Rapid shifts in receptive fields of cells in trigeminal subnucleus interpolaris following infraorbital nerve transection in adult rats

Brain Research 779 Ž1998. 136–148

Research report

Rapid shifts in receptive fields of cells in trigeminal subnucleus interpolaris following infraorbital nerve transection in adult rats Bradley G. Klein ) , Carl F. White, Jeanette R. Duffin Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061-0442, USA Accepted 2 September 1997

Abstract Transection of the infraorbital nerve in adult rats results in an array of chronic functional anomalies in trigeminal brainstem subnucleus interpolaris, including changes in normal receptive field organization. This work examined whether long-term maintenance of acute modifications, such as unmasking or strengthening of normally ineffective inputs to interpolaris cells, might contribute to the previously described chronic abnormalities. Using glass micropipettes, extracellular isolation of 37 interpolaris cells, with infraorbital receptive fields, was maintained following intraorbital transection of the infraorbital nerve. Receptive fields and dynamic response properties were characterized immediately before and after the cut and throughout the post-transection isolation period. Orthodromic latencies to trigeminal ganglion shocks and antidromic activation from thalamus or cerebellum were also examined. Of the 37 cells, 21.6% exhibited receptive field shifts to non-infraorbital regions after cutting the infraorbital nerve. Using the normal probability of observing an interpolaris cell with more than one trigeminal division in its receptive field, the probability of observing this shift by chance was 0.0013. No such changes were observed for 12 control cells, recorded for durations equal to or greater than total recording times for the shifting cells, with the nerve intact. The representation of local circuit, thalamic-projecting and cerebellar-projecting cells was similar in the total sample; however, all neurons exhibiting transection-induced receptive field shifts were projection neurons. In comparing the sample of cells that exhibited receptive field shifts with those that did not, prior to infraorbital nerve cut, there was no difference in mean latencies and thresholds for activation from the stimulating electrodes or in mean depth at which the cells were isolated. In addition, no difference was evident in receptive field size, effective receptor surface, dynamic response characteristics or spontaneous activity. These data suggest that maintenance of acute receptive field changes, following infraorbital nerve cut, may contribute to some types of chronic functional alterations observed after such damage. q 1998 Elsevier Science B.V. Keywords: Acute plasticity; Receptive field; Trigeminal; Infraorbital nerve; Subnucleus interpolaris; Extracellular recording

1. Introduction Clinical and experimental studies have documented that damage of peripheral nerves results in a wide variety of abnormalities throughout mammalian somatosensory pathways w36,47,52,59,64x. Understanding the physiological, anatomical and biochemical substrates of this reorganization is essential for ultimately developing clinical strategies for the control of damage-induced sequelae. Toward this end, we and others have examined the chronic functional consequences of infraorbital nerve ŽION. transection for the trigeminal primary afferent pathway w48x, and the post-synaptic targets of these neurons w37,58x within subnucleus interpolaris ŽSpVi. of the mature rat. Many of the )

Corresponding author. Tel.: q1 Ž540. 2317398; Fax: q1 Ž540. 2317367; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 1 0 8 - 6

chronic functional alterations in the primary afferent pathway to SpVi Že.g. abnormal somatotopy, unusual receptive fields, modification of dynamic response characteristics and peripheral receptor relationships. appear to reflect anatomical changes which have been described for these first-order neurons following peripheral transection w1– 3,16,35,42,48–50x. For the cells of SpVi, many of the chronic functional anomalies reported following ION damage can be most easily explained by a simple transfer of abnormal primary afferent response properties. However, some functional anomalies of SpVi cells, such as receptive fields ŽRFs. exhibiting intermodality convergence or some forms of interdivisional convergence w37x, are not adequately explained by existing information on chronic trigeminal primary afferent reorganization. The possibility that alteration of SpVi cell morphology could account for some anomalous response properties

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observed in adults, following neonatal ION damage, has recently been addressed w31x. Following such damage, alteration of SpVi cell response properties is qualitatively similar to that seen following adult lesion w25,37x. Nevertheless, no significant change in cell morphology was observed, casting doubt on whether this would be a viable mechanism for functional change, within SpVi, in rats lesioned as adults. There is evidence that central afferents to the trigeminal brainstem such as those from the posteromedial barrel subfield of somatosensory cortex w33x, the serotonergic raphe nuclei w8,9,23,41x or other trigeminal brainstem subnuclei w18x can modulate response properties in cells of this region. Thus, damage-induced reorganization of these projections in adult rats might also contribute to some of the functional alterations resulting from ION transection. It has been shown that such damage in adult rats does in fact alter the serotonergic afference to SpVi w38,39x, although the contribution of this change to coincident alteration of response properties in SpVi remains to be demonstrated. Another possible substrate for the chronic functional changes in rat SpVi, following ION damage, is the longterm maintenance of acute functional alterations. The unmasking or strengthening of normally ineffective inputs to central neurons have been proposed as a means by which shifts in RFs could occur with a very short latency following peripheral damage or disruption of normal afferent activity. Such rapid alterations of receptive fields in adult mammals have been observed in both forebrain w6,44x and brainstem w13,29,45,65x. In the spinal cord, dynamic changes in receptive field extent or excitability of dorsal horn cells have been demonstrated with a short latency following alteration of normal peripheral input using the chemical irritants mustard oil and capsaicin w56,60x. In the mature rodent trigeminal system, only a small number of studies have examined acute functional changes in central neurons following deafferentation. In the ventral posterior medial nucleus ŽVPM. of the thalamus, removal of afferents originating in the principal sensory nucleus of the trigeminal brainstem nuclear complex did not result in acute expression of novel RFs, even though afferents from SpVi have access to the same cells w7,51x. In the superior colliculus, Jacquin et al. w29x reported the unmasking of new areas of sensitivity within 15 min after cutting the ION or after subcutaneous injection of xylocaine into the original RF. In somatosensory cortex and the trigeminal brainstem nuclear complex, Waite w58x found no change between somatotopic maps made before and 6 h after ION transection, except for a large, unresponsive area between mandibular and ophthalmic territories. However, only gentle mechanical stimulation was used for mapping, and given the normal intra-animal variability in somatotopy, areas near divisional borders were excluded from the sample. For cells of rat trigeminal subnuclei caudalis and oralis, Yu et al. w66x and Hu et al. w24x have shown that modification of normal peripheral input, using the small-

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fiber excitant mustard oil, can induce RF expansion which includes mechanoreceptive fields. In light of the limited and somewhat equivocal nature of the literature regarding acute shifts in RFs of cells in the trigeminal system following removal or alteration of normal afferent information, we re-examined this issue in SpVi of adult rats subjected to ION transection. To attempt to circumvent the problem of intra-animal variability associated with somatotopic mapping, the same well-isolated SpVi cell was recorded immediately before, during and immediately after cutting the ION. In addition, RFs were evaluated using noxious as well as innocuous mechanical stimuli.

2. Materials and methods Forty-nine adult male Sprague-Dawley rats were used. Forty were used to assess the effects of ION cut and nine were used for control recordings. Surgical preparation, physiological maintenance and physiological recording were executed according to methods previously used by Klein w37x, except where noted. Briefly, the animals were anesthetized with sodium pentobarbital and given atropine sulfate. They were then tracheostomized, placed in a stereotaxic apparatus, paralyzed with gallamine triethiodide and artificially respirated. The caudal medulla was exposed, and skull above the left trigeminal ganglion, crus II of the left cerebellum and the right ventroposteromedial thalamus was removed. Bipolar stimulating electrodes were stereotaxically placed within these latter three areas. The ION was initially exposed, prior to recording, using an intra-orbital approach. This was done by retracting the skin over the supraorbital ridge, carefully cutting the underlying connective tissue between the ridge and the dorsal portion of the eye, and gently retracting the eye to expose the ION. Care was taken to avoid damaging major blood vessels and other cranial nerves within the orbit. After exposing the nerve, a microscissors was placed around it, without cutting, to ensure that free access had been achieved. Following this exposure, all retraction was released, and eye and skin were returned to their resting position. This procedure served several purposes. It allowed access to the ION with no disturbance of the normal receptor surfaces served by the ION, and with minimal disturbance of non-infraorbital receptor regions. It also served as a surgical control condition for the post-lesion phase of the experiment. Most important, it allowed access to the ION during the experiment, with minimal mechanical disturbance. This allowed the same neuron to be recorded before and after the nerve cut Žsee below.. Extracellular recordings were made with glass microelectrodes Ž30–70 M V . filled with either 6% horseradish peroxidase ŽHRP. in 0.05 M Tris-buffered 0.3 M KCl or Tris-buffered KCl alone. Neural activity was amplified with a high-voltage electrometer, played over an audio

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monitor and displayed on a storage oscilloscope. Trigeminal ganglion shocks Ž50 ms at 1 Hz. were used as search stimuli for locating brainstem units. SpVi cells were distinguished from fibers on the basis of waveform, latency and following-frequency in response to ganglion shock. Criteria for identifying antidromic activation from cerebellar or thalamic electrodes included invariant response latency, high following-frequency Ž250–500 Hz. or collision of responses elicited from shocking the ganglion and the putative efferent target. RF mapping for individual cells was carried out in a fashion similar to that used in a study of acute collicular RF changes following ION transection in hamster w29x. When a unit in the trigeminal brainstem nuclear complex was located by electrical stimulation of the ganglion, it was determined whether it was a cell or a fiber and its orthodromic or antidromic response latencies were checked from all stimulating electrodes. Initial localization of regions of the receptor surface that activated the cell were done with strong, non-noxious, mechanical stimulation using cotton swabs or calibrated Von Frey-type hairs ŽSemmes–Weinstein pressure anesthesiometer, Stoelting.. This stimulus intensity corresponded to an anesthesiometer value of 8.6 g, which was not painful when applied to the back of the experimenter’s hand, and was considered the upper limit for low-threshold stimulation. If it was insufficient to activate the cell, a noxious pinch with serrated forceps was used in an attempt to locate the RF. Stimulus intensity defined as noxious was based upon subjective reports of pain when the stimulus was applied to the back of the human hand. The cell was considered responsive if it could be activated by a stimulus on at least 50% of the stimulus presentations. Only cells with an infraorbital RF component were studied. After initial localization of the infraorbital RF region a rapid RF assessment was made, followed by two consecutive, more detailed determinations, each taking approximately 15 min to complete. Responsiveness was determined using the mechanical stimuli noted above, all delivered by hand, according to the following procedure. The vibrissae were rapidly stroked with a cotton swab or held down for 3 s, in order to identify a rapidly adapting or slowly adapting response to low-threshold mechanical stimulation. If vibrissa stimulation elicited a response, the stimulus intensity that produced a response, 50% of the time Žminimum effective stimulus intensity., was used to delineate the size of the RF. If low-threshold vibrissa stimulation failed to elicit a response, regions between the vibrissae, as well as other common fur in the infraorbital region, were rapidly stroked or slowly deflected laterally with the tip of a 27-gauge hypodermic needle or a cotton swab. If infraorbital guard hair movement produced a response, the minimum effective stimulus intensity was applied with the hypodermic needle to delineate the RF. If no guard hair response to low-threshold stimulation was identified, the infraorbital skin was rapidly or slowly de-

pressed with the 8.6-g Von Frey-type hair Žstrong nonnoxious mechanical stimulation.. If a response was elicited at this maximal low-threshold intensity, the Von Frey-type hair was used in a non-calibrated fashion to determine the minimum effective stimulus intensity, which was then used to delineate the RF. If no response was elicited by any of the aforementioned low-threshold stimulation, the skin and the vibrissa follicles were rapidly or slowly pinched with the serrated forceps, using the intensity described as painful to the human hand. If this noxious stimulus elicited a response, it was used to delineate the extent of the RF. Cells with low-threshold RFs were also checked for differential responsiveness to noxious stimulation. Heat stimuli were not used for determination of RFs, nor was electrical stimulation of receptor surfaces. Each detailed RF assessment also included a search for non-infraorbital RF components. The same procedure was used as that for delineating infraorbital RFs, beginning with strong, non-noxious mechanical stimulation for initial localization of the responsive region. Non-infraorbital regions of the head were checked, in addition to the neck, shoulders, chest, ears and forelimbs. Intra-oral tissues were not examined. If non-infraorbital responsiveness was observed and if it appeared to be contiguous with the previously defined infraorbital RF Že.g. stimulus spread., the cell was not used in the experiment. Non-infraorbital vibrissae Že.g. supraorbital vibrissae. have been previously shown to be spatially non-contiguous components of infraorbital RFs of some SpVi projection neurons w25,37x. In such cases, the non-infraorbital RF component was subjected to the same scrutiny as the infraorbital component. Cells with significant spontaneous activity, which interfered with accurate RF determinations, were not used in the experiment. RF boundaries were plotted on scaled drawings of the rat head. The waveform of the response was traced from the storage oscilloscope and a full photographic record of response properties was made. Following completion of the last detailed RF analysis, and immediately before cutting the nerve, latency and threshold to stimulating-electrode shocks were determined and waveform and RF were quickly checked. Immediately following intra-orbital transection of the ION a timer was started, and the ability of stimulation of the original RF to activate the cell was tested. In no case was stimulation of the original RF capable of driving the cell following ION cut. The waveform, and orthodromic or antidromic response latencies to ganglion, thalamic and cerebellar shocks were also immediately examined to assess whether the same cell was being recorded. In all cases, attempts to identify new RFs were initiated approximately every 15 min, in the manner described above for RF mapping, until the cell was no longer isolated. When a novel RF was observed, its time of appearance was noted and a photographic record of cell activation, by RF stimulation and by stimulating-electrode Žtrigeminal ganglion, thalamus, cerebellum. shocks, was made.

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To explore the influence of elapsed experimental time upon the probability of detecting novel RFs we carried out a series of long-duration control recordings Žsee Section 3.. Twelve additional cells were recorded from nine rats which were prepared and treated identically to the experimental rats discussed above, except that the ION was not transected during the course of the recordings. In order to determine the probability that the observed number of cells that shifted their RFs was due to chance, the binomial probability distribution was used. ION transection in each animal was considered a single trial with two possible outcomes: the single cell studied in that animal either shifted its RF or it did not. In normal rats, we previously observed that the maximum probability of an SpVi cell having more than one trigeminal division represented in its RF was 6% w37x. To make our analysis as

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conservative as possible we calculated the binomial probability distribution for the appropriate number of trials, assuming that 6% represented the expected probability of finding an additional RF component after cutting the nerve. The distribution was then used to calculate the sum of the probabilities for obtaining a number of RF shifts that were equal to or greater than that observed in our experiment. Mean values for response characteristics were compared using two-tailed independent groups, or paired t tests Ž a s 0.05.. If the assumptions of the test were significantly violated, a non-parametric test was employed ŽWilcoxon signed rank or Mann–Whitney rank sum, respectively.. If mean differences were less than the absolute error of measurement, statistical tests were not used. Although multiple t-tests were used in some cases, increasing the overall a to more than 0.05, this was not a significant

Fig. 1. Responses of a cell which exhibited a shift in RF following ION transection. Pre-lesion responses to orthodromic electrical stimulation from the ganglion ŽA. and antidromic stimulation from the cerebellum ŽB and C. are illustrated. The vibrissae that elicited responses prior to the lesion are circled in the figurine and rapidly adapting responses to light touch are shown in D–F. Post-lesion responses to ganglion ŽG. and cerebellar ŽH and I. stimulation are also shown. Note the similarity, before and after transection, for latency and waveform of the responses. The post-lesion, ophthalmic guard hair field is hatched in the figurine, and light touch of the indicated regions elicited the corresponding responses shown in J and K.

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Table 1 Mean latency Ž"S.D.. and threshold Ž"S.D.. to electrical stimulation, prior to infraorbital nerve cut, and mean changes after transection, for cells that shifted RFs and those that did not Stimulating electrode

Post-cut status

Pre-cut latency Žms.

Post-cut change in latency Žms.

Pre-cut threshold ŽmA.

Post-cut change in threshold ŽmA.

Ganglion

No shift Ž29. Shifted Ž8.

1.06 " 0.16 1.13 " 0.35

0.01 0

0.95 " 0.32 0.73 " 0.27

0.03 0

Cerebellum

No shift Ž9. Shifted Ž6.

0.57 " 0.07 0.68 " 0.31

0.02 0

1.84 " 0.45 2.49 " 1.24

0.05 0.04

Thalamus

No shift Ž10. Shifted Ž2.

1.13 " 0.87 1.10 " 0.71

0.02 0

2.33 " 0.89 1.85 " 0.07

0.05 0

Values for the ganglion are for orthodromic activation and those for cerebellum and thalamus are for antidromic activation. Sample size is shown in parentheses.

factor in interpreting the results since no significant differences were detected with these t-tests. Percentages were compared with the Pearson chi-square test Ž a s 0.05, df s

1 in all cases.. If expected frequency was less than 5, the Yates corrected chi-square statistic was used. Recording-site locations were confirmed by intra- or

Fig. 2. RFs and effective receptor surfaces, before and after ION transection, for all cells that exhibited an RF shift. All reponses were rapidly adapting and elicited by light touch, except in G, where the pre-lesion response was slowly adapting and the post-lesion response required a noxious pinch.

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extracellular ejection of HRP, or by clipping and leaving the non-HRP filled electrode tip within the brainstem Žsee Klein w37x.. If HRP ejection was used, rats were perfused transcardially with salinerheparin followed by buffered aldehydes, and reaction product was visualized using a cobalt-intensified diaminobenzidine reaction Žsee Jacquin et al. w30x.. For clipped electrode tips, rats were perfused with buffered saline followed by 10% formalin. Tissue was then stained with cresyl violet. HRP reaction product or electrode tips were used to assess whether cells were within the boundaries of SpVi, according to previously described criteria Žsee Jacquin et al. w30x; Phelan and Falls w46x..

3. Results A total of 40 cells were recorded both immediately before and immediately after ION transection. For three cells, isolation was lost following nerve transection, before the RF could be fully characterized. These cells were therefore eliminated from the analysis. Of the 37 cells included in the analysis, eight Ž21.6%. exhibited RF shifts to non-infraorbital regions after cutting the ION. The binomial probability of observing a non-infraorbital shift following the cut, by chance, in eight or more of the 37 cells, was 0.0013. Fig. 1 illustrates the receptive fields and electrical responses of one such cell that was activated by vibrissae deflection before ION transection and non-maxillary guard hairs after the cut. Mean latencies and thresholds for activation from the stimulating electrodes, prior to cutting the nerve Žsee Table 1., were compared with their corresponding values immediately following transection. No significant changes in these parameters were observed for cells that shifted RFs or for those that did not, suggesting that isolation of the same cell was maintained through the cut. Furthermore, prior to nerve transection, no significant differences in latency or threshold of activation were detected when cells that eventually shifted RFs were compared with those that did not. This suggested that neither latency or threshold of activation could reliably predict whether a cell would shift its RF following ION transection. In addition, the

Fig. 3. Distribution of total recording times for the control cells and for the cells that shifted or failed to shift their RFs following nerve transection. For the shifting cells, total recording time is comprised of pre-lesion RF mapping duration plus the post-lesion latency for identifying the RF shift. For the non-shifting cells, total recording time is equivalent to the pre-lesion mapping duration plus the post-lesion period during which adequate recording isolation was maintained.

dorsoventral location of cells within the brainstem did not appear to predict lesion-induced RF shifts since there was considerable overlap of the distributions of recording depths for cells that shifted RFs Žmean s 1574 mm, S.D.s 720 mm. and those that did not Žmean s 1736 mm, S.D.s 558 mm.. There was no significant difference between the means of these distributions. The numbers of local circuit Ž n s 10., cerebellar-projecting Ž n s 15. and thalamic-projecting cells Ž n s 12. recorded in the total sample were quite similar. However, analysis of the proportion of cells within a given projection class that exhibited RF shifts after the nerve transection revealed that all of the shifting cells were projection neurons Ž40.0% of cerebellar-projecting, 16.7% of thalamic-projecting, 0% of local circuit.. This suggested that projection status may be a pre-disposing factor for RF shifts after the lesion.

Table 2 RF size, effective receptor surface and dynamic response characteristics, prior to ION transection, for cells that shifted RFs and those that did not Characteristic

Did not shift RF

Shifted RF

Number of vibrissae in RF Žcerebellar-projecting. % Vibrissae-sensitive % Guard hair-sensitive % Rapidly adapting % Slowly adapting % Spontaneously active

4.7 " 4.1 96.6 3.4 79.3 20.7 44.8

3.2 " 2.6 100.0 0 87.5 12.5 50.0

Number of vibrissae in the RF is presented as mean " S.D. The number of thalamic-projecting neurons that shifted their RFs after the cut did not permit a meaningful statistical comparison on this parameter and as noted above, no local circuit neurons exhibited RF shifts.

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Fig. 2 illustrates RF location and the effective receptor surface, before and after ION transection, for those cells that exhibited lesion-induced RF shifts. Prior to transection, all of the cells responded to stimulation of infraorbital vibrissae. Two of the eight cells ŽFig. 2B and E. also included non-mystacial vibrissae in their RFs. Following ION cut, all cells were unresponsive to stimulation of

infraorbital regions but developed novel responses to stimulation of hairy skin or guard hairs in non-maxillary territories. In the two cases where non-mystacial vibrissae were part of the RF prior to transection, these vibrissae continued to elicit responses after the lesion. However, the novel non-maxillary RFs observed after the cut were discontinuous with these vibrissae. Although the number of

Fig. 4. RFs of the four cells which exhibited minor RF expansions during control recordings. All responses were elicited by light touch and were rapidly adapting.

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neurons exhibiting RF shifts was small, there was considerable overlap of pre-lesion RF topography between cells that shifted RFs and those that did not. Thus, RF location, prior to nerve damage, did not appear to be an obvious predictor for a cell shifting its RF following transection. Prior to cutting the nerve, all of the cells illustrated in Fig. 2 exhibited a rapidly adapting response to light touch of the infraorbital vibrissae, except for one cell ŽFig. 2G. where the response was slowly adapting. No differential responsiveness to noxious stimulation was observed for any of these cells. Following nerve transection, only the slowly adapting cell illustrated in Fig. 2G changed its dynamic response character, requiring a noxious pinch for activation and exhibiting a rapidly adapting response. The cells sampled in the study were primarily vibrissae-sensitive and rapidly adapting. In Table 2, the samples of cells that shifted RFs and those that did not are compared with respect to RF size and the representation of effective receptor surface and dynamic response characteristics, prior to ION transection. There was no significant difference between the two groups of neurons with regard to any of these parameters. As noted in Section 2, four pre-lesion RF determinations were made over the course of about 30 min for each of the 37 cells discussed so far. Therefore, prior to reporting our control recordings, it should be noted that for all of these cells no discontinuous novel RFs were observed, compared with the initial RF estimate, by the end of the 30-min pre-lesion mapping period. As with our control recordings discussed below, there was some change in RF size during these pre-operative determinations, contiguous with the initial RF boundaries. However, such changes did not venture across approximate trigeminal divisions. Our control recordings further explored the influence of elapsed experimental time upon the probability of detecting novel RFs. Here, RF mapping was performed with the ION exposed but intact, for periods which well exceeded the 30-min pre-lesion characterization time noted above. The response characteristics of these 12 control cells were quite similar to the pre-lesion response properties of the cells that shifted RFs after cutting the ION. All but one of the 12 cells was activated by light touch of the mystacial vibrissae and all but two exhibited rapidly adapting responses. No differential responsiveness to noxious stimulation was observed. All but three of the cells were projection neurons. As noted above, for rats where the ION was cut, pre-lesion mapping was carried out over a period of approximately 30 min. For the cells which exhibited RF shifts following the lesion, the change was observed between 15 min and 1 h following the cut. Fifteen minutes is the minimum time noted, since this was the time required to complete one detailed mapping. As can be seen in Fig. 3, for all but one of the control cells, recording times were within or greater than the range of total recording times Žpre-lesion RF mappingq post-lesion latency for RF shift.

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for the cells that shifted RFs following ION cut. This was also true for all but two of the cells that failed to shift their RF’s after nerve transection. The mean total recording time for cells exhibiting post-lesion shifts was 57.9 ŽS.D.s 16.0. min, for the control cells it was 78.8 ŽS.D.s 36.8. min and for cells that did not exhibit post-lesion RF shifts it was 76.6 ŽS.D.s 23.2. min. During these control recordings, the only changes observed were minor, contiguous expansions of the original RF boundaries, which can be seen in Fig. 4. In no case did these expansions cross the vicinity of trigeminal inter-divisional boundaries. For the four cells which exhibited expansions, all were projection neurons. Three of the four cells were activated by infraorbital vibrissae deflection and one cell was activated by movement of infraorbital guard hairs. All responses were rapidly adapting. Additional RF components were always the same type of receptor surface Žvibrissae or guard hair. and had the same dynamic response characteristics as the original RF. These results suggest that experimental procedures, other than ION transection, did not play a significant role in facilitating RF shifts. Furthermore, the overlap of the distributions of total recording times for cells that shifted RFs after the lesion and those that failed to shift suggests that failure to detect RF shifts cannot be primarily attributed to differences in recording time between these samples of cells.

4. Discussion In summary, our results indicate that for adult SpVi cells with infraorbital RF regions, cutting the ION can reveal previously ineffective, non-infraorbital RF components within one hour of the transection. Furthermore, of the pre-lesion response properties we examined, efferent projection status appeared to be the most likely predictor of whether a cell would change its RF after ION cut. Before discussing the results, it is appropriate to consider some of the technical aspects of the study which may have influenced the data. As noted above, only hand-held mechanical stimuli were used to identify RFs. Although many previous studies of acute RF changes following altered peripheral input have relied on similar procedures w6,13,29,45x, hand-held stimulation can introduce variability in quantitative aspects of stimulus delivery and may obscure the time-locked nature of a response with respect to stimulus onset. Thus, as noted above Žsee Section 2., it was necessary to eliminate some types of cells from the experiment, prior to nerve transection, in order to reduce the probability of detecting a false-positive RF shift. These were cells with high spontaneous activity or cells with an infraorbital RF that appeared to be activated by stimulus spread from a contiguous non-infraorbital region. Excluding these cells may have resulted in an underestimate of the number of neurons capable of acute RF changes fol-

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lowing nerve transection. Use of more accurate, reproducible, automated stimulation of receptor surfaces, capable of triggering the oscilloscope, would have provided more confidence for the inclusion of these cells. However, given our RF mapping protocol Žsee Section 2., we feel that the use of automated mechanical stimulation would not have altered the primary conclusion of the experiment that was based upon the cells that were included in the analysis. Since the experimenter was aware of the experimental condition of the animals, another possible technical complication was that of experimenter bias. However, the limitation of excluding certain cell groups to reduce erroneous identification of RF shifts, noted above, may have worked to reduce the possibility of experimenter bias by limiting the ambiguity in RF localization and mapping. An additional factor likely to have reduced ambiguity in RF determination, and hence the possibility of experimenter bias, was the multiple RF determinations built into the experimental protocol. The protocol was set up to be as objective as possible in order to circumvent bias problems. However, use of a design where the experimenter would have been unaware of the animal’s treatment condition would have dispensed with this issue in the most effective manner. Since many of our previous studies of normal and lesion-induced RF properties in rat trigeminal brainstem have employed sodium pentobarbital, the same anesthetic regimen was used in the present experiment. Since choice of anesthetic is often cited as responsible for differences in RF properties of populations of neurons, across studies, it was felt that using sodium pentobarbital would permit more consistent comparison among our current and previous data. However, it should be kept in mind that the magnitude of changes observed in the present experiment may have been dependent upon the anesthetic employed, although the direction of such changes would be hard to predict based upon previous reports. The data from control recordings employed in the present experiment argue against changes in level of anesthesia as being responsible for the RF shifts observed after nerve transection. The acute RF shifts observed in the present experiment suggest a short latency physiological substrate that could contribute to some types of chronic RF anomalies observed in SpVi following adult ION transection w37x. For example, a simple acute shift from an infraorbital to a non-infraorbital RF, after ION transection, might appear as an increase in the proportion of non-infraorbital RFs. Furthermore, if the pre-lesion infraorbital RF included a non-infraorbital vibrissa, as has been reported in SpVi of normal rats, an acute post-lesion shift to a different non-infraorbital RF could be interpreted as a discontinuous RF or even as an example of inter-divisional convergence, depending upon the new RF’s location. It should be stressed, however, that the contribution of acute, lesion-induced RF shifts to longer-term alterations of somatosensation are

extremely difficult to interpret given the complex array of anatomical, physiological, biochemical and transcriptional changes that have been demonstrated to occur, at various latencies and at various levels of the neuraxis, following peripheral nerve damage. For example, GAP-43, a growth factor that may play a role in anatomical plasticity of intact or damaged axons after nerve transection or crush, has been shown to increase in peripheral and central processes of damaged axons within as few as 3–4 days following nerve damage w53,61x. Furthermore, peripheral and central sprouting of intact axons can be demonstrated anatomically within about 1 week following deafferentation w12,62,63x. Periodic assessment of RF characteristics of single cells, over the course of the first few days following an acute RF shift, would help to clarify the relevance of such shifts for longer-term RF reorganization. The value of such an analysis would be greatly facilitated by complementary time course studies of anatomical and biochemical changes that occur, within a single brainstem or spinal cord region, following nerve transection. Perhaps evaluation of the contribution of acute RF shifts to longer-term functional reorganization could also be facilitated by using a chronic, reversible insult to the primary afferent pathway. It would be interesting to determine the effect of reversing the insult after chronic reorganization has occurred. The rapid RF shifts in SpVi reported above also support the notion that acute functional alterations at the pontomedullary level may be substrates for rapid changes observed in adult rodents at more rostral levels of the trigeminal neuraxis following alteration of trigeminal primary afference. As noted above, it has been shown that lidocaine injection in the maxillary gum induces rapid spatial and temporal shifts in RFs of cells in the VPM of the thalamus w44x, and rapid RF shifts have also been observed in superior colliculus of hamster following either ION transection or lidocaine injection into the original RF w29x. In VPM, the RF analysis appears to have been restricted to the whiskerpad and therefore, spatial shifts appeared to be restricted to this region. However, in superior colliculus, larger shifts to non-infraorbital regions were observed, similar to those reported in the present experiment. Further support for a caudal substrate for more rostral acute RF changes can be found in a recent abstract where cells in trigeminal brainstem, VPM and primary somatosensory cortex were recorded simultaneously in a single animal during lidocaine injection around the whisker pad. Acute shifts in whiskerpad RFs were reported in all three regions, although analysis was restricted to the whiskerpad and details of the magnitude of the shift were not provided w15x. Acute RF changes in the non-trigeminal somatosensory neuraxis have also been reported at brainstem w13,45,65x, as well as thalamic w43x and cortical levels w6x. However, it should be noted that acute RF shifts at more caudal levels do not always predict similar changes at more rostral levels of a sensory pathway w14,17x and that

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coincident acute alterations at different levels may be qualitatively and quantitatively different. Given the scope of the present experiment, we can only speculate on the possible mechanisms responsible for the RF shifts. Reversible deafferentation studies, using anesthetic injections, suggest a rapid unmasking of normally silent inputs can contribute to such rapid RF shifts in somatosensory cortex of the flying fox, rat VPM thalamus and cat cuneate nucleus w6,44,45x. Several authors have suggested that such silent inputs are actively suppressed in the normal animal in a tonic fashion. Support for this idea has come from iontophoretic studies utilizing antagonists of inhibitory receptors. For example, in cat primary somatosensory cortex and rat barrel cortex, bicuculline interference with the GABA A receptor led to RF expansion w20,40x. In rat spinal cord, strychnine interference with glycine receptors unmasked saphenous inputs to cells in sciatic territory. However, the nature of the tonic drive for the inhibitory neurons is unclear. Calford and Tweedale w6x suggest that spontaneous activity of spinal primary afferents in cat is insufficient to maintain such a tonic drive. Alternatively, Biella and Sotgiu w5x suggest that the modest spontaneous activity of rat spinal primary afferents is sufficient to drive glycine neurons to suppress long-ranging saphenous input to sciatic neurons. Their data suggest that this long-ranging input comes from second-order neurons in saphenous territory. With regard to the trigeminal mechanisms that underlie the RF shifts observed in this experiment, there is previous functional evidence suggesting normally silent inputs to trigeminal brainstem neurons. Hu, Sessle and colleagues w11,21,22,24,54x demonstrated that electrical stimulation of receptor surfaces, topographically distant from the RF mapped with mechanical stimuli, could activate cells in subnuclei caudalis and oralis of rat and cat. They suggested that electrical stimulation may have resulted in a synchrony of activity in afferents not normally activated by natural stimulation. However, it is currently unclear how that long-range silent afference might be affected following nerve damage. There are many influences upon the environment of trigeminal brainstem neurons which may modulate their RF properties. These include central monoaminergic inputs w38,39,41x, cortical afferents w33x, inter-subnuclear projections w18,27x and the presence of GABAergic interneurons w19x. Acute deafferentation effects upon trigeminal brainstem neural activity could be mediated through any of these systems. It is unlikely that the acute deafferentation removed a direct influence of infraorbital primary afferents upon the central territory of primary afferents mediating the novel RF. Bulk labeling studies of rat infraorbital and mandibular nerves have demonstrated little or no overlap of these central projection territories within the trigeminal brainstem w32x. Furthermore, it has been shown that the central terminals of A-beta vibrissa afferents, within the trigeminal brainstem, are tightly clustered, and that at least within the

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principal sensory nucleus, there is little or no overlap between arbor fringes of neighboring whiskers w4x. In addition, in SpVi, the well circumscribed shape and transverse area encompassed by the central terminals of these vibrissae afferents does not differ from primary afferents having different peripheral receptor associations Že.g. nonwhisker facial hair, glabrous skin, hairy skin, guard hair. w55x. However, this does not preclude the possibility that other fiber types within the infraorbital nerve ŽA d and C-fibers. exhibit greater overlap of arbor fringes. It is more likely that the functional interactions responsible for the acute RF shifts, observed in this experiment, are mediated by local circuit interneurons or interneuron networks. For example, structure–function analysis of local circuit neurons, within SpVi has shown that the dendritic fields of non-vibrissae sensitive neurons can span transverse areas greater than 500 mm, and that these dendrites can stray into topographic regions that do not match their primary afferent input w28x. These cells can also have widespread, local axon collaterals which stray outside of their topographically appropriate region. Thus, such neurons could conceivably bridge the structural and functional gap between the central territories of some infraorbital primary afferents and regions containing nonvibrissae sensitive, non-infraorbital cells. SpVi has been shown to contain a large population of homogenously distributed GABAergic cells w19x. If a portion of these GABAergic cells were interneurons that span distinct topographic regions, infraorbital deafferentation could affect inhibitory input to a topographically different area. Furthermore, polysynaptic connections among local circuit interneurons could bridge more extensive inter-topographic regions. Little information is available regarding such circuitry in the trigeminal system. There is also little information regarding the operation of inhibitory glycinergic systems in the trigeminal brainstem, although glycinergic terminals w10x and glycine receptor chloride channels w57x have been shown to exist in the spinal trigeminal nucleus. It should be kept in mind that even if the circuitry exists for establishing suppression of afference to a given area by a topographically distinct region, the limited spontaneous activity of trigeminal primary afferents w34x may pose a problem for a tonic suppression model. Nevertheless, it does seem that suppressive mechanisms should exist within the trigeminal brainstem since many SpVi cells exhibit activation by a single receptor modality, and singular dynamic response properties, even though their dendrites reside in areas which appear to contain primary afferent populations that are heterogeneous with respect to the peripheral receptor surfaces that they serve w28x. If some of the proposed mechanisms noted above are viable, it is still reasonable to ask why only some of the cells sampled in the present experiment exhibit RF shifts. No RF shifts were identified in local circuit neurons and only some of the projection neurons exhibited such changes. It is possible that since rat SpVi projection neu-

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rons generally have a more extensive dendritic field than local circuit neurons w26x, the projection neurons may have a greater probability of contacting neural elements that may be extending away from their topographically appropriate area or bridging topographically distant regions. Some technical aspects of the experiment could be responsible for failure to identify RF shifts for some of the projection neurons. For example, some types of stimuli were not included in the RF mapping protocol, such as radiant heat or chemical irritants. Furthermore, intra-oral stimulation was not used for RF mapping. Thus, in some instances, we may have simply failed to provide an effective, topographically distant stimulus. Finally, it is possible that topographically inappropriate connections provide a safety mechanism to partially compensate for the loss of topographically appropriate primary afference. As such, it might be inefficient, from a design point of view, to have a back-up for every sensory neuron in a given topographic region. Irrespective of the underlying mechanism, the present experiment suggests that the trigeminal system can exhibit acute functional reorganization, within the caudal brainstem, following peripheral nerve damage. This finding adds to an ever increasing body of literature demonstrating the substantial plasticity, not only of the developing nervous system, but of the mature nervous system as well. Clarifying the mechanisms of such plasticity within the trigeminal brainstem should shed light on normal trigeminal brainstem circuitry, while pointing to potential therapeutic interventions for damage-induced somatosensory anomalies. Acknowledgements We would like to thank Mary Nickle for her excellent assistance with animal care, and Drs. R.W. Rhoades and M.F. Jacquin for their advice in the early stages of this project. This work was supported by grant DE 08966 ŽB.G.K.. from the National Institutes of Health. References w1x H. Aldskogius, J. Arvidsson, G. Grant, The reaction of primary sensory neurons to peripheral nerve injury with particular emphasis on transganglionic changes, Brain Res. Rev. 10 Ž1985. 27–46. w2x J. Arvidsson, Transganglionic degeneration in vibrissae innervating primary sensory neurons of the rat: A light and electron microscopic study, J. Comp. Neurol. 249 Ž1986. 392–403. w3x J. Arvidsson, K. Johansson, Changes in the central projection pattern of vibrissae innervating primary sensory neurons after peripheral nerve injury in the rat, Neurosci. Lett. 84 Ž1988. 120–124. w4x A. Baer, P.J. Shortland, D.H. Gutmann, M.F. Jacquin, Spatial relationships of terminals from primary afferent pairs with same- or neighboring-whisker receptive fields, Soc. Neurosci. Abstr. 21 Ž1995. 106. w5x G. Biella, M.L. Sotgiu, Evidence that inhibitory mechanisms mask

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