Forelimb amputation-induced reorganization in the cuneate nucleus (CN) is not reflected in large-scale reorganization in rat forepaw barrel subfield cortex (FBS)

Abstract

We examined reorganization in cuneate nucleus (CN) in juvenile rat following forelimb amputation (n=34) and in intact controls (n=5) to determine whether CN forms a substrate for large-scale reorganization in forepaw barrel subfield (FBS) cortex. New input from the shoulder first appears in the FBS 4 weeks after amputation, and by 6 weeks, the new shoulder input comes to occupy most of the FBS. Electrophysiological recording was used to map CN in controls and in forelimb amputees during the first 12 weeks following deafferentation and at 26 and 30 weeks post-amputation. Mapping was confined to a location 300 μm anterior to the obex where a medial-to-lateral row of electrode penetrations traversed through a complete complement of cytochrome-oxidase stained clusters (called barrelettes) that are associated with the representation of the glabrous forepaw digits and pads and adjacent non-cluster zones that are associated with the representation of the wrist, arm, and shoulder. Following amputation, non-cluster zones became occupied with new input from the body/chest and head/neck, while the cluster zone remained largely devoid of new input except at the border. A regression analysis comparing controls and amputees over the first 12 weeks post-amputation found significant differences for the total area of new input from the body/chest and head/neck in the non-cluster zones, while no significant differences were found for any new input into the cluster zone. When the averaged areas of a body-part representation were reexamined as a percentage of the averaged zonal area, a non-significant increase in new input from the body was observed within the cluster zone during post-amputation weeks 2–3 that returned to baseline in the subsequent weeks. In contrast, significant differences in averaged area of body-part representations for body/chest and head/neck were found in non-cluster zones over the first 12 weeks post-amputation. The present findings suggest that reorganization occurs only within the non-cluster zones whereby new input from the body/chest and head/neck moves in and occupies the deafferented territory immediately after amputation. Additionally, the lack of significant differences in new shoulder input in either cluster or non-cluster zones over the first 12 weeks after amputation suggests that CN provides an unlikely substrate for large-scale reorganization in the FBS.

1. Introduction

The cuneate nucleus (CN) receives and processes incoming somesthetic input from the primary afferents of the forelimb (Andersen et al., 1962; Andersen et al., 1964a; Andersen et al., 1964b) before relaying this information, in part, to the ventral posterior nucleus (VPL) of the thalamus(Alloway and Aaron, 1996; Berkley et al., 1980; Kemplay and Webster, 1989; Massopust et al., 1985). The organization of CN has been described in monkey (Florence et al., 1989), cat (Nyberg, 1988), raccoon (Rasmusson, 1989), and rat (Beck, 1981; Li et al., 2012; Maslany et al., 1990; Nord, 1967), and it is generally agreed that the rostrocaudally oriented CN is partitioned into rostral, middle, and caudal regions (Berkley et al., 1986; Bermejo et al., 2003; Dykes et al., 1982; Maslany et al., 1992). Recently, the details of the somatotopic organization of CN in rat were elucidated using fine-grain electrophysiological mapping (Li et al., 2012). The middle region was further partitioned into medial, central, and lateral zones. The central zone containing cytochrome oxidase (CO)–stained clusters, termed barrelettes, was mapped, and the individual labeled clusters were associated with the representation of the glabrous forepaw digits and digit and palmar pads; the medial zone was mapped to the ulnar representation of the wrist, forearm, and upper arm, while the lateral zone was mapped to the radial representation of the wrist, forearm, and upper arm. A lateral tail region was identified that received input primarily from the shoulder, head/neck, and ear. This somatotopy in the forelimb-intact rat provided a useful starting point from which to compare CN reorganization following deafferentation.

CN organization and the resulting reorganization in rat have been studied following limb amputation (Crockett et al., 1993; Lane et al., 1995), dorsal rhizotomy (Sengelaub et al., 1997), and nerve transection (Crockett et al., 1993). Time of deafferentation has varied from embryonic (Killackey and Dawson, 1989; Rhoades et al., 1993), neonatal (Lane et al., 1995; Lane et al., 2008), and adult (Sengelaub et al., 1997), while the time of assessment ranged from days, weeks, and months post-deafferentation (Sengelaub et al., 1997). The resulting reorganization has been reported using electrophysiological mapping of receptive fields (Rhoades et al., 1993), transganglionic labeling (Maslany et al., 1990; Maslany et al., 1991), receptor expression mapping (Foschini et al., 1994), and metabolic uptake measurement (Crockett et al., 1993). There is also evidence that CN reorganization plays some role in cortical reorganization (Bowlus et al., 2003; Killackey and Dawson, 1989; Lane et al., 1995; Lane et al., 2008).

The forepaw barrel subfield (FBS) in primary somatosensory cortex in rat contains CO-stained clusters (called barrels) that are associated with the representation of the glabrous forepaw digits, digit pads, and palmar pads (Waters et al., 1995); this cluster arrangement of CO labeling in rat SI is similar to that reported in rat CN (Li et al., 2012). The representation of the wrist lies within a nebulously stained field immediately posterior to the FBS and is bordered successively by the representations of the forearm, upper arm, and shoulder, hereby described as the “original shoulder”. Following forelimb amputation in juvenile rats, new input from the shoulder moves in to occupy the deafferented cortical space left vacant in the FBS (Pearson et al., 1999). The new input first appears 4 weeks after amputation, and by 6 weeks post-amputation, the shoulder representation occupies large regions of the FBS (Pearson et al., 2003). The new shoulder representation does not derive from the original shoulder representation or from the shoulder representation in second somatosensory cortex (SII) (Pearson et al., 2001). This finding led us to speculate that subcortical loci in the ventral posterior thalamus (VPL) and/or cuneate nucleus (CN) are likely responsible for the expression of delayed large-scale cortical reorganization in the FBS.

In the present study, we used extracellular recording techniques in rat to examine the input to CN during the first 12 weeks following forelimb amputation and at 26 and 30 weeks post-amputation in order to compare the temporal pattern of reorganization with that previously reported in the FBS (Pearson et al., 2003). We hypothesized that CN would display a pattern of reorganization similar to that previously reported in the FBS, but the time of first appearance of the new input from the shoulder in CN would occur prior to or simultaneously with its expression in the cortex. Our data show that CN reorganization begins within one week after amputation. New input from the body/chest and/or head/neck appears in the medial and lateral zones. In contrast, significant new input from the shoulder and reorganization within the central zone are absent. These results run counter to our prediction that CN forms a substrate for delayed large-scale cortical reorganization.

2. Results

A total of 39 juvenile Sprague-Dawley rats was used in this study. Of that number, 34 rats had their left forelimb amputated between 6 and 8 weeks of age. The deafferented ipsilateral CN and adjacent brainstem, in the vicinity of the obex, were physiologically mapped between 1 and 12 weeks and at 26 weeks and 30 weeks post-amputation. In these forelimb amputee rats, 631 electrode penetrations were made and receptive fields were examined at 4,675 sites. An additional 5 juvenile Sprague-Dawley rats that did not undergo forelimb amputation served as controls and were similarly mapped by making 58 penetrations and examining receptive fields at 829 sites. The total number of electrode penetrations and total number of recording sites examined for intact and forelimb amputees are shown in Table 1.

Table 1.

Table showing number of electrode penetrations and recording sites for experimental groups

Experimental Groups (number of rats per group)	Total Penetrations	Total Receptive Field Recording Sites	Penetrations (+300 μm)	Receptive Field Recording Sites (+300 μm)
Controls (n=5)	58 (11.6)	829 (165.8)	55 (11)	744 (148.8)
1-WD (n=4)	76 (19.0)	533 (133.3)	36 (9.0)	281 (70.3)
2-WD (n=4)	75 (18.8)	628 (157.0)	40 (10.0)	290 (72.5)
3-WD (n=5)	82 (16.4)	631 (126.2)	48 (9.6)	379 (75.8)
4-WD (n=3)	60 (20.0)	497 (165.7)	33 (11.0)	250 (83.3)
5-WD (n=4)	78 (19.5)	582 (145.5)	40 (10.0)	299 (74.75)
6–8-WD (n=6)	103 (17.2)	737 (122.8)	52 (8.67)	377 (62.8)
9–12-WD (n=6)	122 (20.3)	791 (158.2)	60 (10.0)	433 (72.2)
26, 30-WD (n=2)	35 (17.5)	276 (138.0)	21 (10.5)	181 (90.5)

Data given parenthetically for penetrations and recording sites are group means.

2.1. Normal organization of CN

A relationship exists between the physiological and morphological organization of the glabrous forepaw representation in CN. In the present study, we focused on the region approximately +300 μm anterior to the obex that contained CO–labeled clusters, called barrelettes, that were associated with the representation of the glabrous digits and digit and palmar pads (Li et al., 2012). While these CO-stained clusters are found throughout an 800-to-900 μm rostrocaudal segment of CN, cross sections taken around +300 μm generally contained a complete complement of forepaw barrelettes that could be directly compared to barrel-like structures in the forepaw barrel subfield (FBS) in SI cortex (Waters et al., 1995).

Examples of 4 intact animals with well-defined barrelettes in CN lying approximately +300 μm anterior to the obex are illustrated in photomicrographs and corresponding line drawings in Fig. 1. The locations of the barrelettes within CN, the general shape of CN, and the location of CN in relationship to the surrounding gracilis nucleus (GN) and spinal trigeminal nucleus (STN) are shown. In each example, the barrelettes are well formed and occupy the central region of CN. On the dorsomedial corner, beginning at the dashed line in the line drawings, CN extends toward and appears to abut or blend into the neighboring GN. The dorsolateral side of CN forms a tail-like structure that can be seen extending toward the brainstem surface and the neighboring STN. These are common features of coronal sections at this level of CN.

Fig. 1.

Photomicrographs and reconstructed line drawings, through the middle region of cuneate nucleus (CN) which lay approximately 300 microns anterior to the obex showing the location of mapping. A: CO-stained coronal section showing the location of CN and overlying fasciculus cuneatus (fc) and gracilis nucleus (GN) and spinal trigeminal nucleus (STN). A-1: Line drawing from “A” highlighting the CO-stained clusters (barrelettes) within CN. B–D: Photomicrographs and line drawings (B-2, C-3, D-4) from 3additional rats illustrating the arrangement of barrelettes in the CN.

For each of the forelimb-intact control rats, a detailed physiological map of the forelimb and surrounding body representation(s) was generated by making rows of closely spaced electrode penetrations and sampling at depths of 50 or 100 μm throughout the penetration down to a depth of 700 μm. Penetrations were then reconstructed in relationship to the underlying morphological map to produce a standardized map for subsequent comparison with forelimb amputees. An example from one intact rat is illustrated in Fig. 2. The photomicrograph in Fig. 2A shows a view of the brainstem surface with the locations of the surface point of entry of 7 electrode penetrations used to generate the physiological map. This row of penetrations was located +300 μm anterior to the obex. The inset in Fig. 2A shows a photomicrograph of a cross-section through the area studied that nicely illustrates the CO-stained clusters in the central region. This 100-micron-thick section is shown Fig. 2B in relationship to the surrounding GN and STN. We subdivided the middle region of CN into 3 zones: a central zone that contains CO-stained barrelettes, a middle zone adjacent to GN, and a lateral zone that extends toward the dorsomedial tail-like region and continues medially where it overlies the cluster containing central zone. Electrophysiological recording was used to explore these zones, and the resulting physiological map is illustrated in a matrix-like format in Fig. 2C. Electrode penetration no. 1 recorded receptive fields on the hindlimb and trunk, and this penetration was localized to GN. Penetration no. 2 passed through the medial zone where receptive fields on the ulnar forearm and upper arm, ulnar wrist, and digit and palmar pads were encountered; one dorsomedial site received input from the shoulder and body. Penetration nos. 3 and 4 passed through the central zone where receptive fields were localized to the glabrous digits and pads; sites responsive to dorsal digit input were found superficially in the lateral zone. Penetration no. 5 passed through the lateral zone where receptive fields were found on the radial wrist, radial upper arm, and shoulder; deeper in the penetration, receptive fields were found on dorsal and glabrous digits. A caricature of CN has been superimposed on the matrix diagram, but appears distorted due to the inherent distortion in the individual cell sizes of the matrix, which is based on the number of receptive fields encountered at each matrix site.

Fig. 2.

Detailed map of CN and adjacent regions in a forelimb-intact control rat. A: Surface view of the brainstem showing the point of entry of 7 electrode penetrations aligned in a medial-lateral row. The location of the obex is outlined with a dashed line in relationship to the mapped region. The rostral (R) to caudal (C) orientation is shown with the arrow. Inset shows a photomicrograph of CN with the CO-stained clusters. B: CO-stained section through the brainstem showing the locations of CN (solid outline), overlying fc, GN (dashed outline), and STN. Recording sites are indicated with black circles. Seven medial-to-lateral electrode penetrations were used to map CN. CN is demarcated into lateral (L), central (C), and medial (M) zones. C: A matrix format is used to plot receptive fields. Penetration number and medial-lateral (M-L) coordinates are shown for each penetration. Receptive fields were recorded at 50-micron steps along a penetration and each penetration began at 100-microns below the surface. Within the matrix, the solid lines indicate recording sites in CN and the filled region within denotes recording sites corresponding to locations within the barrelettes. Receptive field nomenclature used in Fig. 2 (see Fig. 10 for complete nomenclature): A = abdomen; B = back; C = chin; E = ear; FA = forearm; H = head; Hip = hip; HL = hindlimb; J = jaw; N = neck; S = side; SH = shoulder; UA = upper arm; W = wrist. Sub-nomenclature for the forepaw: D = digit; 1–5 = digit number; v = ventral; d = dorsal; P = pad; TH = thenar pad; HT = hypothenar pad. Sub-nomenclature for the body: r = rostral; c = caudal. Sub-nomenclature for the wrist and arm: r = radial; u = ulnar.

A summary map of the forelimb representation that incorporates receptive field data obtained from the 5 forelimb-intact rats is shown in Fig. 3. The receptive fields from each animal have been superimposed on a standardized schematic drawing of CN derived from a smoothed averaged outline of the 5 forelimb-intact CN maps, and this is shown in Fig. 3A. The central zone consists of CO-stained clusters and their immediate surround that is readily demarcated. The lateral edge of the medial zone has been arbitrarily established by placing a 126° line (arrow) that passes through the dorsomedial extent of the central zone and runs parallel to the lateral border that is formed at the CN/GN junction. This line also forms the medial border of the lateral zone. At the lateral edge of the lateral zone, another line is drawn at a 57° angle that forms the base of dorsolateral tail region. Electrode penetrations passing through the medial zone encountered receptive fields on the ulnar aspect of the upper arm, forearm, and wrist, while scattered sites were found in the dorsal-most part that were responsive to input from the shoulder. The barrelette-containing central zone received input almost exclusively from the glabrous digits and pads; a few sites at the edges received input from radial wrist, forearm, upper arm, and dorsal digits/hand. The lateral zone received input largely from the radial wrist, forearm, and upper arm, but sites were also encountered that were responsive to input from the shoulder. This standardized map was then used to plot receptive fields in forelimb-intact controls. Our interpretation of the organization of CN is summarized in Fig. 3B.

Fig. 3.

Interpretative figure from 5 forelimb-intact rats showing locations of recording sites and receptive fields recorded at those sites plotted on a standardized drawing of CN. A: Locations of receptive fields plotted on a summary map. Arrows are drawn to partition CN into medial and lateral zones. The arrow demarcating the lateral border of the medial zone is angled at 126° and runs nearly parallel to the lateral border of GN while the arrow demarcating the lateral border of the lateral zone is drawn perpendicular to the base of the tail region. The central zone is ovoid shaped. Receptive field nomenclature is shown in boxes at right and left. B: Our interpretation of receptive field organization in the forelimb-intact rat. The medial zone contains the representation of the ulnar wrist, ulnar forearm, ulnar upper arm, and shoulder. The central zone contains the representation of the glabrous forepaw digits, forepaw digit pads, and forepaw palmar pads. The lateral zone contains the representation of the radial wrist, radial forearm, radial upper arm, shoulder, dorsal digits, and dorsal hand.

2.2. Cuneate reorganization in forelimb deafferents

From a total of 631 penetrations, 330 penetrations were recovered that passed through clusters of labeling in CN approximately 300 μm rostral to the obex, and receptive fields were measured at 2,490 locations from these penetrations.

Receptive fields of CN neurons in forelimb amputees were examined systematically during the first 5 weeks post-amputation (n=20) and between 6 and 8 weeks (n=6) and 9 through 12 weeks (n=6); one additional rat was mapped at 26 weeks and another rat mapped at 30 weeks post-amputation. The experiments described below were selected to illustrate those maps that in our estimation best represented the averaged body part representation within the barrelette-containing central zone following selected periods of forelimb amputation. Sites that included the suture or stump were noted on the matrix maps, but were not included in the areal measurements.

2.3. Temporal pattern of reorganization following forelimb amputation

2.3.1. One-week deafferented (1-WD) rats

Within the first post-deafferentation week, few sites within the CN were responsive to new input. An example from a 1-WD map is illustrated in Fig. 4. In this rat, 6 electrode penetrations were used to map CN and their entry points into the brainstem, in relationship to the obex, are shown in Fig. 4A. The inset shows the CO-rich clusters found within the central zone. Reconstruction of the recording sites (black circles) is illustrated in the coronal section in Fig. 4B; receptive fields were examined at 100-μm steps along the penetration and continued to a depth of 800 μm. Note that in penetration nos. 1 and 6, the path of the electrode was clearly demarcated from blood coagulation as the electrode passed through the brainstem. The receptive field recordings made at each step along a penetration are shown in matrix format in Fig. 4C. Inspection of the matrix revealed that the majority of sites within the former forelimb representation were unresponsive to peripheral input with the exception that neurons at a depth of 300 μm in the medial zone responded to input from the skin immediately around the suture (SU). Two additional 1-WD rats had similar unresponsive sites throughout all 3 zones in CN. However, these findings were in contrast to those from the fourth 1-WD rat, for which a row of electrode penetrations passed through the lateral border of the central zone where receptive fields were encountered for the shoulder and neck.

Fig. 4.

Detailed map of CN and adjacent regions in a 1-week deafferented rat. A: Surface view of brainstem showing the location of electrode penetrations in relationship to the obex (dashed line). Inset shows CO-stained clusters in CN. B: CO-stained section through CN. Black circles indicate recording sites. Medial (M), lateral (L), and central (C) zones are shown along with lines of demarcation. C: Reconstruction of electrode penetration in a matrix format. Note that recording sites within CN were unresponsive to peripheral input. Shaded region depicts the central zone. See Fig. 10 for nomenclature.

2.3.2. Three-week deafferented (3-WD) rats

In 3-WD rats (n=5), new input was observed in all three zones. An example from one 3-WD rat is shown in Fig. 5. The locations of the surface point of entry of electrode penetrations and barrel-like structures within the CN are shown in Fig. 5A. The recording sites and distribution of middle, central, and lateral zones are shown in Fig. 5B. The receptive fields recorded at 100-micron steps through the penetration are shown in the matrix format in Fig. 5C. In this example, the new input completely occupied the medial and lateral zones and encroached on the medial and lateral borders of the central zone. While this arrangement was most typical, 1 of the 5 rats had responsive sites distributed throughout the middle portion of the central zone.

Fig. 5.

Detailed map of CN and adjacent regions in a 3-week deafferented rat. A: Surface view of the brainstem showing point of entry of 9 electrode penetrations into the brainstem in relationship to the obex (dashed line). Inset shows the CO-stained clusters in CN. B: Recording sites (black circles) in CN and surrounding nuclei. Medial (M), lateral (L), and central (C) zones are shown along with lines of demarcation. C: Reconstruction of electrode penetration in a matrix format. Note that the medial and lateral zones were filled in with new input. Shaded region depicts the central zone. See Fig. 10 for nomenclature.

2.3.3. Four- and five-week deafferented (4-WD, 5-WD) rats

A total of 73 electrode penetrations (mean: 9.5 per animal) was used to map CN at + 300 μm to the obex in seven 4- and 5-WD rats; receptive fields were examined at 549 sites (mean: 79 per animal) at +300 μm. A representative example is shown in Fig. 6 for one 5-WD rat. While the medial zone is completely occupied with new input, few sites were responsive to new input in the central and lateral zones.

Fig. 6.

Detailed map of CN and adjacent regions in a 5-week deafferented rat. A: Surface view of the brainstem showing point of entry of 10 electrode penetrations into the brainstem used to map CN and surrounding nuclei. The penetrations are shown in relationship to the obex (dashed line). Inset shows the CO-stained clusters in CN. B: Recording sites (black circles) in CN and surrounding nuclei. Medial (M), lateral (L), and central (C) zones are shown along with lines of demarcation. C: Reconstruction of electrode penetration in a matrix format. Shaded region depicts the central zone. See Fig. 10 for nomenclature.

2.3.4. Temporal summary

The results for the forelimb-intact controls and deafferented groups are shown in the receptive field plots in Fig. 7. The receptive fields are partitioned into body, shoulder, and head/neck subdivisions, and each receptive field is plotted onto a standardized map of CN. Inspection of the map plots shows that even in the controls, receptive fields for each body part can be found in the medial and lateral zones. In the 1-WD rats, the central zone contains a few sites on the lateral border where shoulder and head/neck receptive fields were found. In the 2-WD rats, more sites were found in the central zone, but these were confined to the lateral edge. However in the 3-WD rats, many sites were observed in the central zone that received input from each of the body parts; the medial and lateral zones also contained new receptive fields that were distributed throughout their zones. In contrast, the 4-WD and 5-WD rats had few examples of new input in the central zone and those that were seen were relegated to the medial and lateral borders. Interestingly, new inputs in the central zone in the 6–8-WD rats were only observed at the medial and lateral border regions, while 9–12-WD had a few new fields in the dorsal part of the C zone. The one 26-WD rat and one 30-WD rat also had new receptive fields localized to the medial and lateral borders of the central zone.

Fig. 7.

Summary diagram displaying the locations of receptive fields for shoulder, body/chest, head/neck recorded in medial (M), lateral (L), and central (C) zones and tail (T) region for controls and forelimb deafferents. A: Recording sites where receptive fields were encountered for forelimb intact controls. Note that receptive fields for the forelimb are not included to emphasize input into CN from non-forelimb sites (see summary Fig. 3 for complete map of forelimb). It is important to note, that even in controls, receptive fields for the shoulder and body were observed within the medial and lateral zones. Receptive fields for the shoulder and head/neck were found in the tail region. B: Receptive fields from the shoulder, body/chest, and head/neck for deafferent weeks 1 (1-WD) through 5-WD and combined weeks 6–8-WD, 9–12-WD, and 26, 30-WD are illustrated on the map. Note that new input in the central zone was mainly localized to the border regions; however, during 2-WD and 3-WD, new input was scattered throughout this zone.

2.4. Dataset and data analysis

The dataset for the total area (μm² as measured at +300 μm anterior to the obex) of the cuneate nucleus; total areas of medial, central, and lateral zones; and total area of the new input from the body, shoulder, and head into each zone for both controls and forelimb deafferented rats is presented in Table 2. Inspection of Table 2 shows the existence of a great deal of variability in body part maps among individual members within an experimental group, and the data were often skewed by one individual. Also evident is the fact that in control rats, receptive fields for the shoulder were encountered in each zone so any finding of “new” shoulder input in the deafferented rat must be carefully interpreted. Also conspicuous is the near absence of responses for the head/neck representation in the medial zone for both controls and amputees.

Table 2.

Table showing total areas for CN, CN zones, and body/chest, shoulder, head/neck representations within each zone in (μm²)

Experimental Groups	Total Cuneate Nucleus	Total Medial Zone	Body/Chest Medial	Shoulder Medial	Head/Neck Medial	Total Central Zone	Body/Chest Central	Shoulder Central	Head/Neck Central	Total Lateral Zone	Body/Chest Lateral	Shoulder Lateral	Head/Neck Lateral
Control	156,089	35,262	-	3,491	-	99,181	-	-	-	32,145	-	4,740	1,542
Control	149,262	29,044	-	4,788	-	74,126	-	-	-	30,247	-	9,940	-
Control	156,356	27,403	-	2,417	-	87,224	-	-	-	38,514	-	14,177	-
Control	145,171	19,652	1,896	2,768	-	101,881	-	-	-	21,736	-	13,029	-
Control	141,836	26,378	2,939	5,927	-	70,849	-	743	-	40,564	-	18,840	-
1-WD	148,165	17,960	-	2,587	-	85,269	-	-	-	36,307	-	-	-
1-WD	164,988	18,555	-	10,935	-	92,464	716	6,747	15,150	44,315	1,429	16,098	28,839
1-WD	125,171	17,511	5,741	-	-	65,060	750	-	-	35,219	-	-	-
1-WD	149,142	27,962	3,824	-	-	72,374	-	-	-	40,832	-	654	654
2-WD	162,264	23,516	1,602	2,745	-	83,557	-	-	-	48,244	-	18,092	18,092
2-WD	159,046	18,083	10,222	-	-	80,865	-	16,335	15,710	50,179	-	28,523	18,991
2-WD	161,523	18,975	18,975	16,464	-	69,465	33,093	5,772	4,516	59,479	8,268	36,322	36,663
2-WD	146,393	18,555	12,060	-	-	65,730	-	-	-	40,079	-	5,598	7,519
3-WD	182,821	20,367	3,454	-	-	99,333	-	5,915	34,731	50,434	-	24,749	43,027
3-WD	123,506	10,293	6,762	4,453	-	74,117	2,297	-	-	35,308	8,224	6,595	4,884
3-WD	114,701	27,350	22,453	3,597	-	51,773	7,829	7,829	-	29,269	4,395	3,994	-
3-WD	128,594	11,229	11,229	-	-	74,008	39,573	-	-	34,606	7,555	13,705	13,705
3-WD	137,107	24,668	24,019	2,173	-	66,426	7,124	-	-	37,822	4,465	17,385	17,881
4-WD	137,455	15,167	7,828	5,827	-	77,081	2,359	-	552	35,419	48	4,752	18,184
4-WD	129,014	19,470	16,945	-	-	60,752	420	-	--	41,832	7,412	9,697	9,697
4-WD	118,432	17,482	15,349	4,511	-	55,188	429	-	-	38,142	6,168	8,605	8,605
5-WD	144,363	16,459	16,459	5,597	2,431	72,269	2,252	1,827	-	45,095	13,595	15,122	11,194
5-WD	146,855	21,936	16,202	8,105	-	75,003	675	1,465	2,360	42,236	-	14,436	16,054
5-WD	127,745	19,722	11,863	6,674	-	62,820	-	3,133	3,133	36,387	-	18,981	18,981
5-WD	135,177	26,632	26,632	26,632	-	63,524	5,952	5,952	-	38,814	3,408	5,920	2,512
6–8-WD	169,497	21,481	21,481	9,493	-	106,515	6,501	8,893	-	49,424	6,460	33,581	15,923
6–8-WD	173,335	32,611	19,158	19,158	-	93,917	-	2,865	11,661	36,936	-	16,127	23,029
6–8-WD	139,473	14,974	14,974	1,972	-	75,245	3,953	331	331	41,378	5,190	9,948	18,801
6–8-WD	118,748	21,206	18,080	2,158	-	61,438	3,208	-	-	29,754	313	3,775	10,797
6–8-WD	133,851	11,291	4,317	-	-	66,657	225	-	-	46,320	-	3,337	3,983
6–8-WD	129,091	18,914	18,914	3,806	-	69,425	25,887	4,190	131	37,228	5,625	19,342	13,723
9–12-WD	132,515	15,243	14,952	-	-	68,942	2,241	813	5,121	39,825	6,401	10,351	17,435
9–12-WD	120,812	14,345	14,345	10,457	-	67,752	20,107	17,339	5,435	32,126	9,556	29,710	19,534
9–12-WD	128,354	10,246	9,385	9,385	-	72,060	-	-	-	37,320	1,036	7,698	10,051
9–12-WD	110,260	9,144	9,144	-	-	56,994	14,951	-	1,873	37,057	5,980	7,476	15,776
9–12-WD	127,971	16,356	6,645	4,527	1,286	60,238	-	7,059	28,305	40,312	3,700	27,019	33,488
9–12-WD	117,927	16,902	16,902	1,311	-	67,142	14,830	-	11,922	24,484	9,090	3,932	11,566
26-WD	135256	17,504	14,890	-	-	75,528	-	8,176	8,176	33,567	2,610	15,663	18,119
30-WD	147648	25,763	25,763	7,151	2,606	77,799	19,944	24,059	39,345	36,567	2,682	19,133	32,555

2.4.1. Comparison of the total size of the CN and total sizes of the medial, central, and lateral zones

An ANOVA was performed on the total area of CN, and no significant differences in total size of CN (p≥.105) or total size of the central zone (p≥.32) were observed between control and deafferented animals. However, significant group differences in total area were found in the total area of the lateral zone (p≤.047) and near significant difference for the medial zone (p≤.06), although no significant differences were found between groups in post hoc comparisons.

2.4.2. Total areas of the shoulder, head/neck, and body representations in CN over post-deafferentation weeks

The total areas of the shoulder, head/neck, and body (back, side, abdomen, chest) representations in each zone were measured in control and amputees over post-deafferented weeks. The data are plotted in a scatter plot format and analyzed using regression analysis and Pearson Product-Moment correlation and presented in Fig. 8. A regression line was plotted for each group.

Fig. 8.

Analysis (Pearson Product-Moment Correlation and linear regression analyses [t-ratio]) of the sizes of the shoulder, head/neck, and body representations within central, lateral, and medial zones in controls (C) and 1-to-12 week forelimb deafferents. A: Size of shoulder, head/neck, and body representations in the central zone for control and forelimb deafferents over post-deafferent weeks. B: Size of shoulder, head/neck, and body representations in the lateral zone for control and forelimb deafferents over post-deafferent weeks. C: Size of shoulder, head/neck, and body representations in the medial zone for control and forelimb deafferents over post-deafferent weeks. Note that the number in parentheses along the base line denotes the number of rats in a control or post-deafferentation week that were unresponsive to input from a particular body part representation.

Medial Zone

No significant differences in the total area of the shoulder and head/neck representations in the medial zone were found over post-deafferentation weeks. However, the body representation did show a significant difference and positive correlation (P≤.0001, t-ratio=4.49, r=0.60) over post-deafferentation weeks.

Central Zone

No significant differences in the total area of the body, shoulder, and head/neck representations in the central zone were observed over post-deafferentation weeks.

Lateral Zone

No significant differences in the total area of the shoulder representation in the lateral zone were observed over post-deafferentation weeks. In contrast, significant differences and positive correlations were observed for the body (P≤.003, t-ratio=3.24, r=0.49) and head/neck (P≤.01, t-ratio=2.98, r=0.45) in the lateral zone over post-deafferentation weeks.

2.4.3. Total mean areas of the shoulder, head/neck, and body representation as a percentage of their respective mean zonal areas over post-deafferentation weeks

The total averaged areas of the shoulder, body, and head/neck were calculated as a percentage of the total averaged area of each zone and these results are presented in Fig. 9. Regression analysis and Spearman Rank correlation were used to analyze the data. While these results are similar to the total areas of the body-part representations presented above, the averaged data nonetheless provide a useful day-by-day overview over post-deafferentation weeks.

Fig. 9.

Analysis (Pearson Product-Moment Correlation and linear regression analyses [t-ratio]) of the mean size of the shoulder, head/neck, and body representations as a percentage of mean zone area in controls and forelimb deafferents (1 to 12 weeks). A: Size of shoulder, head/neck, and body representations as a percentage of central zone area for control and forelimb deafferents over post-deafferent weeks. B: Size of shoulder, head/neck, and body representations as a percentage of lateral zone area for control and forelimb deafferents over post-deafferent weeks. C: Size of shoulder, head/neck, and body representations as a percentage of medial zone area for control and forelimb deafferents over post-deafferent weeks. Asterisks denote a significant regression.

Medial Zone

The percent body representation within the medial zone had a significant increase (P≤.0001, t-ratio=5.74) and positive correlation (r=0.67) over post-deafferentation weeks that reached a 90% occupancy during deafferent weeks 9–12. The shoulder representation occupied 14% of the medial zone in controls and increased to approximately 19% during 1-WD through 4-WD. In 5-WD, 51% of the medial zone was occupied by the shoulder, and subsequently dropped back to 24% in 6–8-WD and jumped to 33% during 9–12-WD. These changes were not significant. Rarely were inputs from the head/neck found in the medial zone.

Central Zone

An increase in the area of the body representation during 2-WD and 3-WD was observed in the central zone that subsequently decreased during the next 2 weeks before elevating again in weeks 6 through 12. Modest increases in percent occupancy were observed for the shoulder and head/neck representations during 2-WD and 3-WD. However, these differences were not significant for any of the representations within the central zone.

Lateral Zone

Approximately 40% of the lateral zone was occupied by the averaged shoulder representation in control rats. During 1-WD, the shoulder representation plummeted and then the percent occupancy gradually increased over post-deafferent weeks, although these increases were not significant. The head/neck representation showed a steady significant increase (P≤.001, t-ratio=0.51) and positive correlation (r=0.53) in percent occupancy during post-deafferentation weeks. The body representation began to increase at 2-WD and remained at a 15%–20% occupancy over the subsequent post-deafferentation weeks; these differences were significant (P≤.003, t-ratio=3.24) and had a positive correlation (r=0.54) over post-deafferentation weeks.

3. Discussion

The present study extends our previous detailed description of the physiological organization of CN in forelimb-intact juvenile rats (Li et al., 2012). The primary goals were to (a) determine the consequences of forelimb amputation on the functional organization of CN, (b) examine the time course for reorganization, and (c) compare our findings in CN with our previously reported findings of delayed large-scale cortical reorganization in forelimb barrel field cortex.

We previously reported that 4 weeks after forelimb amputation new input from the shoulder first appeared in deafferented forepaw barrel subfield cortex, and by 6 weeks the new shoulder input occupied a large part of the FBS (Pearson et al., 1999), the new shoulder input did not originate from the original shoulder cortex nor from the shoulder representation in SII (Pearson et al., 2001), and the new input did not appear until the fourth week after deafferentation (Pearson et al., 2003). From these results, we hypothesized that the substrate for delayed cortical reorganization very likely derived from subcortical circuits in the thalamus or CN. If this were the case, subcortical reorganization should appear prior to or around post-deafferentation week 4. In the present study, the left forelimb was amputated in juvenile rats and CN and surrounding regions were physiologically mapped to systematically examine the time course for reorganization during the first 12 weeks after amputation. Mapping was conducted at a location approximately 300 μm anterior to the obex, where a complete complement of CO-stained clusters was easily visualized in a single 100-micron thick coronal section; here, CN was readily separated into cluster and non-cluster regions. The cluster region corresponds with the central zone of CN. The non-cluster regions correspond to medial and lateral zones of CN that were aligned to the neighboring GN and tail region of CN, respectively. The areas of these 3 zones and the areas of the shoulder, head/neck, and body/chest representations within the zones were then quantified.

The present findings indicate that during the first 12 weeks following forelimb amputation, sites within medial and lateral zones become responsive to new input from body/chest and head/neck, while the central zone remains largely unresponsive. When new input was observed in the central zone, it was mostly confined to the outer regions adjacent to the medial and lateral zones; an exception was seen during the second and third post-deafferentation week, when new input from the shoulder, body/chest, and/or head/neck was transiently distributed throughout the central zone. Within the medial zone, there was a significant increase in new input from body/chest over post-deafferentation weeks and within the lateral zone there was a significant increase in new input from both body/chest and head/neck. Interestingly, no significant differences were found for new input for any body part representation in the central zone. Most importantly, we found no evidence for reorganization of the shoulder representation in CN over the time course of this study. We interpret these findings to suggest that CN does not provide new shoulder input to deafferented forepaw cortex and is therefore not a substrate for large-scale cortical reorganization.

3.1. Organization of the cuneate nucleus into zones

The organization of CN in rat has been previously described (Bermejo et al., 2003; Maslany et al., 1990; Maslany et al., 1992; Nord, 1967). Recently, we reported the functional organization of CN by making closely spaced electrode penetrations and recording receptive fields of neurons throughout CN and neighboring nuclei (Li et al., 2012). The centrally located CO clusters were associated with a complete somatotopic representation of the glabrous forepaw digits and pads. The territory outside the clusters was associated with the representation of the dorsal digits and dorsal hand and ulnar and radial representations of the wrist, arm, and portions of the shoulder. These data permitted us to produce a standard map that separated CN into cluster and non-cluster zones. In the present study, demarcation lines were added to the standard map that allowed further separation of CN into medial and lateral zones that were associated with the representation of the ulnar wrist, arm, and shoulder and radial wrist, arm, and shoulder, respectively. One line, angled at 126°, was abutted against the dorsomedial edge of the cluster region and ran approximately parallel to the border separating CN from the adjacent GN. The second line was placed dorsolateral to the base of the tail region. For each experiment, CO-stained coronal sections through the recording sites were reconstructed and dorsomedial and dorsolateral lines were placed on the reconstructed morphological map. The physiological map was then superimposed over the morphological map using lesion sites and blood coagulated electrode tracks as fiducials. We then reconstructed the recording sites from 5 forelimb intact control rats and noted that several sites in the medial and lateral zones received inputs from the body/chest and head/neck. The appearance of these anomalous receptive fields, in forelimb intact control rats, would have to be taken into account for any interpretation of reorganization in forelimb amputated rats.

3.2. Technical comments

Unlike the FBS (Dawson and Killackey, 1987; Waters et al., 1995; Welker and Woolsey, 1974) where the forelimb is represented in layer IV along a horizontal plane, the forelimb map in CN is represented along a dorsal-to-ventral plane whereby different body parts are represented along the depth of the penetration (Li and Waters, 2010). In the present study, physiological maps of CN were generated in forelimb intact and forelimb amputated rats by systematically advancing the electrode in 50- or 100-μm steps through the brainstem and recording receptive fields; electrode penetrations were spaced at a distance of 100 μm apart, where possible. Physiological recordings were then superimposed on morphological maps to plot the locations of penetration sites in relationship to the zones within CN. The size of a receptive field at any location along a penetration included the point where the electrode was located during the actual recording of the receptive field and the half distance to the next recording site in that penetration as well as the half distance to the recording site in the adjacent penetration. Therefore, a receptive field territory could encompass tissue never actually penetrated by the electrode but nonetheless included within its actual measurement. Depending on the location of a neighboring electrode penetration, the receptive field territory could even crossover into an adjacent CN zone. In the present study, examples of cross over were commonly encountered in both controls and forelimb deafferents, and in those cases, the area of encroachment was minimal and did not appear to alter the interpretation of the data.

Technical problems were also inherent in reconstructing closely spaced electrode penetrations, the largest of which was an inaccurate placement of the electrode penetration. In the present study, electrolytic lesions were used sparingly during the actual mapping to eliminate tissue damage in an unmapped region. However, lesions were always placed at the beginning and end of a row of electrode penetrations. In addition, lesions were also made at selected sites within a penetration, but these were generally done at the end of the experiment, and only at sites where the receptive field coincided with that recorded in the originally mapped site. We used settings on the microdrive to make closely spaced penetrations that were then transferred to a grid matrix. However, the grid was not always perfectly uniform due to an effort to avoid large surface vessels; in those cases where the electrode spacing was adjusted, every effort was made on the succeeding penetration to reposition the penetration back into the grid. Nonetheless, a slight displacement of an electrode track could inadvertently move the electrode penetration into an adjacent zone during reconstruction, producing anomalous receptive fields within a zone.

We separated the middle region of CN into medial, central, and lateral zones by placing angled lines at dorsomedial and dorsolateral locations. As mentioned, the medial and lateral zones are associated with the representation of the ulnar and radial wrist, arm, and shoulder. However, there is a region directly above the central zone that receives input from the dorsal digits and dorsal hand; this region is devoid of CO-stained clusters. No attempt was made to separate this area into a separate dorsal zone, and it was therefore included as part of the lateral zone. Since the medial edge of the lateral zone was adjacent to the medial zone, input from the body could encroach on the lateral zone producing another source of anomalous receptive field input.

Forelimb amputation leaves a sensitive stump, which is then covered by fascia and sutured skin from the adjacent area. While we cannot be certain that stimulation applied over the stump region did not activate both the overlying skin and stump, this region was always probed by lightly brushing the skin with a camel-hair brush. It is possible that some of the cutaneous responses resulted from activation of the stump, but in most cases we were able to differentiate stump responses from cutaneous activation of the skin by lightly tapping the stump area with a wooden probe. This technique was also used to study cortical reorganization following forelimb amputation (Pearson et al., 1999).

One concern is that the unexpected absence of new shoulder input in the central zone and the non-significant differences in new shoulder input in medial and lateral zones following forelimb amputation may be due to a limitation in sampling. To examine reorganization in CN, we elected to focus on mapping receptive fields of neurons along a single mediolateral row of closely spaced electrode penetrations at approximately 300 microns anterior to the obex that contain a well-demarcated morphological map of the forelimb representation. This mediolateral location permits consistency in sampling across both forelimb intact controls and amputees and the results can be readily compared to previously published maps of shoulder reorganization within the barrels in deafferented forelimb cortex (Pearson 1999, 2003). In all experiments, multiple rows of penetrations were made. It is important to underscore that where multiple rows of penetrations were made, receptive fields of neurons in the immediate adjacent row(s) were similar to those examined at +300 microns. For the purposes of this paper, however, only that row of electrode penetrations that was fully aligned to the underlying morphology map was selected to analyze and reconstruct. The fasciculus cuneatus overlies CN and contains axons from primary afferents of the forelimb and shoulder although cell bodies may also be found in this superficial region leading some investigators to include this region as a part of the rostral CN region (Bermejo et al., 2003). Since we did not distinguish between recordings made from axons or cell bodies while recording in the fasciculus, it is unknown whether the shoulder receptive fields belonged to axons of cell bodies in the adjacent lateral or tail regions of CN or to more caudal sites within the central zone. Nonetheless, if shoulder reorganization occurred in the central zone of CN, it would likely be reflected, in part, within the CO-rich central zone, which was not the finding for any post-amputation period examined in the present study.

3.3. Differential reorganization within zones

Our results clearly indicate that reorganization occurs differentially within the 3 separate zones. Almost immediately following amputation, there is a significant increase in new input from the body entering the medial zone and a significant increase in new input from the body and head/neck entering the lateral zone over post-deafferentation weeks. These findings are in contrast to the modest non-significant new input entering the central zone during post-amputation weeks. During post-deafferentation weeks 2–3, there is a slight increase in the area of the body representation within the central zone, but this increase does not reach significance during the period of study. Whether this increase during weeks 2 and 3 is meaningful or reflects the potential bias from the results of one rat remains to be determined. It is noteworthy, that no increases in new shoulder representation were found in any zone despite the fact that new shoulder representations are present in the FBS beginning in post-amputation week 4 (Pearson et al., 2003). These findings of a paucity of new shoulder input to the central zone appear similar to CN physiological maps obtained at a comparable level to the obex following neonatal (Lane et al., 2008) or embryonic (Rhoades et al., 1993) forelimb amputation.

3.4. Does reorganization in CN form a substrate for cortical reorganization

A number of similarities and differences exist between the present study and our previous report of delayed large-scale reorganization in FBS following forelimb amputation (Pearson et al., 2003). In deafferented cortex, we measured inputs only from the shoulder, while in deafferented CN, we also examined and measured inputs from the head/neck and body/chest. As a result, we do not know whether the reorganization of body parts other than the shoulder are expressed in barrel cortex. The shoulder representation in barrel cortex is located approximately 3 mm posterior to the forepaw representation, and we never encountered inputs from the shoulder or arm in the FBS in forelimb intact rats. In CN, the shoulder is represented in the adjacent lateral zone along with representations from the radial wrist and arm, but was never encountered in the barrelette-containing central zone. At 6 weeks following forelimb amputation, islands of new input from the shoulder were scattered throughout all of FBS, and the areas of these new representations were significant over post-deafferentation weeks. In contrast, few sites in the central zone in CN became responsive to new shoulder input at 6 weeks post-amputation nor were significant differences in new shoulder input found in any CN zone over post-amputation weeks. In cortex, new input from the shoulder was observed in the wrist and arm representations in week 2, and by 4 weeks, new shoulder input was recorded in the FBS. In CN control rats, sites responsive to shoulder input were recorded in lateral and medial zones, and at 2–3 weeks post-deafferentation, a transient increase in new shoulder input was found in the central zone that returned to levels similar to control rats in subsequent weeks. In no cases within the central zone were inputs from the shoulder, or for that matter body/chest and head/neck, significantly different over post-deafferentation weeks, although significant differences in the sizes of head/neck and/or body/chest representations were observed in medial and lateral zones.

It is possible that primary axons from the shoulder sprouted into the central zone but were functionally unexpressed. Similar findings of a mismatch between the appearance of sprouted hindlimb afferents into CN and their functional expression have been reported (Rhoades et al., 1993); however, even at 30 weeks post-amputation, few neurons in the central zone responded to input from the shoulder and those that did were relegated to the border region.

In the present study we reported reorganization in CN beginning within 1 week after forelimb amputation, but the absence of significant new input from the shoulder in any zone argues against the role of CN as a substrate for cortical reorganization. This failure of cuneothalamic projecting neurons, particularly in the central zone to become responsive to new input from the shoulder following forelimb amputation was not anticipated. If the central zone in CN does not reorganize to permit the expression of new shoulder input onto cuneothalamic relay neurons in the forepaw central zone, how does the new shoulder input gain access to the FBS following forelimb amputation? One possibility is that cuneothalamic projections (Alloway and Aaron, 1996; Kemplay and Webster, 1989; Massopust et al., 1985; Webster and Kemplay, 1987; Wree et al., 2005) from neurons receiving input from the shoulder in lateral and tail-zones of CN in forelimb-intact rats send wide-spread projections to the somatopically organized VPL (Li et al., 2006). Preliminary evidence from injections of BDA into physiologically identified sites in the shoulder region of CN suggests that shoulder-responsive neurons send strong projections to the shoulder representation in VPL but also label axonal branches in the trunk and forelimb representation (Li et al., 2006). Similarly, microstimulation delivered to forepaw VPL antidromically activated neurons in both forepaw and shoulder regions of CN (Li et al., 2006). The fact that these shoulder inputs in forepaw VPL in forelimb-intact rats are not sufficiently strong to drive these cells suggests that their expression is likely under inhibitory control from the reticular nucleus (Li et al., 2005), since rodent VPL does not contain inhibitory interneurons (Barbaresi et al., 1986). While these cuneothalamic studies were conducted in rats with intact forelimbs, we predict that in the forelimb deafferent, neurons in forepaw VPL shed their inhibitory control and become responsive to new input from the wrist, arm, and shoulder (Li et al., 2005). This new shoulder input in the deafferented forepaw VPL is in turn relayed to the deafferented FBS, suggesting that VPL forms the substrate for large-scale cortical reorganization (Li et al., 2006).

4. Experimental procedure

4.1. Animals

Single and multiunit extracellular recordings were used to map the forelimb representation in CN and immediate surrounding regions in rats (n=39) between 8 and 30 weeks of age. Of this number of rats, 34 had a left forelimb removed and these deafferents were mapped 1 week (1-WD) to 30 weeks (30-WD) following amputation. The remaining 5 rats, with intact forelimb, were mapped and served as controls. These experiments conformed to the Principles of Laboratory Animal Care (NIH publication No. 86–23, revised 1985) and were approved by the Animal Care and Use Committee, University of Tennessee Health Science Center.

4.2. Forelimb amputation

Forelimb amputation was previously described in rats (Pearson et al., 1999). Under aseptic conditions, rats between 6 and 8 weeks of age were anesthetized with Nembutal (35 mg/kg, i.p.), the skin and external shoulder muscles were reflected around the humerus, and the limb was amputated at the glenno-humeral joint. The forelimb nerves were then ligated with surgical sutures (000). The brachial artery was cauterized near the brachial plexus. The skin surrounding the wound was closed using surgical sutures and bupivacaine (0.7%) was topically applied to the wound prior to closure for local analgesia. Postoperatively, animals were given buprenorphine (0.01–0.05 mg/kg SQ, BID) for the first 48 postoperative hours for systemic analgesic effects. An antibiotic, Crystiben (Penicillin G) at dose of 1.5 mg/kg, was also administered at the end of the surgery. Rats were monitored until they recovered from anesthesia. On the following day, they were returned to their home cage with ad libitum access to food and water until physiological mapping. Animals were thereafter monitored daily in their home cage.

4.3. Physiological mapping

The details of the animal preparation and physiological recording were previously described (Pearson et al., 2003; Waters et al., 1995). Forelimb deafferented rats were physiologically mapped 1 to 30 weeks after amputation. Rats were anesthetized with Ketamine/Xylazine (100 mg/kg) and supplemented with a 10% dosage to maintain areflexia. The hair on the head, neck, limbs, trunk, and face was shaved, and the rat was placed on a water-circulating heating pad to maintain body temperature between 36°–38°C. The animal’s head was secured in a stereotaxic frame, and sterile saline (0.9%) was administered (i.p.) at hourly intervals for fluid maintenance. The bone overlying the brainstem was removed to expose the brainstem in the region of the obex, the dura was opened, a recording chamber was placed around the opening, and the brain surface was covered with warmed silicone fluid. A digital image of the brainstem surface was viewed on a computer screen and used to mark the location of the surface point of entry of electrode penetrations.

A carbon fiber electrode attached to a Canberra-type microdrive was used to record unit responses from neurons within the brainstem. Responses were amplified and fed into a storage oscilloscope and audio monitor. A wooden probe or fine-tipped brush was used to examine the cutaneous receptive field of neurons along an electrode penetration; deep responses from muscle and joint were measured by palpating the muscle or stretching the limb. Receptive fields were measured at 50-or 100-μm intervals along a penetration, and the measured receptive fields were drawn on a map of the body surface (see Fig. 10). The receptive field was defined as the location on the skin surface where minimal stimulation evoked a maximum response. Sites over the stump region were always measured by using a brush to lightly stimulate the skin surface. In most cases, tapping with the wooden probe activated deeper responses from the underlying stump. Every effort was made to separate cutaneous responses from the overlying skin from the deeper responses evoked from the stump.

Fig. 10.

Line drawing of the body map along with nomenclature used for plotting receptive fields. A: Body map nomenclature as follows: A = abdomen; B = back; C = chin; CH = chest; E = ear; F = face; H = head; Hip = hip; HL = hindlimb; J = jaw; N = neck; S = side; SH = shoulder; T = tail; Ton = tongue; Vib = vibrissae; ST = stump; SU = suture. Sub-nomenclature for the back, side, abdomen: r = rostral; c = caudal; sub-nomenclature for the neck: r = rostral; m = middle; c = caudal; sub-nomenclature for the hindlimb: p = proximal; d = distal.

Receptive field mapping commenced by inserting the recording electrode 100 μm below the surface of the brainstem in the vicinity of the obex. Sites along a penetration were mapped until 2 successive unresponsive sites were encountered or until the electrode reached a depth of 800–900 μm. Individual electrode penetrations were spaced approximately 100 μm apart in the medial-to-lateral plane as determined from micrometer readings on the microdrive. Every effort was made to avoid large surface vessels, and where a vessel was present, the electrode was placed adjacent to the vessel; in these cases, the penetration was less than 100 μm. Penetration sites and recording sites within a penetration were plotted on the computer screen image of the brainstem surface, and transferred to a grid matrix. Forelimb representational boundaries were established at penetration sites that were unresponsive and/or at penetration sites yielding input from an adjacent body part. Electrolytic lesions (cathodal current, 5 μA × 5 s) were made at the beginning and end of each row of penetrations and at a depth of 100 μm in selective penetrations.

4.4. Tissue processing

Following mapping, animals were given a lethal overdose of Nembutal and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde in 0.15M sodium phosphate buffer solution (NaPBS, pH 7.4, 21°C). The brainstem was removed and fixed in 4% paraformaldehyde at 4 °C and refrigerated overnight. The brainstem was sectioned coronally at 100-μm thickness using a Vibratome. Sections were placed in buffer solution (KPBS, pH 7.4, 21 °C), reacted with cytochrome oxidase (CO), and mounted on gelatin-coated glass slides, air dried overnight, and coverslipped. Intracellularly labeled neurons were reacted with DAB and the background tissue was countered-stained with CO, as previously described (Arnold et al., 2001; Li and Waters, 1996; Li et al., 2002).

4.5. Map reconstruction

Sections were digitized and reconstructed in Photoshop. The borders of CN and neighboring gracilis and spinal trigeminal nuclei were identified from CO-stained sections and used to generate a morphological map. A physiological map was produced from the receptive field data collected from each electrode penetration, and this map was superimposed on the morphological map by aligning the locations of lesions in the 2 maps that served as fiducials. The mismatch between morphological and physiological maps never exceeded 25 μm at any of the lesion sites. Electrode penetrations and receptive field(s) recorded along these penetrations were then extrapolated from the lesion data and plotted in relationship to the underlying morphological map. Electrode tracks could often be seen where blood had coagulated, and these tracks were also used for receptive field reconstruction. Data collected for this study were obtained at approximately 300 μm anterior to the tip of the obex where a complete map of CO-stained clusters representing the forelimb was present.

4.6. Data grouping

Animals were grouped according to the time of amputation and mapping. The 1-week deafferent group (1-WD) had 4 rats that were mapped 1 week after amputation. The 2-week deafferent group (2-WD) had 4 rats that were mapped 2 weeks after amputation, and the 3-week deafferent group (3-WD) had 5 rats that were mapped 3 weeks after amputation. The 4-week deafferent group (4-WD) had 3 rats that were mapped 4 weeks after amputation, and the 5-week deafferent group (5-WD) had 4 rats that were mapped 5 weeks after amputation. The 6-through 8-week deafferent group (6–8-WD) had 6 rats – 2 rats that were mapped 6 weeks after amputation, 2 rats that were mapped 7 weeks after amputation, and 2 rats that were mapped 8 weeks after amputation. The 9- through 12-week deafferent group (9–12-WD) had 6 rats – 1 rat that was mapped 9 weeks after amputation, 1 rat mapped at 10 weeks after amputation, 3 rats that were mapped 11 weeks after amputation, and 1 rat that was mapped 12 weeks after amputation. The 26-week deafferent group (26-WD) and 30-week deafferent group (30-WD) each had 1 rat. All rats were amputated between 6 and 8 weeks of age.

4.7. Analysis

Areal measurements of physiological maps and total areas of CN and total areas of medial, central, and lateral zones were made using Image J (NIH). Measurements were then placed in a relational database (DataDesk), and all measures were compared using Pearson Product Moment or Spearman Rank Correlations and linear regression analyses (t-ratio).

Highlights.

• We report reorganization in rat cuneate nucleus (CN) following forelimb amputation.

• Electrophysiology was used to map CN up to 30 weeks after amputation.

• Results compared to our findings of reorganization in rat forepaw barrel cortex.

• CN reorganization forms an unlikely substrate for reorganization in barrel cortex.