Changes in the organization of the ventroposterior lateral thalamic nucleus after digit removal in adult raccoon

THE JOURNAL OF COMPARATIVE NEUROLOGY 364~92-103 (1996)

Changes in the Organization of the Ventroposterior Lateral Thalamic Nucleus

After Digit Removal in Adult Raccoon

DOUGLAS RASMUSSON Department of Physiology and Biophysics, Dalhousie University,

Halifax, Nova Scotia B3H 4H7, Canada

ABSTRACT The ventroposterior lateral nucleus of the thalamus was studied in seven raccoons that had

undergone amputation of the fourth digit between 2 and 5 months previously. Extracellular recordings were made in a series of closely spaced penetrations through the thalamus in chloralose anesthetized animals. The responses to cutaneous stimulation of the forepaw were used to reconstruct the somatotopic organization of the thalamus and to identify recording sites believed to be located in the digit zone that had lost its peripheral input. Twelve penetrations that passed through both of the adjacent fifth and third digit regions were analyzed in detail to delineate this deafferented region. None of the recording sites in this region were completely silent, indicating that the deafferented thalamus had undergone significant reorganization of its inputs. At most sites, the neurons had receptive fields on the skin surrounding the amputation wound and including one of the adjacent digits. Approximately half of the sites had low thresholds in the range of normal thalamic neurons. These results indicate that the ventroposterior thalamus is capable of substantial reorganization, which may account for much of the reorganization seen in somatosensory cortex. L 1556 Wiley-Liss. Inc.

Indexing terms: plasticity, thalamus, denervation

Since the first clear demonstration that peripheral denervation leads to significant reorganization in the primary somatosensory (SI) cortex of the adult mammal (Kalaska and Pomeranz, 19791, the phenomenon has been studied in a variety of species (cf. reviews in Kaas, 1991; Snow and Wilson, 1991). These studies have shown that a deafferented region of SI cortex becomes responsive to inputs from regions of skin surrounding the denervation. This phenomenon is often called “cortical reorganization,” and numerous theoretical discussions have concentrated on possible changes at the cortical level (Merzenich, 1987; Pearson et al., 1987). Although it has been recognized by many investigators that some or all of the results seen when recording from the cortex may be due to changes occurring at subcortical regions, it is surprising how few studies have examined the subcortical relays in the dorsal column- medial lemniscal system. The most extensive study on the dorsal column nuclei (Kalaska and Pomeranz, 1982) found reorganization in the cat only when denervation of the forepaw was done at an early age. Two studies published since 1991 suggested that the ventroposterior lateral nucleus (VPL) of the thalamus is able to undergo extensive reorganization. In one study, histochemical changes were seen in the VPL of monkeys that had undergone extensive reorganization at the cortical level (Rausell et al., 1992), whereas,

in the other study, physiological evidence of reorganization was provided in monkeys after transection of the median and ulnar nerves (Garraghty and Kaas, 1991). In addition, recent studies have reported short-term changes in the receptive fields (RFs) or response properties of neurons in both the VPL (Nicolelis et al., 1993; Rasmusson et al., 1993a) and the cuneate nuclei (Pettit and Schwark, 1993). The purpose of the present experiment was to examine the VPL of the raccoon by using a denervation procedure (amputation of a single digit) that has been well studied by several investigators and has been shown to produce clear reorganization in the raccoon SI cortex (Kelahan et al., 1981; Rasmusson, 1982; Kelahan and Doetsch, 1984). The somatotopy of the representation of the forepaw in the raccoon VPL has been extensively described by several investigators (Welker and Johnson, 1965; Warren et al., 1986; Wiener et al., 1987a,b). It is characterized by distinct subnuclei for each of the digits with the fifth digit (D5) represented in the most lateral subnucleus and the first digit (D1) represented most medially. Each digit is repre-

Accepted July 11, 1995 Address reprint requests to Douglas Rasmusson, Department of Physiol-

ogy and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.

C 1996 U’ILEY-LISS, INC.

REOKGANIZATION OF VPL IN ADULT RACCOON 93

sented in a progression from proximal to distal on the digit as one moves more ventrally in the VPL. To search for changes in this nucleus, the progression of RFs was examined in a series of closely spaced penetrations through the VPL in animals that had undergone digit amputation 2-5 months prior to recording. The results demonstrate that the VPL thalamus undergoes a reorganization of its somatotopy similar to that seen in the cortex, with the deafferented zone being taken over by inputs from adjacent digits. A preliminary report of some of these data was presented previously (Rasmusson et al., 199313).

MATERIALS AND METHODS Data were obtained from seven adult raccoons of either

sex weighing between 4.4 and 11.0 kg at the time of initial surgery. The fourth digit (D4) of either the right (n = 4) or left forepaw (n = 3J was amputated using sterile surgical techniques. The animal was initially anesthetized with 100-200 mg ketamine hydrochloride (i.m., Ketalean; MTC Pharmaceuticals), and the trachea was intubated so that halothane (2-4%) could be administered. Heart rate was monitored throughout surgery to facilitate the regulation of the depth of anesthesia. D4 was amputated at the metacarpophalangeal joint. Each digital nerve was ligated with 3-0 suture before cutting, and the tendons and soft tissue were cut via cautery. The wound was closed with three or four sutures, and a local antibiotic spray (Neospo- rin) was applied. The animal was given a single intramuscu- lar dose of long-lasting antibiotic (15 mgikg cesazolin sodium; Keszol, LillyJ. Recovery and healing of the wound were uneventful in all cases.

At a postamputation interval ranging from approximately 2 to 5 months (69-158 days), the animal was prepared for electrophysiological recording. The raccoon was again preanesthetized with ketamine so that an intrave- nous catheter could be inserted into the radial vein of the arm contralateral to the amputation. The anesthetic u-chloralose was then administered in an initial dose of 2 ml (5%. solution, i.v.) followed at approximately hourly intervals by supplemental doses of 0.8 ml. A corticosteroid (Solucortef, Upjohn) was also administered in two doses (50 mg, i.v.) a t the time of craniotomy and approximately 2 hours later. A tracheotomy was performed on all animals.

The animal was secured in a Kopf stereotaxic instru- ment, and an incision and craniotomy were made over the cortex contralateral to the amputation. A well of dental acrylic was secured to the skull around the craniotomy, and the dam was filled with warmed mineral oil (37°C). The dura was removed, and the cortex and vascular pattern were photographed to record the location of microelectrode penetrations.

Penetrations were made at an angle of 0-45” from vertical in the coronal plane. Recordings were made using Parylene-C-coated Tungsten microelectrodes (2-5 IZM impedance; A-M Systems, Everett, WAJ. Because these elec- trodes are fairly rigid, no guide tubes were used. The signal was amplified using a high input impedance amplifier (CWE Inc., Ardmore, PA) and was monitored via an audio speaker and an oscilloscope. The microelectrode was advanced with a piezoelectric microdrive (Burleigh, Fishers, NY) that gave continuous feedback as to the depth of the microelectrode.

The entry of the microelectrode into the thalamus was indicated by a dramatic increase in background firing and often by the presence of neurons firing in bursts. The

microelectrode was then advanced in 200-500 pm steps until cells with cutaneous RFs were encountered. After this, the microelectrode was advanced in either 100 pm or 250 pm steps, and the characteristics of the neural responses at each recording site were noted. A penetration was continued until neural activity or responses to cutaneous stimulation were absent for at least 1 mm. Subsequent penetrations were spaced at small distances (250-500 pm) medially or laterally until the region surrounding the lateral representation of the forepaw was completely mapped. The electrode was then moved 500 pm rostra1 or caudal, and another row of penetrations was made. Two or three rows of penetrations were made in each animal.

The information obtained at each recording site consisted of the depth from the cortical surface, the minimal RF size as determined using a small-tipped glass probe or von Frey hair (Stoelting; Wood Dale, ILJ, and a qualitative estimate of the threshold. Low-threshold cutaneous responses were considered to be equivalent to those of neurons in normal regions of the thalamus or the SI cortex. High-threshold responses required stronger than normal stimulation but still had an identifiable, but usually not small, RF. “Tap” responses required a sharp tap to one or more digits or to the palm but showed no clear RF.

A small electrolytic lesion was made at the bottom of at least one penetration in each row of penetrations. At the end of the experiment, the animal was given pentobarbital (130 mg, i.v.) and was perfused transcardially with buffered saline followed by 10% buffered formalin. Blocks containing the thalamus were postfixed for several days and were then placed in 15%, sucrose for at least 24 hours. Frozen sections, 60 pm thick, were cut in the frontal plane, mounted on chrome alum-subbed slides, and stained with thionin for confirmation that the penetrations were within the VPL. Assignment of each recording site to cytoarchitecturally defined subnuclei was impossible without numerous lesions that would have disrupted the physiological recordings. Thus, penetrations were reconstructed using the depth of recording sites relative to the entry into the thalamus and the spacing between penetrations. Recording sites with RFs on the same body part were then grouped to reveal the functional map of the VPL.

RESULTS Somatotopic organization of the VPL

The somatotopic organization of the raccoon VPL has been described previously (Welker and Johnson, 1965), and the digit subnuclei can be seen easily in frontal sections stained for Nissl substance (Fig. 1A) as well as for myelin, cytochrome oxidase, or acetylcholinesterase (Wiener et al., 1987a). Discrete crescents of densely packed neurons are separated by bands of myelin. The lateralmost crescent contains the representation of the hindlimb, whereas the forepaw digks are represented from digit 5 laterally to digit 2 medially (Fig. 1B). The representation for digit 1 swings caudally; therefore, it is not seen in this section. From the orientation of these digit regions, it can be appreciated that a vertical penetration might cross from one diBt region to another, but it might also remain within one region for a long distance or might even pass between two regions. Angled penetrations, on the other hand, are more likely to pass through three or more regions (Welker and Johnson, 1965).

94 D. RASMCSSON

B

5mm - \ Fig. 1. Photomicrograph (A) and schematic drawing (B) of a frontal

section through the left thalamus illustrating the organization of the normal raccoon ventroposterior lateral nucleus of the thalamus (VPL). Numbers indicate forepaw digits (digit 5 is the ulnarmost digit). The digit 1 representation is caudal to this section. HL, hindlimb representation; AV, anterior ventral nucleus; MD, mediodorsal nucleus; VPI, ventroposterior inferior nucleus, Rt, reticular nucleus.

A total of 114 penetrations were made in the thalami of these animals. Vertical penetrations (n = 69) were particularly informative about the dorsal-ventral organization of the VPL. The initial cells with low-threshold cutaneous responses that were encountered in these penetrations were excited from the hairy skin of the back, trunk, or forelimb except at the lateral edge of the VPL, where they were excited from the hindlimb. With an increase in depth, the RFs progressed distally down the forelimb, then down the palm, and, finally, down a digit. Ventral to the represen-

tation of the glabrous tip of the digit, there was often a region that responded to movement of the claw of that digit.

The somatotopy within the palm and digit zones can be seen in the data from a representative animal in Figure 2. The RF locus at each recording site within four vertical penetrations is indicated in Figure 2A, and representative RFs (Fig. 2A, asterisks) are shown in Figure 2D. A recon- struction of boundaries between zones that had RFs on the same body part is shown in Figure 2B. The region represent- ing the hairy skin of the forearm and wrist was dorsal to the palm region in each case and is not shown in Figure 2. The large pads of the palm are traditionally labeled from A to E as shown in the first diagram of Figure 2D. The ulnar-to- medial progression can be seen for both the digits (D5 to D1) and the palm (pads D-A). The change in RFs with increasing depth is indicated by arrows in each diagram of Figure ZD, illustrating the proximal-to-distal progression within both the palm and the digit regions. The dorsal- ventral extent of the palm region was approximately 1-2 mm except medially, where it tended to be much narrower. The representation of individual palmar pads could not be restricted to distinct zones, because many RFs extended across the border between two pads. The distal palm and pads, however, were consistently in register with the digits; i.e., the palm RFs just dorsal to each digit region were always on the palmar pad adjacent to that digit. The shift of RFs from palm to digit zones was gradual; in only three vertical penetrations was there a clear silent region of several hundred microns interposed between palm and digit representations. This is consistent with the absence of any cytoarchitectonic division between palm and digit representations (Fig. 1A).

Several instances in which these vertical penetrations passed from one digit representation to another are illustrated in Figure 2, particularly in the medialmost penetration (penetration 8) in which the RFs jumped from D1 to D2 to D3. The consistent proximal-to-distal progression within each digit region can be seen best by comparing the RFs of several penetrations. For example, penetration 5 passed through the dorsal part of the D3 zone, where it encountered cells with an RF on the proximal digit pad, whereas a more medially placed penetration (penetration 8) encountered cells with an RF on the distal part of D3 at a greater depth. The medial-lateral somatotopy within a digit zone is more subtle but can be seen from a comparison across penetrations 7,6, and 5 in Figure 2D: The ulnar side of D5 is represented in the lateral penetration (penetration 71, whereas the radial side is represented medially (penetration 5).

Somatotopy in and around the deafferented D4 subnucleus

Vertical penetrations were not particularly useful in delineating the deafferented region due to the orientation of the digit zones with respect to the angle of the penetrations. Only if a penetration passed through both D3 and D5 representations could one be confident that it had also passed through the deafferented D4 region. Out of 44 vertical penetrations in the lateral part of VPL, only four met this criterion. Consequently, penetrations that were angled to pass through several digit regions were used to identify the deafferented region and to determine the characteristics of the neural responses in this region.

If no reorganization had taken place within the thalamus, then the D5 and D3 digit zones should have been separated by a silent zone. This was not the case. In all such

REORGANIZATION OF VPL IN ADULT RACCOON

A Pen # 7 6 5 8

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Fig. 2. Summary of receptive fields (RFs) seen in four vertical penetrations in an animal studied 101 days after amputation. The spacing between penetrations was 300 pm. A The location of the RF is indicated for each recording site within each penetration (penetrations 7, 6, 5, and 8 ) . Those RFs with an asterisk are illustrated in D. For the palm RFs. the label indicates the pad that contained the center of the RF. B: Summary map reconstructed from the data in A showing the zones for different regions of the forepaw relative to the penetrations

penetrations, the neurons in the intervening deafferented D4 region were found to be responsive to new inputs from the adjacent digits and the palm surrounding the wound. An example is shown in Figure 3. Seven parallel penetrations separated by 400 pm were made at a 45" angle. The response characteristics were determined every 100 pm within each penetration. The two lateralmost penetrations (Fig. 3, penetrations 17 and 18) passed through both D5 and D3 retrions: therefore. thev must have traversed the

Pen. #5 Pen. # 8

(dashed vertical lines; penetrations 7, 6, 5 , and 8 ) . C: Drawing of the histological section containing these penetrations to illustrate their approximate location within the thalamus. Abbreviations as in Figure 1. D: Representative RFs from each of the four penetrations. The arrows indicate the progression of RFs with increasing depth in each penetration. The palmar pads are identified as shown in the left drawing in D; pads A-E are as defined by Welker and Seidenstein (1959). P, center of palm; T, thenar eminence.

deafferented D4 zone. The RF sequences for these two penetrations are illustrated in Figure 3C, with low- threshold fields shaded black and with high-threshold fields stippled. Normal D5 and D3 regions are characterized by small RFs and low-threshold responses. The locations of the RFs in normal D5 and D3 zones are also consistent with their somatotopic organization, e.g., proximal RFs on both D5 and D3 are dorsal to distal RFs within a penetration and when comuaring medial (uenetration 17) with lateral (Den-

96

A

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D. RASMUSSON

B

Figure 3

REORGANIZATION OF VPL IN ADULT RACCOON 97

Fig. 4. Another example of two adjacent penetrations that passed through D5 and D3 zones in an animal studied 155 days after amputation. The penetrations were separated by 500 pm and were inserted at a 35" angle. RFs are coded as in Figure 3 according to their

threshold to cutaneous stimulation: low threshold (black) or high threshold (stippled). The putative deafferented zone (thicker bar) was very small in this animal, with only two recording sites in each penetration lying between D5 and D3 zones.

etration 18) penetrations. The D5 and D3 regions defined in this way are seen to surround a zone (indicated by the thicker bar in each penetration in Fig. 3) that is considered

Fig. 3. Representative RFs in the deafferented and surrounding regions of an animal studied 69 days after digit amputation. A Schematic drawing of the thalamus illustrating the location of seven penetrations (dashed lines 12-18) that were made at a 45" angle with a separation of 400 pm. The boundaries within the VPL of representations of various body parts are illustrated by thin lines. B: Enlargement of boxed region in A illustrating the two lateral-most penetrations (17 and 18) that traversed both D5 and D3 regions and the intervening deafferented region (D). C: The RFs seen a t recording sites in penetrations 17 and 18. The distance between adjacent recording sites in each penetration was 100 pm. Black RFs indicate sites with low threshold to cutaneous stimulation similar to that seen in normal animals. Stippled fields had high thresholds. The region considered to be the deafferented zone is indicated by the thicker bar in both B and C. Note the progression from small low-threshold RFs on D5, to larger high- threshold RFs in the presumptive deafferented zone, and then to small low-threshold RFs on D3. Although amputation was performed on the left hand in this animal, the views of both hand and thalamus have been reversed to facilitate comparison to other figures. FA, forearm; W, wound; LD. lateral dorsal nucleus.

to be the deafferented region. Cells in this region tended to have high thresholds and generally had RFs that were much larger than normal. In many cases, these RFs included the skin surrounding the wound (Fig. 3, W) as well as one or both of the adjacent digits. In the animal illustrated in Figure 3, those RFs labeled W were entirely on the glabrous skin of distal pad C; however, in several other animals, the RF extended onto the hairy skin of the dorsum of the paw. It should be emphasized that the RFs around the wound were found at recording sites that were as much as 1 mm ventral to the normal representation of pad C.

Another example of two adjacent penetrations traversing the deafferented zone is shown in Figure 4. These data were obtained from an animal in which recordings were performed 5 months after amputation, and the penetrations were separated by 500 pm. The putative deafferented zone was encountered at only two recording sites in each of these penetrations. At two sites, the RFs consisted of two or three spatially distinct fields. Three of the four sites were judged to have low thresholds to cutaneous stimulation.

A total of 12 angled penetrations passed through both normal D5 and D3 zones, which were defined, as outlined

98 D. RASMUSSON

above, by the presence of small low-threshold RFs in a sequence consistent with the known somatotopy of the VPL. The size of the intervening D4 zone was estimated from the number of recording sites between I35 and D3 zones. This distance ranged from 100 to 1,400 pm. These distances were then converted to mediolateral width of the digit zone by multiplying them by the sine of the angle of the penetration. The resulting estimated width of the denervated D4 zone ranged from 57 to 803 pm (mean = 335). Calculating the mediolateral width of the D5 and D3 regions in these penetrations in the same manner yielded similar widths of 357 pm and 378 pm, respectively. Statisti- cal comparison of the width of the deafferented zone with the width of each of these two normal digit zones revealed no significant differences (Mann-Whitney test, P > 0.50 in each case).

The results from the four vertical penetrations that passed through both D5 and D3 zones are consistent with the observations made in the angled penetrations. In two penetrations (Fig. 2, penetrations 5 and 61, the RF jumped from D3 to D5 at adjacent recording sites separated by 250 pm. In the other two, there were intervening sites with RFs on a digit and the palm surrounding the wound, similar to the examples shown in Figure 3. Fifteen additional vertical penetrations likely entered the deafferented zone as the RFs shifted from D3 to the palm near the wound. These sites with RFs near the wound were at least 1 mm ventral to the normal palm zone. A deafferented zone could be in- ferred in some of the remaining vertical penetrations from the appearance of large or unusual RFs adjacent to normal D5 or D3 sites. Two penetrations, for example, had sites with RFs on the dorsum of a digit between sites with normal glabrous RFs.

The range of normal VPL RFs for each region of the forepaw that includes the smallest RF and the largest RF is illustrated in Figure 5A. Normal RFs were usually larger on the proximal pad of a digit than on the distal pad and were larger on the palm than on the digits. A few RFs that were centered on one of the digit pads extended slightly onto the other pad, probably due to the spread of mechanical forces around the crease separating these pads, but no normal RFs included the entire ventral surface of a digit. These normal RFs are comparable in size to those reported by other investigators using a variety of other anesthetics (Welker and Johnson, 1965; Warren et al. 1986; Wiener et al. 198713). The RFs at deafferented sites shown in Figures 3 and 4 and additional examples in Figure 5B-D show that the RFs at the deafferented sites were almost always larger than the normal RFs. Many RFs covered the entire ventral surface of a digit (e.g., three RFs in Fig. 3 and RF 1 in Fig. 5B). A number of RFs included skin on the hairy surface of one or more digits (Fig. 5C,D): This was never seen in this region of the normal VPL. Even more striking were the RFs that involved more than one digit or that involved discon- tinuous regions on a digit and around the wound (e.g., Fig. 5 , RFs 3, 7, and 9).

The relative occurrence of low- and high-threshold sites and of RFs of different sizes were evaluated by combining the response data at 62 recording sites that were confi- dently assigned to the deafferented zone. These data were all from the 12 angled penetrations that passed through D5 and D3 zones (Table 1). Neurons at all 62 sites were responsive to stimulation: Thus, there was no indication of a silent region. At slightly more than half of the sites,

neurons had low thresholds in at least part of their RF (33/62; 53%). RF size was evaluated by considering whether the RF was limited to a single digit or to the palm or whether it included two or more of these body parts. The vast majority of sites had responses to stimulation of the skin surrounding the wound (53/62), and approximately equal numbers had RFs that included D5 and D3 (32 and 34, respectively). At only 17 sites (27%) was the RF restricted to a single digit or to the palm. At 33 sites (53%), the RF included two of these parts, and, at 12 sites (19%), it included both digits and the wound. RFs that included both digits D3 and D5 were observed at 17 sites, a situation that is never seen in normal animals. The hypothesis that sites with smaller RFs might tend to have lower thresholds than those with large RFs was not borne out: A comparison of low- and high-threshold sites in terms of whether one, two, or three of these regions were included in their RFs was not statistically significant (x2 = 0.41; P > 0.50). There was also no difference in the frequency of low-threshold sites as a function of the time after amputation at which recordings were made.

DISCUSSION Evidence for thalamic reorganization

The demonstration that the somatosensory system of the adult animal is able to change in response to peripheral nerve damage has generated a great deal of interest. However, an understanding of the mechanisms responsible for this reorganization has been hampered by the lack of convincing studies identifying the locus where the change is occurring. Although many studies have demonstrated this phenomenon in SI cortex of a variety of animals, few have examined subcortical regions. Studies on the cortex are facilitated by the fact that the body is mapped in two dimensions in the cortex due to the near-identity of RFs within a vertical column (Mountcastle, 1957). The somatotopy of subcortical structures, on the other hand, varies in three dimensions, making it necessary to reconstruct the microelectrode tracts to see the somatotopic map of the nucleus and identify the affected region. I t is also easier to gain access to the cortex and to make accurate, closely spaced penetrations by relying on the blood vessel pattern on the cortical surface.

In the present study, penetrations through the VPL thalamus were closely spaced, and the response properties were identified at steps as small as 100 pm. By taking into account the normal progression of RFs within the VPL, it was possible to delineate the region deafferented by D4 amputation using the normal D5 and D3 regions that lie on each side of it. The data show that the deafferented region of the VPL is not silent but is responsive to inputs from adjacent digits and palm, indicating that a significant reorganization occurs within the VPL similar to that seen in SI cortex.

Several key features of the somatotopy of the raccoon VPL were used to define these areas. One was the consistent dorsal-to-ventral progression, with the palm region dorsal to the digit regions and with the shifting of RFs from proximal to distal within both the palm and digit regions. Another feature was the ulnar-to-radial progression when moving from lateral to medial both on the scale of the whole nucleus and within individual digit subnuclei. These organi- zational principles were first demonstrated within the


B A

C D

Fig. 5. A: Representative RFs from normal thalamic regions indicating the size of RFs in various regions of the hand. B-D: Examples of RFs (numbered 1-9) from the presumptive deafferented zone. A and B are drawings of the ventral, glabrous surface; C and D are drawings of

'I'mLE 1. Extent of RFs in Deafferented WL'

Region Low threshold High threshold Total

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the dorsal surface of the hand. The shading of RFs in B-D is used to distinguish RFs a t different recording sites. All of the RFs shown here were classified as having low thresholds.

raccoon VPL by Welker and Johnson (1965) and can be seen in the figures of the present paper. These principles were used to identify the likely deafferented zone in 12 penetrations. The absence of a silent region between D5 and D3 zones in any of these penetrations is strong evidence of reorganization at this level. The most noticeable character- istic of the RFs in the deafferented zone was their large size, which, in almost all cases, involved an adjacent digit and the skin surrounding the wound. A number of RFs extended onto both of the adjacent digits, a condition that is never seen in the VPL of normal animals. Other characteristics that were important in distinguishing this region from

100 D. RASMUSSON

surrounding normal thalamus were the elevated thresholds and the generally weaker responses at many sites. On the other hand, it is remarkable that half of the sites in this region were categorized as having low thresholds despite their abnormally large RFs.

The only other comparable report of thalamic reorganization after peripheral denervation in the adult mammal is by Garraghty and Kaas (19911, who examined three squirrel monkeys in which the median and ulnar nerves had been transected. At intervals similar to those used here (2-5 months), they found that the dorsal surface of the hand had taken over the parts of the VPL that had previously been dominated by the glabrous surface. The changes seen in the thalamus were similar to those seen in SI cortex of the same animals. In addition, they found a rough somatotopy in the reorganized thalamus, with the dorsum of D5 represented lateral to the dorsum of D1. The existence of somatotopic reorganization in the raccoon VPL could not be determined in the present study because of the small area affected and because of the large RFs within this area. Somatotopic reorganization, in fact, would not be expected because of the poor somatotopy in raccoon cortex after digit amputation (Rasmusson, 1982; Kelahan and Doetsch, 1984; Ras- musson et al., 1992). The somatotopy in the monkey is consistent with similar studies in monkey SI cortex and, in this instance, may be influenced by the overlap of the cut and intact nerves in the periphery. Thus, the thalamic region that was affected may be denervated only partially, and the remaining, intact thalamic innervation might regulate the subsequent reorganization to some extent. This idea is supported by evidence in the raccoon, where the degree of cortical reorganization is highly dependent on the amount of overlap of intact and cut nerves (Turnbull and Rasmusson, 1991 1.

Although it is not exactly comparable to peripheral denervation, an interesting finding in humans is that traumatic section of the spinal cord may result in reorganization of the thalamus. Lenz et al. (1994) reported that three of four patients who were undergoing thalamic recording related to chronic pain relief had a larger than normal representation of the body region above the anesthetized body parts within the VPL.

Short-term changes in RF properties of the VPL cells as a result of acute and reversible sensory deprivation have also been reported in several species. Injection of procaine into the RF of a VPL cell resulted in the appearance of a new RF in 27% of a sample of 59 cells in the cat (Nakahama et al., 1966) and of 49% of 55 cells in the rat (Nicolelis et al., 1993). A comparable study on the raccoon VPL (Rasmusson et al., 1993a) found that only inhibitory inputs from adjacent digits were unmasked by applying local anesthetic to the entire digital nerve supply. Although this is similar to the effects of acute denervation on raccoon cortex (Turnbull and Rasmusson, 1990), it is quite different from the appearance of new excitatory RFs as reported in cat and rat. Possibly, this is because digital nerve block in the raccoon would completely silence all afferents, both suprathreshold and subthreshold, to these neurons in contrast to localized procaine injections into the middle of the RF that would not affect surrounding ineffective afferents.

Dorsal rhizotomy has also been shown to affect a number of biochemical markers in the thalamus, producing a down-regulation in y-aminobutyric acid (GABA) type A (GABAA) receptors, a decrease in cytochrome oxidase and parvalbumin activity, and an increase in calbindin labeling

(Rausell et al., 1992). Sensory deprivation produced by trimming the whiskers rather than by nerve damage in the rat also produces a decrease in the staining for both cytochrome oxidase and GABA-related enzymes in the thalamus (Land and Akhtar, 1987).

A comparison of these data on the VPL to studies on raccoon cortex at similar intervals after amputation reveals many similarities between the reorganized thalamus and the reorganized cortex. Over the course of several weeks, the deafferented cortical cells begin to respond to inputs over very large areas with the gradual emergence of more restricted RFs. The thresholds over this period are initially elevated, but they gradually decrease to normal or to near normal levels (Rasmusson, 1982; Kelahan and Doetsch, 1984; Turnbull and Rasmusson, 1991; Rasmusson et al., 1992). Although a comparison of the data at 2 months (Fig. 3) and at 5 months (Fig. 4) postamputation may suggest such an improvement in thalamic reorganization, more data at different postamputation intervals are necessary to test this adequately.

In delineating the deafferented region, the assumption was made that the surrounding digit zones had not been altered by the deafferentation. This seems reasonable, because the cortical fields for the adjacent intact digits do not appear to be altered by digit amputation in this same model (Rasmusson, 1982; Kelahan and Doetsch, 1984). However, it should be emphasized that the way in which normal and deafferented zones were identified in the present study would result in an underestimate of the deafferented zone.

I t might be argued that the changes reported in the thalamus resulted from shrinkage of the deafferented zone, so that the distance from normal D5 to D3 regions would be less than normal. The regon identified as deafferented in that case might be the normal adjacent regions that have shifted closer together. Evidence for shrinkage in the somatosensory pathway following peripheral denervation is limited. Extensive denervation via dorsal rhizotomy in monkeys (Rausell et al., 1992) and total forepaw denervation in cats (Avendano and Dykes, 1995) can produce a substantial decrease in volume of the cuneate nucleus, but this was not apparent in the thalamus (Rausell et al., 1992). Alternatively, if the D4 cells were totally silent after deafferentation, then the microelectrode might be able to record the normal D5 and D3 cells from a distance. These arguments are unlikely to account for the present results, because the width of the deafferented digit zone was calculated to be the same as the two adjacent digit zones, approximately 350 pm, a value that is quite consistent with previous histological studies (Welker and Johnson, 1965). In addition, the responses that were obtained from stimulation of D5 and D3 did not correspond to the properties of intact D5 or D3 cells; most had much larger RFs than normal, and many responded less well to low intensity stimulation. Finally, the sites within the deafferented zone that had RFs on the palm were inconsistent with this possibility, because the palm region is more than 1 mm dorsal to most of these sites.

Site of reorganization Cortical reorganization. One possible explanation for

the changes observed in the VPL is that, in fact, the main reorganization is occurring in the cortex and that the thalamic responses are entirely the result of feedback via the strong corticothalamic pathway. The evidence available


from experiments in which the cortex was blocked suggests that it has only a facilitatory role in the VPL (Yuan et al., 1986; Ghosh et al., 1994). However, because the overwhelm- ing afferent input from the cuneate nucleus is intact in the normal animals used in these studies, it is difficult to use these data to estimate the capacity of corticothalamic neurons to control VPL neurons in the absence of lemniscal afferents. Direct testing of this hypothesis could be accom- plished by examination of the response latencies at both cortical and thalamic levels or by ablation of the cortex in the denervated animal.

Zntrathalarnic reorganization. Alternatively, reorganization could be occurring within the thalamus via some of the mechanisms that have been postulated for cortical reorganization. These include two broad categories of hy- potheses that are not mutually exclusive: one involving the growth of new connections and the other involving the unmasking andlor strengthening of preexisting, but per- haps ineffective, synapses. One advantage that reorganization could have at a subcortical site is that the distances between functional regions are much smaller. Collateral sprouting then becomes a much more attractive possibility than it does in the cortex, where reorganization occurs across regons separated by many millimeters (Rasmusson, 1982; Kelahan and Doetsch, 1984). An interesting hypothesis in light of the anatomy of the thalamus is that the balance between the medial lemniscal (ML) and spinothalamic (STT) or spinocervicothalamic (SCT) pathways may be shifted by peripheral denervation in favor of the STT and SCT tracts. Two important differences between these pathways are 1 ) that the ML system transmits information with much higher spatial and temporal resolution than the STT and SCT pathways and 2) that the STT and SCT tracts carry nociceptive information (Willis and Coggeshall, 1991). The difference in spatial convergence suggests that digit amputation would produce a much more severe effect on cuneate D4 neurons than on dorsal horn D4 neurons. Cuneate neurons might be totally deafferented, whereas dorsal horn cells might lose some, but not all, of their input. Convergence of these pathways within the VPL might then leave the affected D4 neurons to be activated only by the STT and SCT. This hypothesis is attractive in light of data in monkeys with extensive forearm denervation (Rausell et al., 1992 1. A differential change in calcium-binding proteins was found within the thalamus of these animals. Parvalbu- min-labeled neurons, which are associated more with the ML pathway, were found to be depleted in the VPL, whereas calbindin labeling, which is associated with the STT tract, was increased. The importance of nociceptive input to reorganized regions has not been adequately tested. The supposition in most of the work on cortical reorganization appears to be that the reorganized cells take on characteristics of the ML pathway. However, the higher thresholds observed in this study and in some studies of reorganized cortex may be indicative of input from wide- dynamic range neurons in the STT and/or the SCT.

A major difficulty with an explanation of reorganization involving takeover by the STT and the SCT is the extent to which the terminal fields for ML and these spinal pathways overlap. This hypothesis would require fairly extensive overlap to account for the changes without additional extensive intrathalamic changes. Ultrastructural studies have shown that STT and medial lemniscal afferents can terminate upon the same VPL neuron in both the rat (Ma et sl., 1987) and the primate (Ralston and Ralston, 1994).

Comparable studies have not been done on the raccoon, although, at the light microscopic level, there appears to be less overlap in raccoon than in cat and primate. Craig and Burton (1985) found that the distribution of STT terminals in raccoon is sparser and is more along the ventral aspect of the VPL than is the case with ML fibers.

Dykes et al. (1988) pointed out that anatomical overlap does not necessarily mean that there is functional convergence. The demonstration of single neurons with nociceptive input within VPL may indicate that functionally distinct cell types are intermixed. Proof of convergence onto a single cell requires the observation of its response to electrical stimulation of both pathways. In the cat, such a convergence was seen in only 16% of the VPL neurons tested (Yen et al., 1991 ). However, functional convergence may also be obscured by inhibitory mechanisms within the thalamus. Ralston and Ralston (1994), for example, found that ML afferents to the primate thalamus participate in glomeruli and in “triads” involving GABA-positive presyn- aptic terminals, whereas STT afferents end on distal den- drites without close apposition to inhibitory interneurons. The strong postexcitatory inhibition seen in the VPL neurons after ML stimulation (Poggio and Mountcastle, 1963) could easily obscure the slightly delayed input from the STT. Lee et al. (1994a), for example, found that the feedback from the thalamic reticular nucleus in the rat regulates the weight of the input from the spinal trigeminal nucleus (corresponding in many ways to the STT tract) relative to the input from the principal trigeminal nucleus (corresponding to the ML pathway). In addition, the two corresponding inputs to the thalamus are differentially affected by GABAA and GABAH antagonists (Lee et al., 1994b). This could account for the inability of the GABA, antagonist bicuculline to increase RF size of most VPL neurons (Hicks et al., 1986).

The relevance of these data to the raccoon needs to be tested. Neurons of the STT have not been studied physiologi- cally in this animal, but, based on retrograde labeling studies, the STT is smaller than the SCT (Pubols and Haring, 1995). The possible importance of the SCT in the raccoon is also evidenced by its large input from the glabrous skin of the forepaw (Simone et al., 1993) and by a large proportion of neurons with high thresholds to mechanical stimulation (Hirata and Pubols, 1989; Simone and Pubols, 1991). These studies also demonstrated that the RFs of neurons in the SCT pathway of the raccoon are larger than the RFs in its cuneate projection system. However, they might not be large enough to account for the RFs of reorganized thalamic neurons seen in the present study, because the RFs on the digits still appear to be restricted to a single digit. Neurons that are responsive to nociceptive stimulation are also spread widely throughout the VPL of the raccoon (Simone et al., 1993), suggesting that functional STT and SCT inputs are not restricted to regions where the anatomical terminations are dense (Craig and Burton, 1985).

Reorganization below the thalamus The evidence that reorganization is present in the VPL

thalamus raises the possibility that, in fact, this is a reflection of reorganization at even lower levels of the somatosensory pathways. Physiological evidence for long- term reorganization in the cuneate nucleus following peripheral denervation is limited to a study in cat in which changes were seen only if the denervation had been carried

102 D. RASMUSSON

out in young animals (Kalaska and Pomeranz, 1982). An attempt to demonstrate sprouting of adjacent digit afferents into the deafferented digit zone of the cuneate nucleus after digit amputation in the raccoon was not successful (Rasmusson, 1988). However, changes may occur in the cuneate nucleus via mechanisms other than collateral sprouting, such as takeover by the postsynaptic dorsal column inputs. These possibilities remain to be tested.

There have been a variety of reports supporting and refuting claims of reorganization of the somatotopy of the dorsal horn after peripheral nerve damage. These reports have been thoroughly reviewed by Snow and Wilson (1991). In only one example, Wilson and Snow (1987) observed reorganization of the RFs of identified spinocervical tract neurons after digit amputation in the cat. Although some of these data are still controversial, an explanation of thalamic reorganization based on dorsal horn reorganization would still require an understanding of how the STT and SCT pathways interact with the ML within the thalamus.

CONCLUSIONS The demonstration that peripheral nerve damage results

in changes in the organization of the somatosensory thalamus suggests that any emphasis on cortical plasticity is incomplete. Reorganization at the thalamic level does not preclude the possibility of changes at lower levels nor does it prevent further changes at higher levels. The relative capacity of these different levels for change in response to peripheral injury has important implications in the etiology and treatment of various clinical problems.

ACKNOWLEDGMENTS I thank Dr. Deon Louw, Stacey Northgrave, Kathy Clow,

Julie Jordan, and Dr. Roger Croll their for assistance at various stages of this work. I also thank Dr. R.W. Dykes for his comments on the manuscript. This work was supported by grants from the Medical Research Council of Canada and from the Scottish Rite Charitable Foundation of Canada.

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Changes in the organization of the ventroposterior lateral thalamic nucleus after digit removal in adult raccoon