The Journal of Neurophysiology Vol. 81 No. 2 February 1999, pp. 435-446
Copyright ©1999 by the American Physiological Society

C-Fiber Depletion Alters Response Properties of Neurons in Trigeminal Nucleus Principalis

C. L. Kwan¹, J. A. Demaro², J. W. Hu¹, M. F. Jacquin², and B. J. Sessle¹

¹ Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada; and ² Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Kwan, C. L., J. A. Demaro, J. W. Hu, M. F. Jacquin, and B. J. Sessle. C-fiber depletion alters response properties of neurons in trigeminal nucleus principalis. J. Neurophysiol. 81: 435-446, 1999. The effects of C-fiber depletion induced by neonatal capsaicin treatment on the functional properties of vibrissa-sensitive low-threshold mechanoreceptive (LTM) neurons in the rat trigeminal nucleus principalis were examined in adult rats. Neonatal rats were injected either with capsaicin or its vehicle within 48 h of birth. The depletion of unmyelinated afferents was confirmed by the significant decrease in plasma extravasation of Evan's blue dye induced in the hindlimb skin of capsaicin-treated rats by cutaneous application of mustard oil and by the significant decrease of unmyelinated fibers in both the sciatic and infraorbital nerves. The mechanoreceptive field (RF) and response properties of 31 vibrissa-sensitive neurons in capsaicin-treated rats were compared with those of 32 vibrissa-sensitive neurons in control (untreated or vehicle-treated) rats. The use of electronically controlled mechanical stimuli allowed quantitative analysis of response properties of vibrissa-sensitive neurons; these included the number of center- and surround-RF vibrissae within the RF (i.e., those vibrissae which when stimulated elicited 1 and <1 action potential per stimulus, respectively), the response magnitude and latency, and the selectivity of responses to stimulation of vibrissae in different directions with emphasis on combining both the response magnitude and direction of vibrissal deflection in a vector analysis. Neonatal capsaicin treatment was associated with significant increases in the total number of vibrissae, in the number of center-RF vibrissae per neuronal RF, and in the percentage of vibrissa-sensitive neurons that also responded to stimulation of other types of orofacial tissues. Compared with control rats, capsaicin-treated rats showed significant increases in the response magnitude to stimulation of surround-RF vibrissae as well as in response latency variability to stimulation of both center- and surround-RF vibrissae. C-fiber depletion also significantly altered the directional selectivity of responses to stimulation of vibrissae. For neurons with multiple center-RF vibrissae, the proportion of center-RF vibrissae with net vector responses oriented toward the same quadrant was significantly less in capsaicin-treated compared with control rats. These changes in the functional properties of principalis vibrissa-sensitive neurons associated with marked depletion of C-fiber afferents are consistent with similarly induced alterations in LTM neurons studied at other levels of the rodent somatosensory system, and indeed may contribute to alterations previously described in the somatosensory cortex of adult rodents. Furthermore, these results provide additional support to the view that C fibers may have an important role in shaping the functional properties of LTM neurons in central somatosensory pathways.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Systemic application of capsaicin to neonatal rodents predominately depletes their C-fiber primary afferents and also results in a wide variety of changes observable in adulthood (for review, see Buck and Burks 1986; Fitzgerald 1983; Holzer 1991). In the spinal somatosensory system of capsaicin-treated rodents for example, there is a considerable reduction of unmyelinated dorsal root fibers, small-diameter dorsal root ganglion cells, and central terminals of unmyelinated fibers in the superficial laminae of the spinal dorsal horn, as well as in a wide spectrum of neurochemicals associated with C-fiber afferents (e.g., Arvidsson and Ygge 1986; Nagy and Hunt 1983; Shortland et al. 1990). Also, capsaicin-treated rodents may exhibit impaired nociceptive responses to noxious thermal, mechanical, or chemical stimuli (e.g., Kim et al. 1995; Nagy and van der Kooy 1983; Ren et al. 1994) and a marked reduction in the neurogenic inflammation evoked by the application of the C-fiber excitant and inflammatory irritant mustard oil (Gamse et al. 1980).

In the adult rodent spinal and trigeminal (V) somatosensory systems, mechanoreceptive field (RF) enlargement and altered response properties of second- and higher-order low-threshold mechanoreceptive (LTM) neurons also have been reported after C-fiber depletion by neonatal capsaicin treatment (e.g., Kwan et al. 1996; McMahon and Wall 1983; Nussbaumer and Wall 1985; Wall et al. 1982). For example, RF enlargement of vibrissa-sensitive neurons has been reported at various levels of the "barrel" neuraxis including neurons in the somatosensory barrel cortex (Nussbaumer and Wall 1985; Wall et al. 1982) and nucleus principalis of the V brain stem sensory nucleus complex (Kwan et al. 1996). However, only limited information on the RF properties of vibrissa-sensitive neurons can be obtained with the manual methods used for stimulation of vibrissae in these earlier studies. Standardized electronically controlled mechanical stimulation is needed to provide objective assessment of RF size and a quantitative evaluation of the response properties of vibrissa-sensitive neurons (Armstrong-James and Fox 1987; Simons 1983). Therefore the aim of this study was to use such an approach to provide a quantitative evaluation of the effects of neonatal capsaicin treatment on the RF and response properties of principalis vibrissa-sensitive LTM neurons in adult rats in which there was documented evidence of marked depletion of C fibers.

Some of the data have been briefly reported in abstract form (Kwan et al. 1995; Sessle et al. 1995).

	METHODS

Abstract Introduction Methods Results Discussion References

Neonatal capsaicin treatment

The neonatal capsaicin treatment procedure has been described in detail in our previous study (Kwan et al. 1996). The capsaicin injection solution consisted of a solution of 20% capsaicin in ethanol dissolved with Tween 80 and 0.9% saline in a ratio of 1:1:8 by volume, respectively, while the vehicle solution consisted only of ethanol, Tween 80, and saline. Briefly, neonatal rats were immobilized on a bed of ice and then given a single subcutaneous injection of either capsaicin (50 mg/kg, n = 18) or its vehicle (n = 6) within 48 h of birth. After the injection procedure, neonates then were allowed to recover on a warm heating pad and only those showing full recovery from the injection procedure were returned to their mother.

The effectiveness of neonatal capsaicin treatment in reducing the number of C-fiber afferents in each rat was verified by two approaches. The first involved spectrophotometric analysis of the plasma extravasation of Evan's blue dye induced by the topical application of the C-fiber excitant and inflammatory irritant mustard oil (Haas et al. 1992; Jancso et al. 1967; Reeh et al. 1986). Briefly, after each recording experiment, a 1% Evan's blue dye solution (20 mg/kg iv) was infused into the rat and ~0.1 ml of 20% mustard oil was applied to the skin of the left hindleg 2 min later. The skin area of mustard oil application and a skin area of comparable size and location on the contralateral hindleg were excised after 20 min and frozen at 20°C for subsequent colorimetric analysis. Extraction of Evan's blue dye was adapted from the method described by Gamse et al. (1980). The Evan's blue dye in skin samples was extracted with 1 ml of formamide for 24 h at 60°C for colorimetric determination on a spectrophotometer at 620 nm. The amount of dye within a given sample was extrapolated from a standard curve constructed from varying concentrations of Evan's blue dye. The net amount of dye extravasation induced by mustard oil was quantified as the amount of dye extracted from the skin sample to which mustard oil had been applied that exceeded the amount of dye extracted from the contralateral skin sample. The magnitude of plasma extravasation in capsaicin-treated and control rats was expressed as the net amount of dye extravasated per gram of tissue.

The efficacy of neonatal capsaicin treatment in depleting C-fiber afferents also was assessed by electronmicroscopic examination of the infraorbital (IO) and sciatic (SC) nerves of four control rats and five capsaicin-treated rats. After the conclusion of the electrophysiological recording experiment and plasma extravasation procedure, these rats were perfused transcardially with 250 ml of 0.1 M phosphate-buffered saline (pH 7.3) followed by 250 ml of fixative [1% paraformaldehyde, 2% glutaraldehyde, 4% sucrose in 0.1 M phosphate buffer (pH 7.3)]. A 4- to 5-mm section of the left SC nerve 5 mm above the popliteal fossa and a 4- to 5-mm section of the left IO nerve within the IO canal then were excised and postfixed in the same fixative for 24 h. In two control and two capsaicin-treated rats, the nerve branches supplying the vibrissa follicles also were dissected for subsequent electronmicroscopic analysis. Nerve samples were prepared for electronmicroscopic analysis in accordance with the methods of Golden et al. (1993). Briefly, nerve samples were fixed in osmium, dehydrated, and resin embedded. Thin (1 µm) transverse sections were cut and placed on copper grids, stained with lead citrate and uranyl acetate, and viewed with a JEOL 100CX electron microscope. The fiber spectra of each nerve were analyzed from photographic montages made of the nerve in its entire transverse extent at a magnification of ×4,500. A single observer, blinded as to the identity of each sample, counted the nerve fiber composition of each nerve.

To investigate possible morphological changes in the principalis neuropil of capsaicin-treated rats, three capsaicin-treated rats also were prepared for cytochrome oxidase staining procedures with methods identical to those of Jacquin et al. (1993). Transverse (100 µm) sections were taken from principalis and all levels of the trigeminal spinal tract nucleus and processed according to the methods of Wong-Riley (1979). The sections from capsaicin-treated rats were photographed under ×20 objectives and qualitatively compared with analogous sections from normal rats described a previous study (Jacquin et al. 1993).

Electrophysiological experiments

All procedures related to animal preparation, recording of V brain stem neuronal activity, and manual stimulation of the orofacial region have been described previously in detail (e.g., Dallel et al. 1990; Hu et al. 1986; Kwan et al. 1993). Briefly, on the day of the electrophysiological experiment, each animal was anesthetized (alpha-chloralose, 50 mg/kg and urethan, 1 g/kg ip) and heart rate, respiration rate, expired percentage CO₂, and rectal temperature were monitored continuously and maintained within normal physiological ranges. Supplementary doses of anesthetic (25% of induction dose iv) were administered as required. Standardized procedures were used in the electrophysiological study of each animal. The brain stem was exposed, and an epoxy resin-coated tungsten microelectrode (5-20 M at 1,000 Hz) was lowered stereotaxically into the region of the left nucleus principalis (5.1-5.5 mm rostral to obex level, 2.7-3.0 mm lateral to midline) to record the extracellular activity of single neurons. Electrolytic lesions were placed in selected microelectrode penetrations in each rat for subsequent histological confirmation and reconstruction of penetrations and loci of recorded neurons (Hu et al. 1986).

The techniques used to display neuronal activity and ensure neuronal isolation have been described previously (Hu et al. 1986). Manual mechanical stimuli (applied by a camel hair brush, blunt metal probe, or serrated forceps) were delivered routinely to search for activity evoked from the orofacial region as the microelectrode was lowered into the nucleus principalis. Once the activity of a vibrissa-sensitive neuron was encountered, the presence and rate of any spontaneous activity was determined immediately; however, none of the vibrissa-sensitive neurons encountered and studied with electronically controlled mechanical stimuli were spontaneously active. Manual mechanical stimuli were further applied to determine if stimulation of other types of peripheral tissues (e.g., nonsinus hair, glabrous skin/mucosa, subcutaneous structures such as joint or muscle) could activate the vibrissa-sensitive neuron and to determine provisionally if the evoked neuronal response was slowly adapting (SA) or rapidly adapting (RA) (also see Data analysis). The number of vibrissae comprising the RF was assessed provisionally by the insertion of each vibrissa into a modified 27-gauge needle placed at a distance of 5-6 mm from the base of the vibrissa and deflected manually 1 mm in all four cardinal directions (rostral, caudal, dorsal, and ventral). Electrical bipolar stimuli (0.05-2.0 mA, 0.1-0.2 ms, 1-150 Hz) also were applied within the neuron's delineated RF to determine the minimum response latency and verify that the neuron studied was a second-order neuron (Hu et al. 1981).

Vibrissa stimulation and neuronal data acquisition

Each vibrissa-sensitive neuron was studied with mechanical stimuli delivered electronically by a piezoelectric mechanical stimulator (Simons 1983). Briefly, those vibrissae that evoked a neuronal response when stimulated by manual mechanical deflection (see previous section) and nearby surrounding vibrissae were stimulated by the electronically controlled mechanical stimulator. Each vibrissa was inserted into the stimulator placed at a distance of 4-5 mm from the base of the vibrissa and then deflected (amplitude of 250-400 µm and 200-ms duration) for 30 trials in each of four cardinal directions (rostral, caudal, dorsal, and ventral) with stimuli delivered at 1 Hz. Also, the deflection amplitude chosen for the stimulation of each vibrissa was the maximum amplitude that would move only the vibrissa and no other nearby structures when viewed under a magnifying glass. Action potentials evoked by each stimulus were digitally stored with the use of an IBM-clone computer connected to a CED-1401 interface (Cambridge Electronic Design, Cambridge, UK).

Data analysis

The use of manual mechanical stimuli to different orofacial regions permitted the percentage of vibrissa-sensitive neurons responsive to stimulation of other types of peripheral tissues to be determined. The use of electronically controlled mechanical stimulation of vibrissae allowed for the quantitative analysis of the RF and response properties of vibrissa-sensitive neurons. Neuronal responses evoked during the peristimulus time interval (50-50 ms, 1-ms binwidth) from each series of 30 trials were analyzed in a cumulative peristimulus time histogram (PSTH). In accordance with the criteria used by Armstrong-James and Fox (1987), a response was considered to occur if at least three action potentials occurred within the same 1-ms bin in the poststimulus time interval of 3-50 ms over the 30 trials. Data from each PSTH were used to determine the magnitude and the modal latency of responses evoked by deflection of a given vibrissa. The neuronal response magnitude was expressed as the total number of action potentials in the poststimulus time interval of 3-50 ms minus the total number of action potentials in the prestimulus time interval of 0 to 50 ms (i.e., resting activity) divided by the 30 trials of the PSTH. The modal response latency represented the latency of occurrence of the most action potentials over the 30 trials of the PSTH. The latency variability of neuronal response evoked by deflection of individual vibrissa was calculated as the latency of the last response minus the latency of the first response within the poststimulus time interval of 3-50 ms. Each vibrissa within the neuronal RF was classified as being either a center-RF vibrissa or a surround-RF vibrissa depending on the neuronal response magnitude and the modal response latency observed on the PSTH; stimulation of a center-RF vibrissa would elicit a response magnitude of 1 with a clear modal response latency of 10 ms, whereas stimulation of a surround-RF vibrissa would elicit a response magnitude of <1. The term total vibrissal RF refers to the number of center-RF vibrissa(e) plus surround-RF vibrissae.

Other RF properties studied included directional selectivity, and adaptation. The neuronal responses evoked by stimulation of a vibrissa were considered to be very selective if only one of the four directions of stimulation produced a response (see preceding section), moderately selective if two or three of the four directions of stimulation produced a response, and nonselective if all four directions of stimulation produced a response.

The directionality of vibrissa-evoked neuronal responses was investigated further with the use of a vector analysis. This vector analysis combined the properties of neuronal response magnitude and the direction of vibrissal deflection into one measure (i.e., a vector). The neuronal responses elicited by deflection of a vibrissa in each of the four cardinal directions then could be represented as vectors on a polar plot, and these subsequently were summed to produce a single net vector for that individual vibrissa that had a net response magnitude and pointed toward a single direction in space.

The adaptation characteristic of each vibrissa-sensitive neuron was assessed by deflection of the center-RF vibrissa in the direction which elicited the maximal response of the neuron. The neuron was considered to be SA if a response could be elicited over the entire stimulus duration of 200 ms and RA if only a phasic (within the 1st 50 ms after stimulation) response could be elicited.

All statistical comparisons were a priori. ² tests were used to compare capsaicin-treated and control rats in the proportions of RA, SA, and spontaneously active vibrissa-sensitive neurons and vibrissa-sensitive neurons that were responsive to stimulation of other types of peripheral tissues. Student's t-tests or Mann-Whitney U tests were used to compare both groups of rats with respect to the number of center- and surround-RF vibrissae per neuronal RF, the number of vibrissal rows per neuronal RF, and the maximal length of the vibrissal row (i.e., the number of vibrissae within the longest row of vibrissae, stimulation of which was effective in eliciting a response from a neuron). Also, the neuronal response magnitude, the response latency variability, the modal response latency, and the directional selectivity of the response evoked by the deflection of center-RF vibrissae were compared with analogous data obtained with deflection of surround-RF vibrissae in capsaicin-treated and control rats; data of these four variables also were compared between capsaicin-treated and control rats. For the analysis of vector data, only those neuronal RFs that had more than one vibrissae were compared between both groups of rats. Either Student's t-tests or Mann-Whitney U tests were used to compare both groups in the proportions of center-RF vibrissae, surround-RF vibrissae, and total (center- and surround-RF) vibrissae with net vectors oriented toward the same quadrant within the neuronal RF.

The Evan's blue dye concentration data from capsaicin-treated and control rats were analyzed with a two-way analysis of variance (ANOVA) that used the type of rat (control vs. capsaicin treated) and laterality (ipsilateral vs. contralateral) as the two factors for analysis. Post hoc Bonferroni's t-tests were used to compare, between capsaicin-treated and control rats, the concentrations of Evan's blue dye in either the contralateral or ipsilateral skin samples and to compare the concentrations of Evan's blue dye in ipsilateral and contralateral skin samples of either capsaicin-treated or control rats. A two-way ANOVA also was used to analyze the fiber counts within the IO and SC nerves of capsaicin-treated and control rats. Fiber counts from the IO and SC nerves were analyzed in separate ANOVAs with the type of rat (control vs. capsaicin treated) and the type of fiber (myelinated vs. unmyelinated) as the two factors for analysis. Post hoc Bonferroni's t-tests were used to compare the number of myelinated or unmyelinated nerve fibers in the sciatic or infraorbital nerves between capsaicin-treated and control rats. In addition, Student's t-tests were used to compare the number of myelinated and unmyelinated fibers between the SC and IO nerves within control and capsaicin-treated groups of rats.

	RESULTS

Abstract Introduction Methods Results Discussion References

The study was based on data obtained from 38 male Sprague-Dawley rats weighing 250-375 g. The RF and response properties of principalis vibrissa-sensitive neurons were studied in 18 adult rats, which were treated neonatally with capsaicin, and were compared with analogous data obtained from 20 control rats (14 untreated and 6 vehicle-treated rats). Because no significant difference was noted in the extravasation induced by topical application of mustard oil or in the RF and response properties between untreated and vehicle-treated rats, the data from these rats were pooled as control data.

A total of 63 vibrissa-sensitive neurons were electrophysiologically studied from histologically confirmed locations within the nucleus principalis (Fig. 1). Thirty-one of these vibrissa-sensitive neurons were studied in the capsaicin-treated group of rats while the control group consisted of 32 vibrissa-sensitive neurons (21 from untreated rats and 11 from vehicle-treated rats). Data from vibrissa-sensitive neurons of untreated rats and vehicle-treated rats were pooled as control data (see METHODS).

View larger version (12K): [in this window] [in a new window]   FIG. 1. Histological reconstruction of the location of vibrissa-sensitive neurons within the nucleus principalis of control (A, CON) and capsaicin-treated (B, CAP) rats. Vr, trigeminal sensory root; Vmo, trigeminal motor nucleus; VIIr, root of the facial nerve.

Effectiveness of neonatal capsaicin treatment

Spectrophotometric analysis of the Evan's blue dye extracted from the hindlimb skin of capsaicin-treated and control rats revealed that the Evan's blue extravasation induced by topical application of mustard oil to capsaicin-treated rats was significantly (P < 0.05) less than that in control rats (also see Table 1). The concentration of Evan's blue dye in the ipsilateral hindlimb skin of control rats (mean ± SE, 60.7 ± 6.1 µg/g, n = 20) was more than double that in capsaicin-treated rats (27.8 ± 2.7 µg/g, n = 18). The concentrations of dye in contralateral skin samples of control rats (12.5 ± 1.1 µg/g, n = 20) and capsaicin-treated rats (15.1 ± 1.6 µg/g, n = 18) were not significantly different. However, for both capsaicin-treated and control rats, the amount of dye in the ipsilateral hindlimb skin samples was significantly (P < 0.05) higher compared with that in the contralateral hindlimb skin.

An analysis of the fiber spectra of IO and SC nerves from capsaicin-treated and control rats confirmed the efficacy of neonatal capsaicin treatment. In control rats, the proportion of myelinated:unmyelinated fibers in the IO nerve was ~2:1, whereas in the SC nerve there was a lower proportion (almost 1:1) of myelinated:unmyelinated fibers (Table 1). In capsaicin-treated rats by comparison, the proportion of myelinated:unmyelinated fibers was markedly increased in both nerves (Table 1). The IO and SC nerves of capsaicin-treated rats contained significantly (P < 0.05) fewer unmyelinated fibers (Fig. 2 and Table 1), amounting to ~70% reduction in unmyelinated fibers. There was no significant difference between capsaicin-treated and control rats in the number of myelinated fibers in the IO and SC nerves (Fig. 2 and Table 1). Individual deep vibrissal nerves of capsaicin-treated rats also had fewer unmyelinated fibers than control (Fig. 2), and the proportion of myelinated:unmyelinated fibers averaged 10:1 and 6:1 in capsaicin-treated and control rats, respectively.

View larger version (134K): [in this window] [in a new window]   FIG. 2. Photomicrographs of infraorbital (A and B), sciatic (C and D), and deep vibrissal (E and F) nerves from a control rat (left) and a capsaicin-treated rat (right). Scale bar = 1 µm.

The results of cytochrome oxidase staining in transverse sections through principalis of the three capsaicin-treated rats revealed a topographic and patterned array of patches in its ventral portions (barrelettes) that reiterated the pattern of vibrissae on the face (Fig. 3). Compared with staining patterns of normal rats described in Jacquin et al. (1993), no staining differences were noted in the capsaicin-treated rats. Furthermore analysis of staining patterns in similarly prepared sections through spinal V subnuclei also indicated normal topographic representation of barrelettes (Fig. 3).

View larger version (159K): [in this window] [in a new window]   FIG. 3. Brightfield photomicrographs of transverse sections stained for cytochrome oxidase histochemistry from principalis (PrV), subnucleus oralis (SpVo), subnucleus interpolaris (SpVi), and subnucleus caudalis (SpVc) of a capsaicin-treated rat. Where visible, rows of patchy staining that represent the rows of vibrisse on the face are indicated in the adjacent trigeminal tract by letters A-E, with the A row being the most dorsal vibrissal row and the E row being the most ventral vibrissal row. Note that a vibrissae-related staining pattern is most prominent in PrV and that no pattern was observed in SpVo. Note also that the row pattern is oriented mediolaterally in PrV, whereas in SpVi and SpVc the rows are oriented dorsoventrally.

RF features of vibrissa-sensitive neurons

The sampling distribution of vibrissae within the RF of vibrissa-sensitive neurons was similar between control and capsaicin-treated rats. The percentage of vibrissae located in dorsal (A-C rows) and ventral (D-F rows) halves of the vibrissal pad were 35.9 and 64.1%, respectively, in control rats and 46.9 and 53.1%, respectively, in capsaicin-treated rats. The percentage of vibrissae located in the caudal (straddler column to column 3) and rostral (columns 4 to 7) halves of the vibrissal pad were 58.1 and 41.9%, respectively, in control rats and 55.4 and 44.6%, respectively, in capsaicin-treated rats. Furthermore, an examination of the distribution of one-, two- or three-vibrissae neuronal RFs within the vibrissal pad revealed no tendencies for RFs with a particular number of vibrissae to be located preferentially in a specific region of the vibrissal pad in control or capsaicin-treated rats.

Each vibrissa within the neuronal RF was classified as either a center- or surround-RF vibrissa on the basis of the response magnitude and modal latency to deflection with the electronically controlled mechanical stimuli (see METHODS). The RF was significantly larger in capsaicin-treated rats than in control rats. There was a significant (P < 0.01) increase in the mean number of center-RF vibrissae per neuronal RF (2.8 ± 0.3, n = 31 in capsaicin-treated and 1.5 ± 0.2, n = 32 in control) and a significant (P < 0.05) increase in the total number of vibrissae comprising the RF (Figs. 4-6).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Example of a nucleus principalis vibrissa-sensitive neuron of a vehicle-treated rat studied with electronically controlled mechanical stimuli. A: response [(presented as cumulative peristimulus histograms (PSTHs)] evoked by the deflection of each vibrissa in a given direction. Small vertical line on the abscissa of each PSTH indicates the onset of the mechanical stimulus. B: computer-processed record of the neuron's response (top) to deflection of a vibrissa with electronically controlled mechanical stimulus (bottom). C: figurine depicting the location of each vibrissa that evoked a response from the neuron.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Example of a nucleus principalis vibrissa-sensitive neuron of a capsaicin-treated rat studied with electronically controlled mechanical stimuli. A: response (presented as cumulative PSTHs) evoked by the deflection of each vibrissa in a given direction. Small vertical line on the abscissa of each PSTH indicates the onset of the mechanical stimulus. B: computer-processed record of the neuron's response (top) to deflection of a vibrissa with electronically controlled mechanical stimulus (bottom). C: figurine depicting the location of each vibrissa that evoked a response from the neuron.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Capsaicin-induced changes in nucleus principalis vibrissa-sensitive neurons studied with electronically controlled mechanical stimuli. Mann-Whitney U tests were performed to compare the number of center-mechanoreceptive field (RF) vibrissae, surround-RF vibrissae and total vibrissal RF between control (CON) and capsaicin-treated (CAP) rats.

The increase in vibrissal RF size in capsaicin-treated rats was also evident from data revealing expansions across vibrissal rows as well as vibrissal columns. Compared with control rats, capsaicin-treated rats had a significantly (P < 0.01) greater mean number of vibrissal rows per neuronal RF (2.26 ± 0.14, n = 31, vs. 1.69 ± 0.12, n = 32) and a significantly (P < 0.05) longer maximal length of the vibrissal row (3.06 ± 0.24, n = 31, vs. 2.38 ± 0.17, n = 32). Furthermore, capsaicin-treated rats had a significantly (P < 0.001) higher percentage of vibrissa-sensitive neurons that were responsive to stimulation of other types of nearby peripheral tissues (29.0%, n = 31, vs. 3.1%, n = 32); these neurons had larger cutaneous RF areas (130.9 ± 28.9 mm², n = 9 vs. 16.0 mm², n = 1) and, in addition to vibrissae, were usually also responsive to stimulation of down hairs, guard hairs, or subcutaneous tissues.

Response properties of vibrissa-sensitive neurons

Although the present study primarily investigated the neuronal response to the onset of vibrissal deflection, a substantial proportion of neurons had responses related to the offset of vibrissal deflection. A high percentage of neurons had OFF responses to deflection of center-RF vibrissae in control (86.7 ± 6.0%) and capsaicin-treated (95.0 ± 4.2%) rats. Meanwhile, approximately half of the neurons had OFF responses to deflection of surround-RF vibrissae in control (47.4 ± 9.5%) and capsaicin-treated (56.9 ± 7.9%) rats. Compared with control rats, there were no significant differences in the percentage of neurons with OFF responses to deflection of either center- or surround-RF vibrissae in capsaicin-treated rats.

Another variable investigated by electronically controlled mechanical stimulation of vibrissae was the directional selectivity of responses of vibrissa-sensitive neurons. The directional selectivity of neuronal responses evoked by stimulation of a given vibrissa was established to be very selective if only one of the four directions of stimulation produced a response (see METHODS), moderately selective if two or three of the four directions of stimulation produced a response, and nonselective if all four directions of stimulation produced a response. Compared with control rats, neurons in capsaicin-treated rats showed significantly (P < 0.01) higher numbers of center- and surround-RF vibrissae from which nonselective responses were evoked (Fig. 7). There were no significant differences between neurons of control and capsaicin-treated rats in the number of center- and surround-RF vibrissae that evoked moderately or very selective responses (Fig. 7). Compared with surround-RF vibrissae, the number of center-RF vibrissae that evoked very selective and moderately selective responses was significantly (P < 0.01) lower and the number of center-RF vibrissae that evoked nonselective responses significantly higher (P < 0.01) in neurons of both control and capsaicin-treated rats (not shown on Fig. 7). An analysis of the distribution of vibrissae from which very selective responses could be evoked revealed a tendency in control and capsaicin-treated rats for vibrissae that evoked responses only when they were deflected in the ventral direction to be preferentially located in the ventral rows (i.e., D and E rows) of vibrissae. There was no tendency for vibrissae evoking very selective responses to deflection in the remaining three directions or for vibrissae evoking moderately or nonselective responses to be located in a specific region of the vibrissal pad.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Capsaicin-induced changes in directional selectivity of nucleus principalis vibrissa-sensitive neurons studied with electronically controlled mechanical stimuli. Mann-Whitney U tests were performed to compare the number of center- and surround-RF vibrissae, which when stimulated evoked very selective, moderately selective, and nonselective responses between control (CON) and capsaicin-treated (CAP) rats.

Results of a vector analysis of neuronal RFs with more than one vibrissae revealed that capsaicin treatment was associated with changes in the neuron's ability to define a point in space. Neurons of capsaicin-treated rats had a significantly (P < 0.05) lower proportion (60.5 ± 6.7% in control vs. 47.5 ± 4.5% in capsaicin-treated) of vibrissae with net vectors oriented toward the same quadrant compared with that in control rats.

Analysis of PSTHs enabled the quantitative comparison between control and capsaicin-treated rats of the magnitude and latency of responses evoked by deflection of vibrissae within the neuronal RF. The magnitude of responses evoked by deflection of surround-RF vibrissae in capsaicin-treated rats was significantly (P < 0.05) higher than that in control rats (Fig. 8A). However, there was no difference between control and capsaicin-treated rats in the response magnitude related to deflection of center-RF vibrissae (Fig. 8A). For both control and capsaicin-treated rats, the response magnitude related to deflection of center-RF vibrissae was significantly (P < 0.01) higher than that to deflection of surround-RF vibrissae (not shown in Fig. 8A).

View larger version (20K): [in this window] [in a new window]   FIG. 8. Capsaicin-induced changes in response magnitude and response latency variability of nucleus principalis vibrissa-sensitive neurons studied with electronically controlled mechanical stimuli. Mann-Whitney U tests were performed to compare the response magnitude to deflection of center- and surround-RF vibrissae between control (CON) and capsaicin-treated (CAP) rats (A) and the response latency variability to deflection of center- and surround-RF vibrissae between control and capsaicin-treated rats (B).

Another characteristic of the response magnitude that may be affected by capsaicin treatment is that of temporal summation of responses to repetitive stimuli. A qualitative evaluation of the data revealed that the control group did not have any neurons that exhibited changes in response magnitude with repetitive vibrissal deflection, whereas in the capsaicin-treated group, there were two neurons that exhibited an increase in response magnitude to repetitive stimuli; however, this difference between the two groups was not significant.

There was no significant difference between control and capsaicin-treated rats in the modal response latency related to deflection of center- or surround-RF vibrissae. For both control and capsaicin-treated rats, the modal latency related to deflection of center-RF vibrissae (control, 8.4 ± 0.4 ms, n = 173; capsaicin-treated, 9.1 ± 0.3 ms, n = 348) was significantly (P < 0.01) shorter than that related to deflection of surround-RF vibrissae (control, 10.9 ± 0.5 ms, n = 144; capsaicin-treated, 10.7 ± 0.4 ms, n = 205).

The neuronal response latency to deflection of a vibrissae also was analyzed with respect to its variability, which was calculated as the difference in time between the latency of the first and last action potentials within the post stimulus time interval of 3-50 ms. The response latency variability related to deflection of center- and surround-RF vibrissae was significantly (P < 0.05) longer in capsaicin-treated rats compared with control rats (Fig. 8B). In both control and capsaicin-treated rats, the response latency variability to deflection of center-RF vibrissae was significantly (P < 0.01) longer than that to deflection of surround-RF vibrissae (not shown in Fig. 8B).

The shortest latency of responses to electrical stimulation within the delineated RF of vibrissa-sensitive neurons was not significantly different between control and capsaicin-treated rats (1.1 ± 0.1 ms, n = 32, and 1.3 ± 0.1 ms, n = 31, respectively). There were also no significant differences between control and capsaicin-treated rats in the percentages of RA (79.9 vs. 84.0%) and SA (20.1 vs. 16.0%) vibrissa-sensitive neurons.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Technical considerations

The capsaicin dosage (50 mg/kg) administered in this study was similar to the dosage generally accepted as effective in depleting C-fiber afferents (for review, see Buck and Burks 1986; Fitzgerald 1983; Holzer 1991). Our electronmicroscopic analysis of the IO and SC nerves in the capsaicin-treated rats showed that our dosage was indeed effective in depleting the majority of C fibers in both nerves, and this was substantiated further by the marked reduction in the effectiveness of mustard oil in inducing Evan's blue dye extravasation. Nonetheless, ~30% of the C fibers remained, and these likely comprised sympathetic efferents and C-fiber afferents insensitive to capsaicin (Karlsson and Hildebrand 1993; Kenins 1982; Szolcsanyi et al. 1988); the latter probably contributed to the extravasation induced by topical application of mustard oil to the ipsilateral skin compared with the contralateral skin in capsaicin-treated rats. In addition, our ultrastructural analysis in control animals confirmed earlier findings (Darian-Smith 1966; Jacquin et al. 1984; Schmalbruch 1986) that the proportion of unmyelinated:myelinated fibers is normally much smaller in V nerve branches (e.g., IO nerve) than in spinal nerves (e.g., SC nerve).

Another anatomic technique used in this study was the examination of barrelette morphology by cytochrome oxidase stain. This technique allows for a qualitative comparison of barrelette staining patterns, thus only gross changes in barrelette morphology in capsaicin-treated rats are capable of detection. However, this analysis does not rule out any minute morphological changes in barrelettes induced by neonatal capsaicin treatment that can be examined only by electronmicroscopic analysis.

In our efforts to study in detail the changes of RF and response properties of principalis vibrissa-sensitive neurons as a result of C-fiber depletion, it was necessary to use standardized approaches for neuron recording and stimulation in both capsaicin-treated and control rats. A standardized means of mechanical stimulation of individual vibrissae could only be provided with an electronically controlled mechanical stimulator. Such stimulators have been used commonly in studies of rodent mystacial vibrissal-response properties at various levels of the barrel neuraxis, and our deflection amplitude range of 250-400 µm (or 2.8-4.6° deflection angle) is similar to that used in other studies (e.g., Armstrong-James and Fox 1987; Simons 1983; Woolston et al. 1982). Also, our standardized approach of vibrissae sampling in both capsaicin-treated and control rats was reflected in the similar proportions of vibrissae located within dorsal, caudal, ventral, and rostral regions of the vibrissal pad (see RF features of vibrissa-sensitive neurons) between these two groups.

Because we only had the availability of one stimulator in the present study, we did not have the capability to simultaneously stimulate more than one vibrissa. Studies of neurons in cortical barrels (Simons 1983) and thalamic barreloids (Simons and Carvell 1989) have shown that responses to deflection of maximally excitatory vibrissae may be altered (often a reduction) after deflection of adjacent vibrissae. Because several vibrissae may be moved during the rat's natural behavior, it would be interesting to determine if capsaicin-treatment affects this phenomenon.

Another factor worth considering is that our use of anesthesia (urethan) during the recording experiments almost certainly altered the RF properties of principalis neurons in capsaicin-treated and control rats. All prior analyses of this issue in the CNS portions of the barrel neuraxis are consistent in this regard and indicate that RFs are smaller and less complex under anesthesia (Simons et al. 1992). Therefore it is highly likely that our recording conditions underestimated the effects of neonatal capsaicin treatment.

RF properties of vibrissa-sensitive neurons in control rats

Our use of electronically controlled mechanical stimuli to study the RF properties of vibrissa-sensitive neurons allowed for the precise investigation of various features of the neuronal response such as the exact number of vibrissae within the RF and the response latency and magnitude. Data pertaining to the number of vibrissae within the RF suggest that the mean number of center-RF vibrissae elucidated by electronically controlled mechanical stimuli was similar to the mean number of vibrissae found with the use of hand-held mechanical stimuli (Kwan et al. 1996). More importantly, the present study could show that principalis vibrissa-sensitive neurons possessed surround-RF vibrissae that, when stimulated, can elicit a small response of less than one action potential/stimuli (cf. center-RF vibrissae which when stimulated elicit 1 action potentials/stimuli). It is quite possible that increases in input from surround-RF vibrissae may account for the significant capsaicin-induced increase in the number of vibrissae within the RF of principalis vibrissa-sensitive neurons found in our previous study (Kwan et al. 1996). Also consistent with the notion that surround-RF vibrissae provide only subliminal input to the proper barrelette was that the response latency on deflection of surround-RF vibrissae was significantly longer than that of center-RF vibrissae. Other notable contrasts between center- and surround-RF vibrissae include the response latency variability on deflection center-RF vibrissae being significantly larger than that for surround-RF vibrissae and the directional selectivity of response elicited by deflection of center-RF vibrissae being significantly different from that found for surround-RF vibrissae. Finally, our vector analysis of vibrissae ensembles within the RF of principalis vibrissa-sensitive neurons revealed that almost 2/3 of vibrissae within the RF are normally oriented toward the same quadrant. Such data support a role for vibrissae in providing input to the CNS with regard to the location of discrete points in space.

Capsaicin-induced changes in vibrissal RF and response properties

The RF expansion of principalis vibrissa-sensitive neurons in capsaicin-treated rats studied with electronically controlled mechanical stimuli substantiates analogous findings in nucleus principalis and in subnucleus oralis of capsaicin-treated rats in which only manual stimulation of vibrissae was used (Kwan et al. 1996) and is also consistent with findings at other levels of the barrel neuraxis (Nussbaumer and Wall 1985; Sessle et al. 1995). Indeed, the mean number of center-RF vibrissae per neuronal RF in control and capsaicin-treated rats in this study was comparable with the mean number of vibrissae delineated with manual stimulation in corresponding rats of our previous study (Kwan et al. 1996).

Because we did not use approaches (e.g., antidromic activation) to identify the projection status of the principalis neurons recorded, we cannot be certain if both local circuit neurons and projection neurons (relaying directly to, for example, ventrobasal thalamus) were manifesting the neuroplastic changes induced by C-fiber depletion. It is unlikely that neonatal capsaicin-induced effects were expressed especially in local circuit neurons in our study because local circuit neurons represent only ~20% of the principalis neuronal population (Jacquin et al. 1988). It is more likely that projection neurons as well as local circuit neurons were affected by C-fiber depletion. The capsaicin-induced alterations in the directional selectivity of responses of vibrissa-sensitive neurons and the increase in the response latency variability also might have been the result of a decrease in the inhibition that normally serves to sharpen these RF properties (Kyriazi et al. 1996; Simons 1985). We recently have found that neonatal capsaicin treatment does lead to a disturbance in GABAergic control in subnucleus caudalis nociceptive neurons (Chiang et al. 1996) but have yet to test the effects of C-fiber depletion specifically on GABAergic modulation in principalis. Application of the -aminobutyric acid-A (GABA_A) antagonist bicuculline to subnucleus caudalis of capsaicin-treated rats resulted in differential alterations of the RF of nociceptive-specific and wide dynamic range neurons. Such results may be explained by the disinhibition of GABAergic inhibitory mechanisms that are controlled by descending and segmental afferent inputs (Sivilotti and Woolf 1994). Capsaicin-induced changes in caudalis, however, also could contribute indirectly to the alterations of principalis neuronal properties, as noted below, and so not necessarily involve the GABAergic mechanisms operating within principalis itself.

Vibrissal RF expansion in principalis neurons may be due to unmasking of existing convergent inputs (McMahon and Wall 1983; Wall et al. 1982), and it has been suggested that one control of the RF size of central neurons may originate from the tonic inhibition exerted by C-fiber activity (Calford and Tweedale 1991; Nussbaumer and Wall 1985; Pettit and Schwark 1996). Our observation of a significant increase in the percentage of neurons responsive to stimulation of other types of peripheral tissues might be attributed to the unmasking of existing convergent inputs. Furthermore although the increase in the number of surround-RF vibrissae per neuronal RF was insignificant in capsaicin-treated rats, we observed that the response magnitude of surround-RF vibrissae of principalis vibrissa-sensitive neurons was significantly higher in capsaicin-treated rats than in control rats. This may suggest a decrease in the inhibition of input from surround-RF vibrissae and thus also support the unmasking of convergent inputs as a possible mechanism for these capsaicin-induced changes. Another possibility is that the increase in response magnitude may result in part from a change in the temporal summation of responses induced by C-fiber depletion. Our use of a single stimulation frequency rate (1/s) failed to uncover any difference in temporal summation of neuronal responses between control and capsaicin-treated rats. However, temporally dependent changes in the response magnitude as a result of C-fiber depletion should be tested in future studies with the use of varying stimulus parameters.

The capsaicin-induced changes in RF properties of principalis vibrissa-sensitive neurons may be attributed to neuronal alterations within subnucleus caudalis because nucleus principalis does not receive direct C-fiber afferent inputs (Kruger et al. 1988; Sugimoto et al. 1997, 1998). Thus capsaicin-induced alterations in the central termination of C-fiber primary afferents in subnucleus caudalis (which is the V analog of the spinal dorsal horn) and rearrangements within caudalis analogous to those reported in the spinal dorsal horn (Nagy and Hunt 1983; Shortland et al. 1990) might provide a morphological substrate to account for the capsaicin-induced alterations in RF and response properties of LTM (Sessle et al. 1995) and nociceptive (Chiang et al. 1997) neurons in subnucleus caudalis. These changes in caudalis neurons thereby might alter the modulatory influence that caudalis exerts on principalis neurons (Greenwood and Sessle 1976; Hallas and Jacquin 1990) and so contribute to the capsaicin-induced changes described in this paper. Because neonatal capsaicin treatment also affects neurons in subnucleus oralis of adult rats (Kwan et al. 1996) and may likely also affect neurons in subnucleus interpolaris, other possible sources of alterations in ascending modulation of principalis vibrissa-sensitive neurons may be projections of nearby local circuit neurons from these two subnuclei (Jacquin et al. 1989; Westberg and Olsson 1991).

The increase in RF size of V brain stem somatosensory neurons is not limited to principalis vibrissa-sensitive neurons. Other recent data from our laboratory show that the RF of subnucleus oralis LTM neurons (Kwan et al. 1996) and subnucleus caudalis LTM (Sessle et al. 1995) and nociceptive neurons (Chiang et al. 1997) is enlarged significantly in adult rats neonatally treated with capsaicin compared with that in control rats. Furthermore, the observation that intrathecal application of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 in capsaicin-treated rats can significantly reduce the RF size of these caudalis nociceptive neurons (Chiang et al. 1997) provides strong support for the involvement of NMDA mechanisms in the capsaicin-induced RF changes in V brain stem neurons. Moreover, because nucleus principalis is the main V brain stem relay for vibrissal input to the thalamus and cortex (for review, see Killackey et al. 1990; Rhoades et al. 1990; Woolsey 1990), changes of principalis vibrissa-sensitive neurons induced by C-fiber depletion might account for analogous alterations described in the barrel cortex of capsaicin-treated rats (Nussbaumer and Wall 1985; Wall et al. 1982). A similar influence of C fibers on subcortical relays in the spinal somatosensory system (McMahon and Wall 1983) might contribute to the plasticity of the barrel-like arrangement of the cortical representation of the digits (Florence et al. 1996; Goyal et al. 1992).

There are additional mechanisms that need to be considered as possible factors contributing to the altered RF and response properties of principalis neurons. In the spinal system, it has been shown that the descending modulation by the rostral medial medulla of spinal dorsal horn neurons is reduced significantly in capsaicin-treated rats (Zhuo and Gebhart 1994). Thus C-fiber depletion also might produce changes in the descending modulation of principalis neurons. Another possible mechanism for the capsaicin-induced changes in nucleus principalis could be peripheral sprouting of V primary afferents; however, there has been no documented evidence of peripheral sprouting in the spinal or V somatosensory system. Together with preliminary data from electrophysiological recordings of vibrissal primary afferents in the V sensory root of capsaicin-treated rats, which indicates that a one-to-one correspondence of primary afferent to single vibrissa is still present (Kwan, Hu, and Sessle, unpublished data), it is unlikely that peripheral sprouting would have contributed to the capsaicin-induced changes of principalis neurons. Also, given our results from the cytochrome oxidase staining of sections through nucleus principalis and spinal V subnuclei that show that normal topographic representation of vibrissae (barrelettes) still remains in capsaicin-treated rats, the observed capsaicin-induced RF abnormalities are not likely reflected by gross changes in the organization of the principalis neuropil.

In view of the anatomic, neurochemical (e.g., Arvidsson and Ygge 1986; Nagy and Hunt 1983; Shortland et al. 1990), and physiological (e.g., Gamse et al. 1980; Kwan et al. 1996; McMahon and Wall 1983; Nussbaumer and Wall 1985; Wall et al. 1982) alterations of primary afferents and central somatosensory neurons that may occur as a result of C-fiber depletion, it is evident that C-fiber afferents play an important role in the normal development of the somatosensory system. It has been suggested that C fibers may have neuroeffector and neurotrophic influences on the development of sensory neurons in the peripheral and CNS and as such the normal connectivity of large-caliber fiber afferents (which relay LTM input) with central somatosensory neurons may require the presence of C fibers (Fitzgerald 1983; Kruger 1988). Our findings that C-fiber depletion produces significant alterations in the RF and response properties of principalis neurons provide further evidence to support the view that C-fiber afferents indeed may be required for the normal development of the functional properties of central somatosensory neurons.

	ACKNOWLEDGEMENTS

  We are grateful to Dr. D. Simons for providing the necessary materials for our piezoelectric mechanical stimulator. We are also thankful to Mr. K. MacLeod for excellent technical assistance.

  This study was supported by the National Institute of Dental Research Grants DE-04786 (to B. J. Sessle) and DE-07762 (to M. F. Jacquin).

	FOOTNOTES

  Address for reprint requests: B. J. Sessle, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada.

  Received 16 March 1998; accepted in final form 29 September 1998.

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