showing indices of the distributions of single- and multi-unit group data. The scatterplot indicated that the percentage changes in postBMI RF sizes ranged from little change to maximal enlargement of 440% (Fig. 3G, DCN BMI, mean = 155%). The boxplots indicated that BMI caused similar enlargements of single- and multi-unit RFs (Fig. 2, cortex lesion; mean single-unit RF = 166%, mean multi-unit RF = 144%). These findings suggest that RF sizes continued to be regulated by DCN inhibition after loss of cortical inputs. In control tests, cortical inputs were removed, and individual DCN RFs were repeatedly defined before and after DCN microinjection of saline (e.g., Fig. 3H, 4 single- and multi-unit RF pairs). These saline tests, and BMI tests in 20 RFs (10 singleand multi-unit RF pairs), were done using the blind microinjection procedure. The scatterplots of individual RF changes indicated that after blind BMI treatment 85% (17/20) of RFs underwent enlargements that were larger than the largest changes after blind saline treatment (Fig. 3G, compare blind DCN BMI vs. blind DCN saline; mean BMI = 112%, mean saline = − 3%). Comparison of the post-BMI RF enlargements following cortex lesion to the post-BMI RF enlargements when cortex was intact indicated that the mean RF enlargement after cortex lesion was greater than the mean enlargement when cortex was intact (cortex lesion = 155%, cortex intact = 77%). A similar effect held when means from the subgroup of tests that used the blind microinjection procedures were compared (cortex lesion = 112%, cortex intact = 69%). This upward

shifting in the distribution of RF enlargements after cortex lesion was apparent when enlargements for single- and multiunit RF groups were compared (Fig. 2). In this latter regard, the lower ranges of the distributions of both groups overlapped, but the interquartile ranges and upper ranges of the distributions were shifted upward after cortex lesion relative to when cortical inputs were intact (Fig. 2). These shifts were statistically significant for both groups (cortex lesion vs. intact cortex: single-unit RFs, z = −2.30, P < 0.021; multi-unit RFs, z = −2.40, P < 0.016). This suggests that cortical inputs tended to constrain the magnitudes of RF enlargements that were initiated by BMI (see Discussion).

2.2.

Sequential effects of BMI and LID on the same RF

2.2.1.

BMI and LID tests with intact cortical inputs

Under intact cortical input conditions, individual RFs were sequentially tested with a single DCN microinjection of BMI and a subsequent single cutaneous microinjection of LID to directly compare effects of these treatments in the same RF. These tests led to two results: (1) BMI and LID both caused enlargements in the same RF, and (2) skin areas that were involved in the RF enlargements after BMI and LID overlapped across partially matching locations. For example, as shown in Fig. 4A, DCN microinjection of BMI caused RF enlargement (e.g., arrows after DCN BMI). Comparison of the pre-BMI RF to the post-BMI RF at the time of maximal enlargement identified skin locations that were involved in this RF expansion (Fig. 4B, DCN BMI EXPANSION, black area). Following significant RF retraction after BMI (e.g., Fig. 4A, 62 min), a subsequent cutaneous microinjection of LID caused a second RF enlargement (Fig. 4A, e.g., arrows after SKIN LID). Comparison of the post-BMI retracted RF to the post-LID RF at the time of maximal enlargement revealed skin locations that were involved in post-LID expansion (Fig. 4B, SKIN LID EXPANSION, black area). Further comparison of the skin locations involved in the RF enlargements after BMI (Fig. 4B, DCN BMI EXPANSION, black area) and LID (Fig. 4B, SKIN LID EXPANSION, black area) showed that these enlargements overlapped across partially matching skin (Fig. 4B, OVERLAP, stippled). Similar partial matching in enlargements of the same RF after both treatments was seen in all RFs tested (e.g., Figs. 4C–E; 6 singleand multi-unit RF pairs). In a control test, a multi and single-unit RF pair was tested with a DCN microinjection of saline using the blind microinjection procedure followed by a cutaneous microinjection of LID. No enlargement occurred after saline, but the RF enlarged after LID. This contrasted with results from the above BMI and LID tests, which included results from two multi- and singleunit RF pairs in which the BMI microinjection was made using the blind microinjection procedure and where BMI and LID caused sequential enlargements that partially matched (e.g., Fig. 4B).

2.2.2. BMI and LID tests following acute removal of cortical inputs Sequential BMI and LID microinjections were done following cortical lesion to test if sequential enlargements of the same RF required cortical inputs. For example, as seen in Fig. 5A, following cortical lesion (shaded area on hemisphere), a DCN

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Fig. 3 – Effects of BMI on DCN RFs after cortical lesion. (A) As illustrated by this example, each tested RF was repeatedly defined at several-minute intervals before and after a single microinjection of BMI (DCN BMI). Testing was done, in this case, beginning at 387 min after the indicated contralateral cortical lesion (shaded area on dorsal view of cerebral hemispheres). Arrows indicate examples of RF enlargement into skin that was outside the pre-injection RF. RF enlargement was followed by subsequent RF retraction. (B–F) Further examples of cortical lesions and BMI test results after these lesions. RFs were tested as in A, but only one pre-injection RF (pre) and the maximal post-injection RF (post) are shown as a summary (time after lesion at which the pre-injection RF was defined is indicated under the ‘pre’ RF, and time after BMI that the illustrated maximal ‘post’ RF was defined is indicated under the ‘post’ RF). In B–E, BMI caused RF enlargements (arrows), whereas in F no enlargement was seen. C shows results from a single-unit (top) and multi-unit (bottom) RF pair. (G) Scatterplots showing percentage changes in sizes of individual RFs in all BMI and saline tests. Conventions as in Fig. 1. (H) Example of a control test in which saline was microinjected into the DCN, and no RF enlargement was seen. ‘Blind’ in A–C, G, and H indicates that the RF was tested without knowledge of whether BMI or saline was given. All illustrated RFs are single-unit RFs except the multi-unit RF (‘multi’) in C.

microinjection of BMI caused rapid RF enlargement (e.g., arrows after DCN BMI). Skin locations involved in this RF expansion were identified (Fig. 5B, DCN BMI EXPANSION, black area; for explanation see Fig. 4). Following significant retrac-

tion of this RF enlargement (e.g., 40 min), a subsequent cutaneous microinjection of LID caused a second rapid RF enlargement (Fig. 5A, e.g., arrows after SKIN LID). Skin locations involved in this post-LID expansion were identified

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Fig. 4 – Examples of results from sequential BMI and LID tests in the same RF when cortex was intact. (A) Each RF was repeatedly defined at several-minute intervals before and after a single DCN microinjection of BMI (DCN BMI) and a subsequent single cutaneous microinjection of LID (SKIN LID). Continuous time is shown in minutes, with resetting to 0 at each microinjection to also indicate times after each injection. After DCN microinjection of BMI, the RF enlarged (e.g., arrows) and then retracted to a significant degree following which the RF enlarged a second time after a subsequent cutaneous microinjection of LID (L). ‘Blind’ indicates that the investigator defining the RF did not know whether BMI or saline was given. (B) Summary of the results shown in A. Left: the starting RF prior to BMI and the maximal enlargement of the RF after BMI are shown to indicate the RF expansion after BMI (DCN BMI EXPANSION, black area). Middle: the retracted RF after BMI and the maximal enlargement of the RF after LID are shown to indicate the RF expansion after LID (SKIN LID EXPANSION, black area). Skin that became anesthetic due to LID injection (L) is also indicated. Right: comparison of the DCN BMI EXPANSION and the SKIN LID EXPANSION identified overlap and partial matching of skin locations involved in expansions after BMI and LID (OVERLAP, stippled). (C–E) Further examples of partial matching of enlargements of the same RF after sequential BMI and LID treatments (conventions as in B). E shows results from a single- and multi-unit RF pair. All RFs are single-unit RFs except the multi-unit RF (‘multi’) in E.

(Fig. 5B, SKIN LID EXPANSION, black area). Comparison of RFs after BMI and LID revealed overlapping and partial matching of enlargements (Fig. 5B, OVERLAP, stippled). Similar partial matching was seen in most RFs (94%, 17/18; 9 single- and multi-unit RF pairs; e.g., Figs. 5C–D). Thus, following loss of cortical inputs, sequential disruptions of DCN inhibition with BMI and of peripheral inputs with LID initiated sequential enlargements of the same RF, across partially matching skin locations. In control tests, after cortical lesion, two multi- and singleunit RF pairs were tested with a DCN microinjection of saline using the blind microinjection procedure followed by cutaneous microinjection of LID. RF enlargements were not seen after saline but were seen after LID in all RFs. This contrasts with results from a subgroup of the above post-lesion BMI and LID

tests, which included six multi- and single-unit RF pairs in which BMI microinjection was done using blind procedures and where BMI and LID caused partially matching, sequential enlargements in 92% (11/12) of tests.

3.

Discussion

The present study examined cortical influences on DCN RF enlargements after BMI and on the relationship of enlargements after BMI and LID. New findings from this study are: (1) disruption of DCN inhibition with BMI initiated DCN RF enlargements regardless of whether cortical inputs were removed or intact, (2) post-BMI enlargements were greater when cortical inputs were removed, thus suggesting that

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Fig. 5 – Examples of results from sequential BMI and LID tests in the same RF following cortical lesion. Conventions as in Fig. 4. (A) Following cortical lesion, each RF was repeatedly tested at several-minute intervals before and after a single DCN microinjection of BMI and a subsequent single cutaneous microinjection of LID. As seen in this example, after the indicated cortical lesion (shaded area on hemisphere), the RF enlarged (e.g., arrows) and retracted to a significant degree following DCN microinjection of BMI and then enlarged a second time following subsequent cutaneous microinjection of LID (e.g., arrows). ‘Blind’ indicates that the investigator defining the RF did not know whether BMI or saline was given. (B) Summary of the results from A indicating the partial matching of skin locations involved in the RF expansions after BMI and LID. (C–D) Further examples of cortical lesions and partial matching of RF enlargements. C shows results for a forepaw RF (top) defined in the cuneate nucleus and a hindpaw RF (bottom) defined in the gracile nucleus following the indicated cortical lesion. D shows results from a single- and multi-unit RF pair after the indicated cortical lesion. All RFs are single-unit RFs except the multi-unit RF (‘multi’) in D.

cortical inputs constrained the magnitudes of these enlargements, and (3) disruption of DCN inhibition with BMI and disruption of peripheral inputs with LID caused sequential enlargements of the same RF across partially matching skin areas regardless of whether cortical inputs were removed or intact. These findings add to the present understanding in the following ways.

3.1. DCN RF enlargements after BMI when cortical inputs were removed versus intact The present BMI tests confirm previous findings that DCN administration of BMI causes DCN RF enlargements when cortical inputs are intact (Schwark et al., 1999). In addition, the present results provide the new finding that BMI continued to initiate RF enlargements after removal of cortical inputs. Given that the present cortical lesions very likely removed all direct cortical inputs to the DCN (see Experimental procedures), this indicates that sizes of DCN

RFs remained regulated by DCN inhibition that does not require direct cortical inputs. Beyond the present findings, there is no understanding of whether DCN inhibitory influences on sizes of RFs necessitate ongoing cortical inputs. Cortical projections to the DCN mediate excitatory and, via connections with DCN GABAergic and glycinergic interneurons, inhibitory and disinhibitory effects on DCN responses (Lue et al., 1997a; Aguilar et al., 2003). These descending cortical effects work in topographic register with similar DCN effects of ascending peripheral inputs to sharpen spatial and temporal processing of peripheral inputs. There are also indications that inhibition continues to affect DCN responses after loss of cortical inputs (Perl et al., 1962; Bystrzycka et al., 1977), but effects on RF size have not been studied. The present BMI findings suggest that DCN inhibition continued to be an important determinant of DCN RF sizes following disruption of cortical inputs. A recent study indicated that removal of cortical inputs caused no or modest immediate changes in sizes of DCN RFs (Wang and Wall, 2005).

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From that study, it was not possible to distinguish if DCN inhibition contributed to maintaining RFs at their pre-lesion sizes. The present results suggest that DCN inhibition made important contributions. In addition, given that peripheral inputs have direct synaptic connections onto DCN inhibitory interneurons (Lue et al., 1997b; Lue et al., 2001) and that activity of these interneurons is dependent on peripheral input activity (Willis and Coggeshall, 2004), peripheral inputs likely continued to influence DCN inhibitory effects on RF size after loss of cortical inputs. The present results indicate that post-BMI RF enlargements were larger after removal of cortical inputs. We interpret this to suggest that cortical inputs, in part, constrained these enlargements. Thus, it appears that BMI can initiate DCN RF enlargements without cortical inputs but, in addition, that cortical inputs influence the magnitudes of these enlargements. A recent report found analogous cortical effects in LID tests, that is, LID initiated DCN RF enlargements without cortical inputs but cortical inputs constrained the magnitudes of these changes (Wang and Wall, 2005). These findings suggest that cortical inputs have similar influences on these aspects of post-BMI and post-LID RF enlargements.

3.2. BMI and LID effects on the same DCN RF when cortical inputs were removed or intact BMI tests were extended to include subsequent LID tests to also provide direct comparisons of BMI and LID effects on individual DCN RFs and to assess potential cortical interactions on these effects. The results suggest that BMI and LID initiated enlargements of the same DCN RF and that enlargements after both treatments commonly involved partially matching areas of skin. These effects were produced when cortical inputs were removed or intact and, thus, did not require cortical inputs. There have been no other attempts to evaluate BMI and LID effects on the same DCN RF. Previously, studies have assessed DCN RF changes after either BMI (Schwark et al., 1999) or LID (Pettit and Schwark, 1993; Panetsos et al., 1995; Wang and Wall, 2005). Comparisons of results across studies suggested that both treatments produce rapid RF enlargements when cortical inputs are intact. Emerging from early views on unmasking (Dostrovsky et al., 1976; Wall, 1977), these findings have led to thinking that post-LID DCN RF enlargements are related to post-BMI DCN RF enlargements by a common mechanism of action, i.e., that LID, like BMI, may cause enlargements by decreasing DCN inhibition. Findings that LID also causes RF enlargements after removal of cortical inputs (Pettit and Schwark, 1993; Wang and Wall, 2005) raise the possibility that this relationship also applies after removal of cortical inputs. The present results provide evidence for this possibility. With regard to the above thinking, the finding that BMI and LID caused partially matching enlargements in an individual RF when cortical inputs were intact provides evidence that is arguably more direct than evidence from cross-study comparisons. If the above thinking is valid, it is reasonable to predict that BMI and LID should cause enlargements not only of different RFs, as suggested by previous cross-study data, but also of the same RF. The present study provides evidence for

this prediction. After cortical lesion, the findings that RFs enlarged after BMI, and that partially matching enlargements were produced by BMI and LID, provide further evidence that the above thinking may apply when cortical inputs are removed as well as intact. Overall, the present findings of new inputs emerging from common skin locations after BMI and LID provide evidence for spatially correlated changes in the same RF that appear unlikely to be coincidental. We offer the following speculation about how the present findings can contribute to understanding of cortical influences on rapid DCN RF plasticity after BMI and LID. As discussed above, the present results appear consistent with the view that LID may cause DCN RF enlargements, in part, by decreasing DCN inhibition. DCN inhibition of RF size remained active and was disrupted by BMI after removal of cortical input. Peripheral inputs can directly activate DCN inhibitory interneurons, suggesting that some of this inhibition resulted from peripheral activity. Since enlargements of an individual DCN RF can be initiated by both BMI and LID, without and with cortical inputs, initiation of these enlargements does not require cortical inputs. These results appear consistent with the possibility that initiation of DCN RF enlargement may, minimally, involve post-LID changes in peripheral input activity and related decreases in DCN GABA inhibition (Fig. 6). Beside DCN changes, LID also causes equally rapid enlargements of cortical RFs (Byrne and Calford, 1991; Calford and Tweedale, 1991; Panetsos et al., 1995). These cortical changes, in turn, may alter descending signals to the

Fig. 6 – Proposal for how peripheral inputs, DCN inhibition, and cortical inputs may have contributed to rapid DCN plasticity in the present study. The present results suggest that disruption of DCN inhibition with BMI partially simulated the effects that disruption of peripheral inputs with LID initiated in the same DCN RF. Cortical inputs were not required for these changes. This could suggest that LID initiated rapid DCN RF enlargements, in part, by decreasing DCN inhibition without the need for cortex. LID also causes concurrent enlargements of cortical RFs. The present results suggest that cortical changes may alter descending cortical input to the DCN to, in part, constrain magnitudes of DCN RF enlargements. Thus, rapid DCN RF enlargements in the present study may reflect changes in balances of inputs to DCN neurons from peripheral, intrinsic DCN, and cortical inputs.

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DCN. Since post-BMI and post-LID DCN RF enlargements increase in magnitude following removal of cortical inputs, altered descending signals may, in part, constrain DCN RF enlargements. Thus, LID and BMI may initiate enlargement of an individual DCN RF by similar DCN disinhibition mechanisms that do not require cortical inputs, while concurrent changes in descending cortical inputs may contribute to constraining magnitudes of these DCN RF enlargements (Fig. 6). From this view, ascending peripheral inputs, intrinsic DCN inhibitory inputs, and descending cortical inputs may jointly influence this rapid DCN plasticity. In this way, DCN plasticity appears partly independent of, and partly linked to, broader system-wide changes, including descending cortical influences. Interestingly, recent studies in humans suggest that local anesthetic blocks of peripheral inputs cause a range of rapid changes in body feelings and body image. Given local anesthetic actions, some of these changes are expected including, for example, loss of sensitivity and dulled feelings to stimuli in the blocked body part. Other sensory effects are, however, surprising and seem paradoxical to block of peripheral inputs. These include erroneous feelings of position or illusions of ‘swelling’ (increases in size and changes in shape) in blocked body parts and improved 2-point discrimination, mislocalization of stimuli, lowered touch thresholds, and ‘swelling’ in unblocked body parts (Gandevia and Phegan, 1999; Isaacson et al., 2000; Paqueron et al., 2003; Bjorkman et al., 2004; Paqueron et al., 2004a; Paqueron et al., 2004b; Weiss et al., 2004; Bjorkman et al., 2005; Turker et al., 2005). Explanations for these unusual sensory effects have usually focused on cortical unmasking due to decreased intracortical inhibition and changes in cortical receptive fields, maps of body parts, and background activity (Gandevia and Phegan, 1999; Paqueron et al., 2004b; Weiss et al., 2004; Turker et al., 2005). Consistent with these explanations, there is evidence that local anesthetic blocks in humans cause rapid somatosensory cortical functional changes (Tinazzi et al., 2003; Weiss et al., 2004). These changes resemble other rapid cortical changes in humans and animals (Merzenich et al., 1983; Kolarik et al., 1994; Buonomano and Merzenich, 1998; Jones, 2000; Myers et al., 2000; Calford, 2002; Kaas, 2000, 2002; Li et al., 2002; Wall et al., 2002). Subcortical changes are also thought to contribute to cortical and sensory effects of peripheral anesthetic block in humans (Turker et al., 2005), but there is little information on these subcortical contributions. Peripheral anesthetic blocks in humans can cause rapid enlargements in thalamic receptive fields (Dostrovsky, 1999), but analogous tests of RF enlargements in the brainstem have not been feasible. Studies of somatosensory evoked potentials (SEPs) in humans indicate that amplitudes of brainstem potentials do not change after local anesthetic block (Tinazzi et al., 1997, 2003). In view of findings of brainstem RF changes in animals, it was suggested that averaged SEPs may not detect RF changes in single or small clusters of brainstem neurons or that brainstem changes may be subtle (Tinazzi et al., 1997, 2003). If the present results apply to humans, it seems possible that cortical changes and related cortical constraint of DCN effects may attenuate brainstem changes. Alternatively, immediate brainstem reactions to peripheral block may be different in humans and animals.

3.3.

81

Technical limitations

The present results should be considered in view of limits of our methods. We examined cortical influences on DCN inhibition with the help of BMI. Other experimental designs, using other agents (e.g., inhibition agonists or antagonists), merit attention. Cortical input disruptions involved large cortical lesions that were designed to remove all direct cortical inputs to the DCN. Effects of more limited or specific cortical disruptions deserve study. We used stimulation approaches for defining RFs that have proven valuable in previous investigations of rapid RF changes in many laboratories (see Experimental procedures) but that entail subjective judgments. We attempted to minimize subjectivity by using blinded procedures; however, other controls and approaches could be used. All results come from tactile RFs defined in ketamine-anesthetized animals. Finally, the present results pertain to RF size. Other properties of functional organization deserve attention.

4.

Experimental procedures

The results were derived from 31 adult Sprague–Dawley rats, prepared in accordance with approved protocols that have been described previously (Wang and Wall, 2005). Atropine (0.05 mg/kg, im) was given to decrease respiratory tract secretion, and a surgical plane of anesthesia sufficient to suppress eyeblink and limb withdrawal reflexes was initiated and maintained with ketamine (80–90 mg/kg) and acepromazine (1.7 mg/kg). The trachea was intubated, and the animal was positioned on a heating pad with the head stabilized in a stereotaxic holder. The DCN were exposed and covered with silicone to preclude desiccation and dampen movement. In tests requiring disruption of cortical inputs, the contralateral cerebral hemisphere was also exposed. BMI was injected via micropipette to disrupt local DCN inhibition (2.5 mM in most cases, range 0.5–5.0 mM; mean volume = 21 nl). The micropipette and recording microelectrode tips were angled to approach each other and were independently advanced following insertion 300 μm or less apart. Micropipettes were attached to a nanoliter syringe with zero dead space, which was driven by a computer controlled delivery system (WPI Micro4). DCN microinjections of vehicle were used to assess nonspecific effects of the microinjection procedures (0.9% saline; mean volume = 17 nl). In many tests, ‘blind’ procedures were used in which the investigator defining RFs did not know if BMI or vehicle was given. Only one test was run in a given gracile and/or cuneate nucleus in each animal. In further tests, DCN microinjection of BMI was followed by a cutaneous microinjection of LID to test the effects of disrupting the DCN inhibition and peripheral inputs of the same RF. LID microinjection was done at a time when BMI effects had reversed to a significant or complete degree. LID was delivered through a microsyringe that was aimed at a randomly selected location in the starting RF (2% LID; mean volume = 7.3 μl). The injections typically resulted in loss of tactile inputs from part but not all the starting RF. Previous

82

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studies indicate that DCN RFs are not changed by similar cutaneous microinjections of saline (Wang and Wall, 2005). Cortical inputs to the DCN of 15 rats were removed by acute cortical lesions that were produced by previously described procedures (Wang and Wall, 2005). Briefly, following exposure of the dorsal and lateral surfaces of the cerebral hemisphere, somatosensory, motor, and much of the adjacent frontal, parietal, and occipital cortex was cauterized and then aspirated to the white matter. All exposed surfaces were covered with gelfoam, silicone, and gauze pads to preclude desiccation. To document lesions, outlines of the cerebral hemispheres and aspirated cortical areas were traced from dorsal view photographs that were made after recording and removal of the brain from the head. Visually apparent aspirated areas were used to summarize extents of cortical lesions. These areas underestimated lesion extents because marginal cauterized cortex was not included. In each case, the lesion spanned all cortical areas known to project to the DCN (Wise and Jones, 1977; Desbois et al., 1999; Martinez-Lorenzana et al., 2001). These lesions were done with the goal of disrupting all direct cortical inputs to the DCN. Previous studies indicate that these lesions, by themselves, cause no or modest acute changes in the sizes of DCN RFs (Wang and Wall, 2005). Upon isolation of a stable RF, tests were begun 67– 490 min after cortical lesion. RF enlargements were seen in tests at earlier and later post-lesion times. DCN recording procedures have been described (Xu and Wall, 1999a; Wang and Wall, 2005). In brief, recordings were made with tungsten microelectrodes (∼1.0 MΩ) that were coupled to amplification, window discrimination, oscilloscope, and audiomonitor stages. Single- or multi-unit responses or, in other tests, paired single- and multi-unit responses were recorded at individual recording sites. Singleunit spikes with 3:1 or greater signal:noise ratio were isolated with a window discriminator and continuously monitored on an oscilloscope to assure that the same unit was followed. Multi-unit responses were taken without individual spike isolation. During recordings, the gracile and cuneate subdivisions of the DCN were localized using visible tubercles, obex, and locations of RFs on respective lower and upper extremities. To further confirm the DCN location of recordings, recording sites were electrolytically marked (about 4 μA for 5 s). To identify marked sites, the brain was subsequently perfused (saline + 4% paraformaldehyde), and the medulla was cut into serial 50-μm-thick transverse sections on a freezing microtome. All sections were stained with cytochrome oxidase and mounted on slides (Xu and Wall, 1999b). Examination of these sections indicated that all 27 successfully marked sites were in the DCN (e.g., Fig. 7). Stimulation procedures to define RFs were similar to those used in recent DCN studies (Wang and Wall, 2005) and provided RF data that could be compared to results from those studies. Hand-held probes and brushes were used to stimulate low threshold cutaneous mechanoreceptors and repeatedly determine RF borders of tactilely responsive DCN neurons at several-minute intervals. An RF was defined as the total area of skin which elicited neuronal discharges to cutaneous stimulation and, under our test conditions, was judged to reflect a maximal tactile RF. It is possible that use of machine-controlled stimuli and multiple response averaging

Fig. 7 – Cytochrome-oxidase-stained transverse sections showing examples of electrolytically marked recording sites (asterisks) in the gracile (top, G) and cuneate (bottom, C) nuclei subdivisions of the DCN. Orientation for both as indicated.

might have permitted detection of additional weakly responsive skin sites; however, such sites would be equally likely detected before or after treatments and, thus, would not systematically affect RF size tests. More important, the time required for stimulation at individual sites would not permit re-testing all borders of an RF at short time intervals, which was a main goal. The stimulation procedures used for defining tactile RF size are similar to those used in many previous studies of rapid changes in somatosensory RF size (Merzenich et al., 1983; Calford and Tweedale, 1991; Pettit and Schwark, 1993; Ergenzinger et al., 1998; Klein et al., 1998; Dostrovsky, 1999; Calford, 2002; Chowdhury and Rasmusson, 2002; Kawamata et al., 2005). Procedures to quantify RF sizes have been described previously (Wang and Wall, 2005). RFs were located on paw skin. Visible landmarks, i.e., different digits and their phalanges, hairy vs. glabrous skin edges, paw pads, and joint lines, were used to chart RF borders on scaled paw diagrams. In all tests, an RF was repeatedly charted before and after treatment. Measures of RF size were made from these charts with a digitizing pad and NIH Image software. Changes in RF size were assessed by expressing the RF area at the time of largest RF change after treatment as a percentage change from the pre-treatment RF area. Changes in RF size were charted using SPSS programs. Data from all individual RFs that were tested

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were charted in scatterplots; in addition, group data were charted as boxplots which showed the interquartile (25–75th percentile) and total ranges of data for the group. Group differences were tested with Mann–Whitney U tests (P < 0.05 was considered significant).

Acknowledgment We very gratefully acknowledge support of this work by National Institutes of Health Grant HD 39791.

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