evolution of morphological and histochemical changes in the adult cat cuneate nucleus following...

THE JOURNAL OF COMPARATIVE NEUROLOGY 370479490 (1996)

Evolution of Morphological and Histochemical Changes in the Adult Cat

Cuneate Nucleus Following Forelimb Denervation

CARLOS A V E N D A ~ O AND ROBERT w. DYKES Departamento de Morfologia, Facultad de Medicina, Universidad Authoma de Madrid, 28029

Madrid, Spain (C.A.); Departement de Physiologie, Facult6 de Medecine, Universite de Montreal, Montreal, Quebec, Canada H3C 357 (R.W.D.)

ABSTRACT Morphological and histochemical changes were studied in the ipsilateral cuneate nucleus

between one and 52 weeks after forelimb denervation in adult cats. The deafferented nucleus and neighboring fasciculus were noticeably reduced in size within four weeks and decreased further by 13 weeks. The intensity of acetylcholinesterase staining decreased within one week and was further reduced one month after nerve transections. This reduction in acetylcholinesterase staining was transient, approaching control levels within one year. Parvalbumin immunostaining was also altered by the nerve transections; on the deafferented side, the neuropil staining in the cuneate nucleus and fasciculus decreased, but the number of parvalbumin-positive cells was consistently greater than in the contralateral side. These cell counts returned to normal levels within one year. One month after the injury, cytochrome oxidase activity was reduced. This reduction persisted and was even more apparent after one year. In parallel, the cell clusters of the nucleus became progressively less distinct.

These observations in an adult mammal indicate that peripheral nerve injury imposes molecular and morphological changes on second-order sensory neurons which evolve differentially with time. Although some changes developed rapidly after deafferentation, the onset of others was slower; and whereas some seemed irreversible, others eventually regressed. Taken together with the functional studies of others, these findings suggest that early molecular changes observed in cuneate neurons reflect adaptive reactions to lesion-induced alterations in afferent activity. Permanent deprivation of the normal input, however, would eventually lead to chronic, and perhaps irreversible, degenerative changes. o 1996 Wiley-Liss, Inc.

Indexing terms: plasticity, deafferentation, cytochrome oxidase, acetylcholinesterase, parvalbumin

Peripheral nerve injuries or dorsal rhizotomies in adult mammals bring about changes in a variety of neurotransmitters, peptides, and enzymes in the spinal cord, the trigeminal nuclear complex, or the dorsal column nuclei. Lesion- induced changes involving the primary afferents and presynaptic structures have been clearly documented (Sto- ver et al., 1992; Hoeflinger et al., 1993; Zhang et al., 1993a,b). Less is known, however, about the transneuronal effects of peripheral deafferentations on target neurons. Degenerative changes in neurons of Clarke’s column or in dendrites in the dorsal horn of the spinal cord or in the spinal trigeminal nucleus have been reported after peripheral nerve transections (Loewy, 1972; Gobel, 1984; Knyihar and Csillik, 1976; Sugimoto and Gobel, 1984), as well as changes in cytochrome oxidase levels and cell membrane

proteins, including opioid and substance P receptors (Besse et al., 1992; Croul et al., 1992; Goldberger et al., 1993).

Anterograde transneuronal changes have also been reported in the dorsal column nuclei following dorsal root or dorsal funiculus lesions, but only several months or years after denervation. According to Loewy (19731, thoracic spinal cord transections in humans did not produce significant changes in Nissl substance, neurofibrillary patterns, or cell body or nuclear size in the gracile nucleus up to 6 weeks after the lesion; such changes were present, however,

Accepted February 28,1996 Address reprint requests to Dr. Carlos Avendano, Departamento de

Morfologia, Facultad de Medicina, Universidad Autonoma de Madrid, 22029 Madrid, Spain; E-mail: [email protected]

o 1996 WILEY-LISS, INC.

480 C. AVENDANO AND R.W. DYKES

in a case surviving 22 years. In neither case was neuronal cell loss reported. In spinal monkeys surviving 6 months, no overt changes in Nissl staining were found in neurons of the gracile nucleus, although cell body and nuclear areas were significantly smaller compared to controls (Loewy, 1973). In monkeys sustaining long-lasting (over 12 years) cervical dorsal rhizotomies, Rausell et al. (1992) reported marked shrinkage of the cuneate nucleus, with apparent cell loss, and decreased parvalbumin immunoreactivity. These changes were accompanied by a remarkable histochemical reorganization in the ventral posterolateral nucleus of the thalamus.

These findings suggest that manipulations of the sensory input in adults lead to a cascade of structural, metabolic, and neurochemical transneuronal effects in primary, second- ary, and higher-order neurons that may be the substrate for the functional plasticity displayed in central sensory regions following deafferentation (Devor and Wall, 1978; Dykes et al., 1995; Kaas, 1991; Nicolelis et al., 1993). To understand more fully the central consequences of peripheral deafferentations and their time-course we studied the cuneate nuclei of adult cats at times between one week and 52 weeks after unilaterally transecting the major nerves to the forepaw and forearm. This procedure was chosen because it removes most of the normal sensory input to the cuneate nucleus but causes only small reductions in the number of axons in the dorsal roots or the cuneate fasciculus (Aldskogius et al., 1985), thereby sparing the cuneate neurons from the effects of a massive, direct, and synchronous denervation as would follow a dorsal rhizotomy or dorsal column lesion. The present paper describes acetylcholinesterase, cytochrome oxidase, and parvalbumin staining in the cuneate nuclei, three molecular markers which have different functional roles, and are known to be affected in some systems undergoing loss of afferent input. Quantita- tive data based on Nissl-stained, celloidin-embedded sections from separate cases are presented in the companion paper (Avendaiio and Dykes, 1996).

MATERIALS AND METHODS Subjects and surgery

Sixteen adult female cats were used. In 15 cases, following sodium pentobarbital (35 mg/kg i.p.) anesthesia, the right forelimb, shoulder, and axilla were shaved. Under sterile conditions, incisions in the medial and lateral sur- faces of the arm exposed the radial, median, musculocutaneous, antebrachial cutaneous, and ulnar nerves. These were ligated and cut with removal of at least a 5 mm segment of each. The radial nerve transection included both its deep and superficial branches. The musculocutaneous nerve was cut below its innervation of the biceps and the brachialis muscles. The medial cutaneous nerve of the arm remained intact. The wounds were closed with 3-0 silk and the animals were allowed to survive 1 (3 cats), 2 (2 cats), 4 (3 cats), 8 (2 cats), 13 (3 cats) or 52 (2 cats) weeks. After surgery the animals recovered without difficulty and ig- nored the deafferented limb. There was little change in walking because the animals supported themselves on the dorsal surface of the right paw and were able to distribute their weight in a relatively normal manner. At the end of the survival period they were re-anesthetized and prepared for a terminal experiment on the somatosensory cortex described elsewhere (Avendafio et al., 1995; Dykes et al.,

1995). An additional untreated animal was processed according to the same protocol as the deafferented cats.

Tissue preparation After completion of the cortical experiment, the animals

received additional sodium pentobarbital (45 mg/kg, i.p.), the descending aorta was clamped and they were perfused through the ascending aorta. A brief wash of saline (about 300 ml) at room temperature was followed by 500 ml of a cold (6-8°C) mixture of 0.03% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M sodium acetate buffer (pH 6.5). This was followed by 2,500 ml of a cold solution of 2.5% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 8.5). The fixatives were passed at a rate of 70-100 ml/ minute. These were followed by 500 ml of a solution containing 5% sucrose in 0.1 M sodium phosphate buffer, pH 7.4. The brainstem was removed, blocked, and kept for 48 hours in 25% sucrose in 0.1 M sodium phosphate buffer for cryoprotection. After freezing with dry ice, it was mounted and sectioned on a sliding microtome in 20-km- thick coronal sections. Additional series, cut at 40 pm, were taken for control procedures (see below). All sections were collected in cold 0.1 M sodium phosphate buffer (pH 7.4), and kept at 4°C until processed.

Parvalbumin staining A mouse monoclonal antibody against parvalbumin from

Sigma Chemical Co (St. Louis, Missouri) was used for this study. Twenty-micron-thick sections were pre-incubated, free-floating, for 60-90 minutes at room temperature in 0.01 M sodium phosphate-buffered saline (PBS, pH 7.4) containing 2.5% normal horse serum (Vector), 2.5% bovine serum albumin, 0.02% sodium azide, and 0.5% Triton X-100 (Sigma). They were then incubated for 2-4 hours at room temperature, and 16-20 hours at 4"C, in the same solution containing a 1:3,000 dilution of primary antibody against parvalbumin. Immunostaining was carried out by the ABC method. After thorough rinses in PBS (3 x 10 min) the sections were incubated for two hours at room temperature in a 1/200 dilution of rat-adsorbed, horse anti-mouse antibody (Vector) in 0.1 M potassium phosphate buffer (pH 7.5) containing 2% normal horse serum and 2% bovine serum albumin. This was followed by the avidin-biotin procedure (Vector kit) for 1.5 hours at room temperature. The immunostaining was developed for 1-4 minute in a solution of 0.05% of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) containing 0.01% CoCl2, 0.01% NiS04, 0.01% (NH4I2SO4, and 0.005% H202. As negative controls, one section of each batch was not incubated in the primary antibody.

Cytochrome oxidase staining Cytochrome-oxidase (CyO) staining was carried out on a

series of 40-pm-thick sections according to the original protocol of Wong-Riley (1979). Briefly, sections were transferred from the collecting solution (0.1 M sodium phosphate buffer) into an incubation medium containing 0.04% cytochrome C (Sigma), 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma), and 4% sucrose in sodium phosphate buffer 0.1 M, pH 7.3, at 37°C. The sections were protected from light and agitated during incubation for 30 minutes to 2 hours, until good staining was obtained. They were then washed in three changes of sodium phosphate buffer, mounted on chrome alum-coated slides, and dehydrated, defatted, and coverslipped.

DEAFFERENTED CUNEATE NUCLEUS HISTOCHEMISTRY 481

Nissl and AChE staining Alternate 40-pm-thick sections were Nissl-stained with

0.5% cresyl violet, or processed for acetylcholinesterase (AChE) according to a slightly modified version of the protocol described by Geneser-Jensen and Blackstad (1971): the sections were transferred to an incubation medium containing acetylthiocholine iodide (4.1 mM), glycine (10 mM), copper sulfate (2 mM), ethopropazine hydrochloride (0.17 mM), and sodium acetate (25 mM) in distilled water. This solution was titrated with glacial acetic acid to a final pH of 5.0. The incubation was carried out at room temperature in agitation for 45-120 minutes. After 3-6 brief washes in distilled water, the sections were developed for one minute in a 56 mM aqueous solution of sodium sulfide, pH 7.5, washed again in 3-6 changes of water, and then transferred for 30-60 seconds into a 25 mM aqueous solution of silver nitrate to intensify the reaction product. After three washes in water, the excess silver was removed in 5% sodium thiosulfate for 10 minutes. The sections were again washed in three changes of water, and mounted on chrome alum-coated slides from 0.03 M phosphate buffer to be subsequently dehydrated, defatted, and coverslipped. The lesioned side was always mounted on the right side and all photographs presented here show the tissue from the lesioned side on the right with the contralateral, normal side of the same animal on the left for comparison.

Analysis The differences in staining between the normal and

experimental sides were compared using light microscopy. For CyO and AChE staining optical density measures of low-power views of the cell cluster regions of the left and right cuneate nuclei in the same section were obtained with a commercial image analysis system (MClD, Brock Univer- sity, St. Catherine, Ontario). Left minus right differences were obtained for each section and sets of 5 or more sections from each animal were used to make estimates of the differences between the two sides. Parvalbumin-positive cells were counted from camera lucida drawings of the sections using a similar protocol. Practically all immunoreactive cells were heavily stained, and all appeared to be neurons. Changes as a function of time were tested by analysis of variance or protected t-tests as noted at appropri- ate points in the text and legends.

RESULTS Normally in cadaver material the cuneate and gracile

nuclei form obvious intumescences on the dorsal surface of the spinal cord. One month after surgery, however, the elevation on the operated side was noticeably smaller than that on the normal side. Inspection of serial sections confirmed this impression; the size of the cuneate nucleus on the operated side was reduced, as was the immediately adjacent dorsal funiculus. The reduction in size persisted for the duration of the study (Avendafio and Dykes, 1996). The major changes seemed to occur primarily in the cell cluster region, and were most obvious on the lateral side, extending for a considerable distance throughout the rostro- caudal extent of the nucleus, and causing the nucleus to lose its lenticular shape. Figure 1A shows the flattening and narrowing of the nucleus in a single section taken from an animal studied 8 weeks after deafferentation.

Camera lucida drawings of the brainstems studied at different times after injury confirmed the smaller size of the

cuneate nucleus on the side of the nerve transections. Figure 2 illustrates one of these series from an animal studied 8 weeks after nerve transection. Although the effect of the surgery is apparent, we chose not to quantify the differences since these sections were prepared from unem- bedded, frozen material, that is subject to some distortion during mounting; to obtain reliable estimates of the changes in volume, measures were obtained from celloidin-embedded material using unbiased stereological methods (Aven- dafio and Dykes, 1996). Subsequently, the camera lucida drawings of the frozen sections were digitized and used for constructing 3-dimensional images of the cuneate nucleus. Figure 1B,C shows one case studied 13 weeks after the injury, in which the reduction of the lateral side was noticeable in the caudal third of the cell cluster region.

AChE The control cuneate nucleus showed high AChE activity

in the neuropil, particularly within the clusters. Also, a distinct population of fine AChE-positive fibers was inter- spersed among the largely AChE-negative fibers of the dorsal funiculus, forming a halo around the cuneate. This histochemical marker distinguished the intact and the deafferented cuneate nuclei. On the side of the nerve transection, the nucleus was stained more lightly (Fig. lA), mostly because of a lighter staining of the cell clusters. As well, the absolute size of the individual clusters was reduced by comparison with the intact side. The staining differences and size differences were well-developed 8 weeks after nerve transection (Fig. lA), but left-right differences in staining could be seen as early as 1 week after the injury.

Figure 3A illustrates an AChE-stained section obtained 4 weeks after nerve injury. The difference in size between the two cuneate nuclei had not developed to the extent seen at 8 weeks. Nevertheless, the lesioned (right) side was stained less darkly than the normal side, even though the cell clusters were quite distinct. In contrast, there were no noticeable differences in either the size or the staining of the adjacent gracile nucleus, or in the halo of AChE-positive fibers which surround the cuneate. Figure 3B, taken from an animal 52 weeks after the lesion, shows that the morphological differences between the two sides progressed with time, with the clusters becoming less distinct: the normal whorled pattern of the clusters, and their boundary with the surrounding neuropil was less obvious, even though some of the AChE staining returned.

The staining for AChE proved to be the most sensitive measure of the changes occurring within the nucleus. The right-left difference in staining was quantified by optical density measurements. At each time after transection AChE was measured in at least two animals and five regularly spaced sections per animal. The changes were significant and relatively permanent (Fig. 4). Apparent at one week, the difference between the left and right cuneate cluster regions reached its maximum at 2 weeks. One year after the injury the decrease in AChE staining within the cluster region was less obvious than at 2 weeks but it remained reduced to an extent comparable to that seen one week after surgery. Notice, however, that despite the gradual return of AChE staining, there was a progressive blurring of the cell clusters with time; at 4 weeks they were reduced in size and poorly stained but they were still clearly individual, whorled groups of cells and fibers, while, at survival times of 8 weeks or longer, the clusters were smaller and harder to distinguish because their whorled

Figure 1


pattern was less apparent and their boundaries were poorly defined.

A left-right difference in histochemical staining of CyO was not apparent immediately; only 4 weeks after the deafferentation could a subtle difference be detected. By 13 weeks the difference was more evident by visual inspection (Fig. 5A), but this difference did not reach significance in low-power densitometric measures comparing deafferented and normal nuclear regions. Differences in CyO staining remained at 52 weeks after deafferentation (Fig. 5B), and these differences were significant in optical density measurements (P < 0.011, with the deafferented side being an average of 18% less dense than the normal side.

The progressive change in appearance of the cell cluster region was at least as evident in the CyO-stained sections as it had been in the AChE-stained sections, with the clusters having a more abnormal appearance at 52 weeks than at 13 weeks, as if the process had progressed during this interval. This is apparent in Figure 5B, where the boundaries of the cell clusters are difficult to define in an animal studied one year after nerve injury. In contrast, the right gracile nucleus still appeared indistinguishable from the left and seemed to have all of the features of the normal nucleus.

Nissl The staining intensity of the neurons on the left and the

right sides in the Nissl-stained sections did not differ, but the loss of volume in the right cell cluster region and in the overlying fasciculus was obvious (Fig. 6). The neuropil between the clusters in the right side seemed to stain more intensely than in the control side, probably reflecting an increase in glial cells on the side of the lesion, which occurred particularly in the dorsal part of the nucleus and in the cuneate fasciculus.

Parvalbumin Parvalbumin immunostaining in control cases was evi-

dent in fibers of the cuneate fasciculus and the neuropil of the cuneate nucleus, as well as in cell bodies within the clusters. Differences in parvalbumin staining on the two sides became progressively more apparent following deafferentation; there were no clear differences at one week, but by two weeks after nerve transection the neuropil was less intensely stained on the side of the lesion. The decreased intensity of the reaction was apparent between the cuneate cell clusters as well as outside the cluster region, in the adjacent cuneate fasciculus of the deprived side. Staining of

Fig. 1. A: A frontal section through the dorsal column nuclei stained for acetylcholinesterase (AChE). The major nerves serving the right forearm were transected 8 weeks prior to the perfusion of this animal. The right cuneate nucleus (right) shows the effects of this treatment. The size of the cell cluster region is less than on the left, the intensity of the staining is less and the cell clusters are reduced in size. B: A perspective view of a reconstruction of the cuneate nuclei 13 weeks after nerve section. The view from above and behind shows the reduced volume of the cell cluster region (red) on the right side (arrow), without any noticeable change in the reticular region (yellow). C: Same view as in B, with a reconstruction of the surface of the medulla around the nuclei to show the prominence they form on the sides of the fourth ventricle. The flattening of the lateral wall of the nucleus is still apparent in this view.

c-49

W Caudal level

H 1 rnrn

Fig. 2. Camera lucida drawings of serial sections from an animal having undergone nerve transections 8 weeks prior to perfusion. The cuneate nucleus on the right side was ipsilateral to the lesion. Careful inspection of the drawings shows that the cell cluster region of the cuneate nucleus (shaded areas) on the right tends to be smaller than the one on the left. (ECN, external cuneate nucleus; G, gracile nucleus).

the neuropil within the clusters was less markedly reduced (Fig. 7).

The effects seen in the cuneate fasciculus with parvalbumin staining reached a maximum at about 13 weeks. At that time, when the neuropil staining was at its lowest on the lesioned side, the parvalbumin-stained cells of the lesioned side appeared darker and more numerous than on

484 c. A V E N D ~ O AND R.W. DYKES

Fig. 3. A: A frontal section through the cell cluster region of the dorsal column nuclei four weeks after the forelimb nerves had been transected. The effect of the deafferentation is a reduction of the intensity of the AChE staining in the ipsilateral (right) cuneate nucleus by comparison to the adjacent gracile nucleus and to both nuclei on the left. Many of the cell clusters are less well defined than on the normal side. Although not readily noticeable in this section, the volume of the

the normal side. This effect was not limited to the cell cluster region but extended into the reticular part of the cuneate nucleus beneath the clusters. As well, the external cuneate nucleus (not shown) was noticeably lighter on the deafferented side, and the cuneate fasciculus was still less intensely stained than was the rest of the dorsal columns (Fig. 7).

At 52 weeks after surgery, the left-right difference between the parvalbumin staining within the cuneate nuclei was less marked than at earlier times but it was still obvious. There was a return of some immunoreaction in the neuropil on the affected side. The reticular portion of the nucleus was not different from the normal side 52 weeks after the lesion, but differences were still apparent in the external cuneate nucleus, and staining in the cuneate fasciculus had not recovered.

Efforts to quantify these differences with low-magnifica- tion optical density measures of the left and right cell cluster regions of the cuneate nucleus failed to detect statistical significance because the lighter staining of the neuropil on the lesioned side was compensated by the apparently larger size and darker staining of the parvalbu-

cell cluster region is reduced. B A frontal section stained for AChE one year after nerve section. The size of the cluster region on the affected (right) side is smaller than on the normal side. The cell clusters within the affected nucleus are difficult to discern because their boundaries are poorly defined. The intensity of the AChE staining is somewhat greater than that seen at four weeks (more noticeable in the medial part of the nucleus), but it still seems less than normal. Scale = 0.5 mm.

min-stained cell bodies found within the lighter matrix of these areas, as well as by an increase in their number. The magnitude of this increase was estimated by counting the parvalbumin-stained cells on camera lucida drawings of the cell cluster region in 9 animals (Fig. 8). A significant difference (I' < 0.04) was found between the counts on the left and right sides. This result was attributable primarily to the large differences seen 4 to 13 weeks after injury, while a difference was no longer detectable at 52 weeks.

Although paralleled by volume changes of the cuneate (Avendaiio and Dykes, 1996), these numerical changes likely reflected a real increase of parvalbumin-expressing cells rather than an increase in packing density, because the shrinkage of the cuneate affected its mediolateral and not its longitudinal axis. Therefore the numerical estimations of cells on cross sections of the cuneate should give a good approximation for evaluating side differences.

DISCUSSION The neurons of the clusters in the dorsal column nuclei

receive a dominant afferent drive from axons of the dorsal


REDUCTION IN ACETYLCHOLINESTERASE

-12 ' I

1 2 4 0 13 52 TIME IN WEEKS

Fig. 4. A histogram summarizing the differences between the optical density measures of AChE-stained frontal sections of the left and right cluster regions of the cuneate nuclei at selected times following transection of the forelimb nerves. Mean values for each animal were obtained from left-right differences in 5 sections. Each bar illustrates the average (and SEM) of these mean values for 2 or 3 animals with the same survival time. The probability that the optical density measures would be lower in the deeerented side at each of the six survival tinies is '/26 - 0.016. A protected Student's t-test showed that the percentage decrease at two, four, and eight weeks were significantly different from zero (t = 19.33.4.71. 6.44; df = 2 in every case; P < 0.003,0.05,0.008).

funiculi (Andersen et al., 1964), the majority of which are the myelinated central branches of dorsal root ganglion cells. These branches contribute dense clusters of large, excitatory synaptic boutons which generally contact neuronal cell bodies and large- and medium-sized dendrites (Walberg, 1966; Fyffe et al., 1986). In this context, both transganglionic and transneuronal changes could be ex- pected to follow the peripheral nerve lesions, and the markers used in this study have proved to be valuable for analyzing some of these changes.

Transganglionic component of histochemical changes in the cuneate

Some of the substantial neurochemical and morphological changes in the cuneate nucleus may be attributed to changes in the primary afferent fibers arising from the injured ganglion cells. In particular, the decreased parvalbumin-immunoreactivity of fibers in the dorsal funiculus and the cuneate neuropil seems to form part of the constellation of molecular changes occurring in the large- and medium- sized dorsal root ganglion cells and their central branches after peripheral axotomy (Zhang et al., 1993b). Also, a part at least of the volume changes in the nucleus (see also Avendafio and Dykes, 1996) may be caused by loss or alterations in primary afferents. In trying to understand the degree to which a loss of primary afferents might contribute to these changes, it is important to establish first the extent of neuronal degeneration in the dorsal root ganglia. The proportion of dorsal root ganglion cells reported to die after a nerve transection is highly variable

(Aldskogius et al., 1985). In the rat a 23% cell loss was found 60 or more days after sciatic nerve transection (Arvidsson et al., 1986; Himes and Tessler, 1989). Findings in the cat were less consistent: the reported proportion of cells lost in ganglion L7 90 days after sciatic nerve transection ranges between none and 50%, with mean values around 5% (Tessler et al., 1985) or 30% (Riding et al., 1983).

Whatever the actual proportion of cells lost in the dorsal root ganglion after peripheral axotomy, the ensuing axonal loss in the dorsal columns seems to be less important. Riding et al. (1983) found that the number of axons in the dorsal roots after sciatic nerve section in the cat decreased only slightly compared to a more substantial ganglion cell loss, and suggested that the smaller effect in the columns could be due to collateral sprouting of axons from surviving cells. Persson et al. (1991) observed relatively minor ultrastructural changes in the gracile nucleus beginning about 3 weeks after sciatic nerve section and lasting up to 36 weeks. They attributed the small magnitude of these changes to a reshaping of a small percentage of the synaptic contacts subsequent to a mild loss of dorsal root ganglion cells. In contrast, ultrastructural changes were more notable and long-lasting after dorsal rhizotomies (Rustioni and Sotelo, 1974). The massive and synchronous denervation that dorsal rhizotomies cause on the dorsal column nuclei may well bring about activity-dependent and trophic changes eventually leading to a severe chronic degeneration of the nuclei (Rausell et al., 1992).

If the reduction in volume of the cuneate fasciculus cannot be attributed to substantial axon loss, it is possible, however, that affected axons undergo shrinkage of their axoplasm and/or myelin sheath. Also, a partial loss and/or shrinkage of their terminal arbors could contribute to neuropil shrinkage in the cuneate nucleus. None of these possibilities, however, have been tested as yet.

Transsynaptic effects of deafferentation The presence of parvalbumin is normal in long-projection

neurons of subcortical somatosensory nuclei (Jones and Hendry, 1989; Celio, 19901, and has been associated with high levels of metabolic activity and fast firing rates (Baim- bridge et al., 1992). In some regions, particularly the cerebral cortex, parvalbumin co-localizes with GABA (Hen- dry et al., 19891, but this is probably not the case for the dorsal column nuclei, where most gabaergic interneurons distribute between the cell clusters (Rustioni et al., 1984; Heino and Westman, 1991), and the parvalbumin-positive cells are preferentially located within the clusters (Fig. 7). In some neural systems, the direct denervation of parvalbumin-expressing neurons does not provoke changes in their parvalbumin content (Tigges and Tigges, 1991; Baimbridge et al., 1992); however, several years after dorsal rhizotomies in monkeys there was a marked loss of parvalbumin- expressing neurons in the cuneate nucleus, although the remaining cells and neuropil showed a normal density of parvalbumin immunoreactivity (Rausell et al., 1992).

Because most CyO is located in dendrites and cell bodies, and the levels of CyO activity within neurons positively correlate with their functional level of activity (Wong-Riley, 1989), this enzyme is a good marker of transsynaptic

486 c. AVENDA~~O AND R.W. DYKES

Fig. 5. A A frontal section showing cytochrome oxidase (CyO) staining of the cuneate and gracile nuclei 13 weeks after forelimb nerve transection. At this time the loss of staining intensity is more apparent and the cell clusters seem even less well organized than at earlier times. B: A frontal section through the dorsal column nuclei 52 weeks after

nerve section stained for CyO. The cell clusters are even less well defined, but the difference in staining intensity between the left and right sides is less obvious than at earlier times. There is less contrast between the cell clusters and the intervening tissue. Scale = 0.5 mm.

changes in metabolic activity. In the cuneate nucleus of chronically rhizotomized monkeys, Rausell et al. (1992) found normal levels of CyO staining. This finding contrasts with our longest-surviving cases, in which CyO staining of the deafferented cuneate nucleus remained depressed one year after injury. Long-lasting reduction of CyO has also been reported in the neurons of Clarke’s nucleus of the cat after dorsal rhizotomies (Goldberger et al., 1993). A marked difference between Rausell et al.’s (1992) monkeys and our cats-apart from the longer survival of the former-is that the deafferented cuneate in those monkeys contained “many fewer” neurons than normal, whereas there was no detectable neuron loss in the cat cuneate nucleus (Avendafio and Dykes, 1996). It may then be speculated that whichever neurons remain alive in the long run will tend to recover normal levels of CyO activity.

The dorsal column nuclei are rich in AChE, but little is known about the significance of this enzyme in the nucleus. The source of this AChE is unknown as well, and this makes an explanation for the changes in this enzyme hard to find. Nevertheless, neither AChE nor acetylcholine are present in primary afferent fibers innervating the cuneate (Broman, 19941, and the AChE-positive fibers surrounding the cuneate do not seem to change after deafferentation; therefore, it may be speculated that most of the AChE

which is affected by deafferentation would be located in dendrites of cuneate neurons. Since cholinergic innervation of the cuneate is sparse, and does not change significantly after deafferentation (C. Avendaiio, unpublished observations), AChE presence is probably revealing a “nonclassi- cal” role of this enzyme in the cuneate nucleus, such as a nonenzymatic modulator of neuronal excitability, or a protease involved in hydrolysis of different peptides (Apple- yard, 1992).

Time-course of events in the deafferented cuneate and functional considerations

The changes detected with the various histochemical markers used in this study varied with time, suggesting that several different processes may have been at work in the deafferented cuneate, each with its own temporal evolution and with specific repercussions on the nucleus. The decrease in AChE staining started soon, within the first week after injury, and was largest in the second week. The decrease in parvalbumin immunostaining in the neuro- pi1 of the cuneate nucleus and in the dorsal funiculus was detectable only two weeks after injury, but it was more sustained: the loss of staining seemed permanent in the funiculus, and showed only a moderate recovery in the


Fig. 6. Low-power photomicrographs of Nissl-stained sections through the dorsal column nuclei at the level of the cell clusters. The cuneate nucleus and fasciculus on the right, ipsilateral to the nerve

transection, are smaller than those on the left. Glial cells increased in the right side, as well as the packing density of the neurons. Scale = 0.5 mm.

neuropil at 52 weeks. In contrast, the number of neuronal cell bodies in the cuneate nucleus expressing parvalbumin slowly increased after deafferentation, leading to a larger number of immunoreactive neurons at 4-13 week surviv- als. Finally, a slower but more persistent anatomical rear- rangement of the characteristic whorls of neurons forming the clusters and a parallel decrease in CyO histochemical staining followed the injury.

Changes in the expression of a variety of peptides have been shown to occur in the spinal cord a few days after peripheral nerve lesions (Tessler et al., 1985; Klein et al., 1991). An up-regulation of galanin was demonstrated in the primary afferent fibers of the rat cuneate nucleus one week after deafferentation (Hoeflinger et al., 1993). The rapid decrease of AChE staining found here may then be included as well among the early-in this case transsynaptic- effects of deafferentation. Early transneuronal neurochemical changes following peripheral lesions have also been reported at higher levels of the sensory pathways: GAD, GABA, and tachykinin immunoreactivity decrease in the ocular dominance columns of monkey visual cortex within two days after enucleation (Jones, 1990), and muscarinic binding sites in the hindlimb cortex of rats are altered as early as 24 hours after sciatic nerve transection (Hanisch et al., 1992).

It is unlikely that these early changes represent degenerative processes in primary afferents or target neurons. In first place, most of them are reversible. Also, axon degeneration in the dorsal funiculus is probably scarce (see above), and its timing is not consistent with the onset of early neurochemical changes: these begin soon after injury whereas ganglion cell loss and degeneration of primary afferent fibers are apparent only after two weeks in the rat and after at least 4 weeks in the cat (Riding et al., 1983; Tessler et al., 1985). Moreover, it has been shown that when ganglion cell death is prevented by placing the cut end of the nerve in a silicon tube (Melville et al., 19891, the same pattern of histochemical changes occur in the dorsal horn of the spinal cord (Klein et al., 1991).

Alternatively, the histochemical changes in the cuneate may reveal an adaptive reaction of the nucleus to an altered synaptic input provoked by the peripheral axotomy. It is known that permanent or reversible blocking of neural impulse activity results in postsynaptic changes in gene regulation and in the expression of a number of neurotrans- mitter- and energy metabolism-related molecules (Jones, 1990; Persson et al., 1993; Shenget al., 1993; Wong-Riley et al., 1994). The nature of the alteration in neural activity that follows nerve transection is not clear. Although it is often presumed that nerve transection produces an abrupt reduction in the afferent activity travelling centrally from the transected nerves, Lu et al. (1993) studied spontaneous activity arising from the dorsal root ganglion cells of cats with intact and transected peripheral nerves, demonstrat- ing that much of the spontaneous activity is generated in the ganglion itself and does not change after nerve section, and implying that transection will not produce a large change in the average level of background activity entering the spinal cord. It is likely, therefore, that the more significant consequence for cuneate neurons of a peripheral nerve transection is to alter the pattern of incoming primary afferent signals, rather than to reduce or remove those signals.

If the decrease in AChE content in the cuneate nucleus, the increased parvalbumin expression in the deafferented cuneate neurons, and a putative decrease of local gabaergic inhibition in the cuneate (Castro-Lopes et al., 1993; Pettit and Schwark, 1993) are considered together, it is possible to speculate that, at least for a time after deafferentation, there is an orchestrated effort of the sensory system to increase the ascending signals from the partially deprived cells in relay nuclei of the pathway. However, despite the changes observed in the nucleus and evidence of loss of a major portion of their afferent drive, collateral sprouting of primary afferent fibers does not seem to occur in the nucleus (Rasmusson, 1988; Florence et al., 19931, and we saw no morphological evidence that the state of the nucleus had substantially improved 1 year later. Portions of the

488 c. AVENDANO AND R.W. DYKES

Fig. 7. A: Parvalbumin staining in the normal and deprived cuneate nucleus of an animal one week after nerve transection. The appear- ances of the left and right nuclei and of the overlying cuneate fasciculi are comparable. B: At 13 weeks after deafkentation there is a marked

decrease in the cuneate fasciculus and in the neuropil within and between the clusters. C: Fifty-two weeks after dederentation parvalbumin staining remains low in the cuneate fasciculus, although there was moderate recovery in the neuropil of the cuneate. Scale = 0.5 mm.

cortical regions served by the cuneate also remain without new afferent drive 1 year later (Dykes et al., 1995). As noted by Rasmusson (1988) in the context of deafferentation by the fifth digit amputation in adult raccoons, there are potential sources of afferent drive within a few hundred microns of the deprived cuneate cell clusters, yet in that situation and in ours, the cell clusters seem to remain in a deprived state. The rapid onset and the long-lasting conse-

quences of the changes provoked by peripheral nerve injuries may then justify taking rapid measures in clinical neurology, avoiding unjustified delays in repairing severed nerves or initiating soon sensory stimulation therapy. The importance of the time interval between nerve lesion and intervention is clear in the peripheral motor system, in which it is well known that prolonged axotomies are responsible of poor motor recovery (Fu and Gordon, 1995).


INCREASE IN CELLS COUNTED

24 1 w

m E

!! 18 T

I -6 ' I

1 2 4 8 1 3 52 TIME IN WEEKS

Fig. 8. A histogram showing differences in the number of parvalbumin positive cells in the left and right cuneate nuclei in nine cats. Mean and SD are given for two animals at one, four, and 13 weeks after lesion. On average, a total of 1,690 cells were counted on 5 sections from each animal. A t-test showed an increased number of cells on the deafFer- ented side (t = 2.76; df = 5; P < 0.04).

ACKNOWLEDGMENTS The histological material was prepared by Mme. F.

Cantin. The data analysis was aided by Ms. Kirby Dykes. Ms. Julia Martison, and Ms. Isabelle Banville used Autocad to generate the color plates of Figure 1. The rest of the art work was provided by Mr. Giovanni Filosi and the manu- script was typed by Ms. Lise Imbeault. C. Avendano was on a sabbatical leave supported by the Ministry of Education and Science of Spain. Funds were provided by the Medical Research Council of Canada, the Fonds de recherche en sante du QuBbec, the North Atlantic Treaty Organization, and the Scottish Rite Charitable Foundation of Canada.

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