Adaptive responses of monkey somatosensory cortex to peripheral and central deafferentation

PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 0 2 8 - 3

Neuroscience Vol. 111, No. 4, pp. 775^797, 2002 G 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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ADAPTIVE RESPONSES OF MONKEY SOMATOSENSORY CORTEX TO PERIPHERAL AND CENTRAL DEAFFERENTATION E. G. JONES, T. M. WOODS and P. R. MANGER1 Center for Neuroscience, University of California, 1544 Newton Court, Davis, CA 95616, USA

Abstract/This study deals with two kinds of activity-dependent phenomena in the somatosensory cortex of adult monkeys, both of which may be related: (1) mutability of representational maps, as de¢ned electrophysiologically; (2) alterations in expression of genes important in the inhibitory and excitatory neurotransmitter systems. Area 3b of the cerebral cortex was mapped physiologically and mRNA levels or numbers of immunocytochemically stained neurons quanti¢ed after disrupting a¡erent input peripherally by section of peripheral nerves, or centrally by making lesions of increasing size in the somatosensory thalamus. Survival times ranged from a few weeks to many months. Mapping studies after peripheral nerve lesions replicated results of previous studies in showing the contraction of representations deprived of sensory input and expansion of adjacent representations. However, these changes in representational maps were in most cases unaccompanied by signi¢cant alterations in gene expression for calcium calmodulindependent protein kinase isoforms, for glutamic acid decarboxylase, GABAA receptor subunits, GABAB receptors, K-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-D-aspartate (NMDA) receptor subunits. Mapping studies after lesions in the ventral posterior lateral nucleus (VPL) of the thalamus revealed no changes in cortical representations of the hand or ¢ngers until s 15% of the thalamic representation was destroyed, and only slight changes until approximately 45% of the representation was destroyed, at which point the cortical representation of the ¢nger at the center of a lesion began to shrink. Lesions destroying s 60% of VPL resulted in silencing of the hand representation. Although all lesions were associated with a loss of parvalbumin-immunoreactive thalamocortical ¢ber terminations, and of cytochrome oxidase staining in a focal zone of area 3b, no changes in gene expression could be detected in the a¡ected zone until s 40^50% of VPL was destroyed, and even after that changes in mRNA levels or in numbers of GABA-immunoreactive neurons in the a¡ected zone were remarkably small. The results of these studies di¡er markedly from the robust changes in gene expression detectable in the visual cortex of monkeys deprived of vision in one eye. The results con¢rm the view that divergence of the a¡erent somatosensory pathways from periphery to cerebral cortex is su⁄ciently great that many ¢bers can be lost before neuronal activity is totally silenced in area 3b. This divergence is capable of maintaining a high degree of cortical function in the face of diminishing inputs from the periphery and is probably an important element in promoting representational plasticity in response to altered patterns of a¡erent input. G 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: activity-dependent gene expression, representational maps, thalamus, dorsal column nuclei, amputation, plasticity, a¡erent divergence.

heightened stimulation or new usage patterns can also result in enlargements of representations and readjustment of their internal topography (Allard et al., 1991; Clark et al., 1988; Jenkins et al., 1990; Mogilner et al., 1993; Recanzone et al., 1990, 1992a,b,c). In these cases, the neural mechanisms responsible may form a basis for the improvements in perceptual skills that accompany extended sensory experience. Reorganization of the somatosensory cortex following peripheral dea¡erentation occurs in phases, which to some extent depend upon the degree of dea¡erentation. Following modest peripheral sensory perturbations, such as section of a peripheral nerve or amputation of a ¢nger, there is an immediate expansion of the cortical representations of parts with intact innervation adjacent to the dea¡erented region. If the dea¡erentation is su⁄ciently large or a particular combination of peripheral nerves is severed, the expansion may not completely ¢ll the silenced part of the representation (Cusick et al., 1990; Garraghty et al., 1994; Kolarik et al., 1994; Merzenich et al., 1983b, 1984, 1996; Wall et al., 1992;

When input to the cerebral cortex from peripheral somatosensory receptors is reduced by peripheral nerve section or amputation, the cortical representation of the denervated or removed part in the ¢rst somatosensory area is invaded by representations of adjacent parts that retain innervation (Merzenich et al., 1983a,b 1984; Kaas et al., 1983; Kaas, 1991). Deprivation-dependent reorganization of the cerebral cortex is likely to be responsible for perturbed sensory experiences that commonly occur after amputation or dea¡erentation of a limb (Carlen et al., 1978; Doetsch, 1998; Knecht et al., 1996; Melzack, 1990; Ramachandran, 1993; Ramachandran et al., 1992; Sherman et al., 1984). In this case, the e¡ect is clearly maladaptive, but overuse,

1 Present address: Karolinska Institute, Stockholm, Sweden. *Corresponding author. Tel. : +1-530-757-8747; fax: +1-530-7549136. E-mail address: [email protected] (E. G. Jones).

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Xu and Wall, 1996, 1999a,b). After larger dea¡erentations, a phase lasting weeks or months follows, in which the new representation is consolidated and any loss of topographic order restored, followed much later by a phase of further expansion and use-dependent re¢nement of internal topography (Churchill et al., 1998). For the limited, immediate expansions of representations that follow modest degrees of dea¡erentation, a cellular basis has been revealed in the emergence of new peripheral receptive ¢elds by release of neurons from GABAergic inhibition (Alloway and Burton, 1986, 1991; Calford and Tweedale, 1990, 1991a,b). The extent of these reorganizations is probably determined by a cortical ‘distance limit’ in which the extent of a short term expansion of a representation in the cortex (V1.5 mm) is probably constrained by the extent of arborization of existing thalamocortical connections (Merzenich et al., 1984; Rausell and Jones, 1995). Large scale reorganizations, occupying many millimeters of cortex and far greater than the cortical distance limit, occur in the longer term after extensive dea¡erentations such as those caused by multiple dorsal rhizotomies (Pons et al., 1991), lesions of a spinal dorsal column (Jain et al., 1997), or amputations of a hand or limb (Florence and Kaas, 1995). Section of the cuneate fasciculus at the upper cervical level, for example, leads to an immediate silencing of the cortical representation of the hand, followed in about 6 months by its later activation by inputs from the face, an e¡ect originally seen 12 years after total dea¡erentation of the upper limb and upper trunk (Pons et al., 1991). The mechanisms responsible for large scale reorganizations of this kind remain unclear but a combination of cortical and subcortical e¡ects is likely (Jones, 2000), with or without sprouting of new connections (Darian-Smith and Gilbert, 1994; Florence et al., 1998). Synaptic plasticity, especially that involving long term potentiation and long term depression, is commonly proposed as being at the heart of the adaptive response of the cortex to conditions in which a¡erent input is perturbed (Buonomano and Merzenich, 1998). Activity-dependent up- or down-regulation of neurotransmitters and their receptors, which may contribute to alterations in synaptic strength, has also been proposed as a component of the intracortical response (Jones, 1993). This view is based mainly upon results of studies on the primary visual cortex in which rapid and reversible changes in gene expression for components of the GABAergic and glutamatergic neurotransmitter systems occur following even brief periods of monocular deprivation. The capacity of cortical cells to compensate for changes in excitatory input by regulating turnover of AMPA receptors, thus scaling excitatory postsynaptic potential amplitudes and the responses of the neurons to stimulation (O’Brien et al., 1998; Turrigiano et al., 1998), may be one of several profoundly important adaptive synaptic responses underlying cortical plasticity, because it is mediated by brain derived neurotrophic factor (Rutherford et al., 1998), a growth factor that is highly expressed in cortical neurons (Huntley et al., 1992), and up-regulation of which may promote formation of new connections (‘sprouting’).

Synaptic mechanisms of cortical plasticity that do not depend upon sprouting, although they could co-exist with it, are thought to operate upon a substrate of extensively overlapping or ‘divergent’ thalamocortical and/or corticocortical connections, some of whose synapses are silenced and others unmasked by altered activity. The unmasking of pre-existing connections under conditions of dea¡erentation not only occurs in the somatosensory cortex but also in the medullary and thalamic relay sites of the ascending pathways a¡erent to the cortex (Millar et al., 1976; Nicolelis et al., 1993; Pettit and Schwark, 1993; Shin et al., 1995; Nakahama et al., 1966; Panetsos et al., 1997; Xu and Wall, 1997, 1999a,b). Modi¢cations in somatotopic maps occur in the dorsal column nuclei and ventral posterior nucleus of the thalamus after both short and long term dea¡erentation (Wall and Egger, 1971; Dostrovsky et al., 1976; Lombard et al., 1979; Pollin and Albe Fessard, 1979; Kalaska and Pomeranz, 1982; Garraghty and Kaas, 1991a,b; Lenz et al., 1994; Rasmusson, 1996; Rasmusson and Northgrave, 1997; Xu and Wall, 1997, 1999a,b). The modi¢cations that occur in the dorsal column nuclei immediately following section of peripheral nerves can be attributed to divergence and convergence of inputs from the peripheral territories of nerves that remain intact, and these changes are in large part re£ected simultaneously in the cortex (Xu and Wall, 1999a,b) and potentially in the thalamus. Longer term dea¡erentations that lead to large scale reorganization of the cortex are accompanied by reorganization of both the dorsal column nuclei and thalamus of monkeys (Jones and Pons, 1998b; Woods et al., 2000), and comparable changes are seen in the human thalamus after amputations (Lenz et al., 1994). The present investigation looked at dea¡erentation-dependent phenomena in the monkey somatosensory cortex from the perspective of divergence of ascending connections. The point of view adopted, one that the experiments presented will attempt to justify, is that divergence in the pathways from the periphery to the somatosensory cortex is su⁄ciently great that small activity-dependent alterations at lower levels of the pathway (in the dorsal column nuclei and thalamus) will be ampli¢ed in the projection to the cortex, that this divergence is capable of maintaining a considerable degree of cortical organization and function in the face of diminishing inputs from the periphery, and that this divergence should normally be an important element in determining the cortical response to altered patterns of a¡erent input. It will emerge that in its response to peripheral perturbations of sensory input, the somatosensory cortex presents marked contrasts with the primary visual cortex of monkeys. Some of the results have been published previously in preliminary form (Woods et al., 1995, 1996, 1997; Manger et al., 1996b; Jones et al., 1997).

EXPERIMENTAL PROCEDURES

Experimental procedures were approved by The Institutional Animal Care and Use Committee and conducted according to guidelines set forth by the National Institutes of Health.

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Table 1. Peripheral nerve lesions and lack of central e¡ects Number of animals

7 1 1 4 1 1 4 4 a

Lesion

Median N. Ulnar N. Radial N. Median and ulnar Nn. Median and radial Nn. Ulnar and radial Nn. Finger amputations Mandibular N.

Survival times

7^180 days 30 days 45 days 5^30 days 10 days 32 days years 30^45 days

Di¡erence in mRNA or immunocytochemical levels in a¡ected part of area 3b representationa GAD and CaMKII-K

GABAA -RK1, L2/L3, GluR2/3, NR1

V 2.5% (p v 0.1) 0 0 V 1.5% (p v 0.1) 0 V 5% (p 9 0.05) V 1% (p v 0.1) 0

0 0 0 0 0 0 0 0

Comparison of hand and leg representations, or of face and hand representations.

Peripheral nerve lesions, cortical maps and cortical gene expression The brains of 40 macaque monkeys that had had nerves cut in the upper limb or face, or that had been a¡ected by chronic amputations of ¢nger(s) or part of the tail, with survivals of several weeks to a number of years were used. Table 1 lists the range of nerves severed or other procedures and the survival times post sectioning. Nerves were cut under aseptic conditions, the proximal and distal stumps turned back on themselves and tied with silk to discourage reunion. In several of the animals the pattern of representation in area 3b of the normal somatosensory cortex and/or of area 3b contralateral to the dea¡erented region was mapped, using single and multiunit recording techniques that have been described previously (Manger et al., 1996b). The somatosensory cortex from 35 of the brains, either from both sides or from one side that had not been subjected to mapping procedures, was prepared for gene expression studies. Because opening the dura mater and introducing microelectrodes compromises immunocytochemistry and in situ hybridization histochemistry, no physiological mapping was performed on the somatosensory cortex used for expression studies. Surface landmarks were used to localize the relevant parts of the body representation, as described below. Gene expression in area 3b Blocks of the pre- and post-central gyri were cut parasagittally or in a plane tangential to the surface of the gyri at 25 or 30 Wm. Sections were collected in groups for in situ hybridization histochemistry, immunocytochemistry, cytochrome oxidase (CO) histochemistry or Nissl staining. In situ hybridization histochemistry Free-£oating sections were pretreated by successive washings in: 0.1 M glycine in 0.1 M phosphate bu¡er (pH 7.4); 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8); 2Usaline sodium citrate (SSC; 1USSC consists of 0.88% NaCl and 0.44% Na3 C6 H5 O3 W2H2 O). They were then incubated for 1 h in a hybridization solution consisting of 50% formamide, l0% dextran sulfate, 0.7% Ficoll, 0.7% polyvinyl pyrrolidone, 0.5 mg/ ml yeast tRNA, 0.33 mg/ml denatured herring sperm DNA, and 20 mM dithiothreitol (DTT) before transferring to the same solution containing 1U106 c.p.m./ml of one of the 33 P-labeled sense or antisense riboprobes. Sections remained in this solution for at least 20 h at 60‡C. Following hybridization, sections were washed in 4USSC at 60‡C for 1 h, digested with 20 mg/ml ribonuclease A (pH 8) for 30 min at 45‡C and then washed through descending concentrations of SSC containing 5 mM DTT to a ¢nal stringency of 0.1USSC for l h at 60‡C. Sections were mounted on gelatin-coated slides, dried and exposed to Amersham L-Max ¢lm for 4^21 days at 4‡C. After development of the ¢lm, the sections were lipid extracted in chloroform,

dipped in Kodak NTB2 emulsion diluted l:l with water, dried, exposed for 20^40 days at 4‡C then developed, ¢xed and stained through the emulsion with Cresyl Violet. Antisense cRNA probes were generated by in vitro transcription using T3 or T7 RNA polymerase in the presence of [K-33 P]UTP (DuPont-NEN). Sense strand riboprobes were similarly produced, by using T7 or T3 RNA polymerase. All cRNA probes used were 340^491 bases in length, had comparable G-C ratios and similar speci¢c activities. Monkey-speci¢c subunit cDNAs corresponding to the K1, K2, K4, K5, L1, L2 and Q2 GABAA receptor subunits were described in Huntsman et al. (1994). All cDNA sequences are co-linear with known human and rat GABAA receptor subunit cDNA sequences. A monkey GABAB receptor cDNA clone (Mun‹oz et al., 1998) is 418 nucleotides long and corresponds to the pan region of the rat cDNA sequence (Kaupmann et al., 1998) that identi¢es the alternatively spliced forms of the GABAB R1a and R1b receptor mRNAs. A monkey cDNA clone (Benson et al., 1991a) corresponding to the mRNA for the 67-kDa form of glutamic acid decarboxylase (GAD67 ) is 360 nucleotides long and corresponds to a portion of the coding region contained between bases 1324 and 1683 of the published cat cDNA coding sequence (Kobayashi et al., 1987). cDNAs corresponding to the N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits NR1, NR2A, NR2B, NR2D, GluR1, GluR2, GluR3, GluR5, GluR6, and GluR7 were described by Sucher et al. (1995), Akbarian et al. (1996) and Tighilet et al. (1998b). A monkey cDNA for the K subunit of type II calcium/calmodulin dependent protein kinase (CaMKII-K) corresponds to bases 869^1185 of the rat CaMKII-K gene. It also contains a 33base insert beginning at nucleotide 984 of the rat sequence (Benson et al., 1991a). Antisense riboprobes made from this cDNA recognize both CaMKII-K and K-33 mRNAs. CaMKII-L riboprobes were transcribed from a 770-bp human cDNA (Tighilet et al., 1998a,b) which corresponds to bases 550^1320 of rat CaMKII-L (Bennet and Kennedy, 1987). Antisense riboprobes made from this cDNA recognize both CaMKII-L and -LP subunit mRNAs. Quanti¢cation Film autoradiograms were quanti¢ed by densitometry, using a microcomputer imaging device (MCID/M4, Imaging Research, St. Catharines, ON, Canada). Optical density readings were taken in transepts of de¢ned width across the thickness of area 3b, matching the peaks and valleys of density to similarly digitized images across the thickness of the same area in an adjacent Nissl-stained section. Anatomical landmarks were used operationally to de¢ne the face, hand and foot representations, based on previous studies (Powell and Mountcastle, 1959; Pons et al., 1987; Manger et al., 1996a,b, 1997). The upper limb representation was de¢ned as the part of area 3b located 0^10

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Fig. 1. (A) Flattened map of area 3b of a normal monkey showing borders with areas 3a and 1 (thicker gray lines), as de¢ned by cytoarchitecture, and the fundus of the central sulcus (black line). Rows of dots indicate points at which recordings were made from single or multiunit clusters of neurons as an electrode was advanced down the posterior bank of the sulcus (right to left in this £attened map). Thinner gray lines delimit approximate subregions of area 3b in which neurons with receptive ¢elds on the indicated part of the contralateral body surface were recorded. (B) Locations of parts of area 3b in which face, hand and leg representations are located, as used for measuring relative levels of mRNAs and immunocytochemically stained neurons. (C) Flattened map of the representation of the index ¢nger (D2) in a normal monkey, as derived from microelectrode mapping similar to A but at higher resolution. Note extent of D2 representation and locations of representations of its various parts. (D) Map similar to C but derived from a monkey in which the middle and distal phalanges had been amputated 22 months previously. Note shrinkage of overall D2 representation but expansion of representation of its remaining proximal parts into anterior regions normally occupied by representations of its distal parts. (Data from Manger et al., 1997.)

mm medial to the level of the anterior tip of the intraparietal sulcus; the lower limb representation was de¢ned as the part of area 3b lying 0^10 mm medial to the level of the medial tip of the postcentral sulcus; the face representation was de¢ned as the part of area 3b lying 0^10 mm lateral to the level of the tip of the intraparietal sulcus (Fig. 1). All densitometric readings for a particular mRNA were made on sections hybridized at the same time and exposed for the same period of time on the same piece of ¢lm. Optical density readings were converted to readings of radioactivity by reference to 14 C standards (Amersham) exposed on the same piece of ¢lm. These standards were calibrated before reading density values on ¢lms and the densities were then converted to units of concentration (nCi/g). The imaging device sampled mean gray level values of all of the pixels within a transept and converted them to a concentration value based upon an integrated optical density. Levels of mRNA expression in an una¡ected body part representation (lower limb or face) were compared with levels in the potentially a¡ected (upper limb) representation. Density

levels in the a¡ected representation were compared in each case with those in the una¡ected representation using Student’s twotailed paired t-test. Levels of mRNA expression in the upper limb representation are reported as a percentage of the levels in the lower limb representation. Control sections hybridized with sense riboprobes showed no labeling above background. Immunocytochemistry Sections adjacent to those processed for in situ hybridization histochemistry were washed three times in 0.1 M phosphate bu¡er. This was followed by pre-incubation in 0.1 M phosphate bu¡er containing 1% Triton X and 3% pre-immune horse, rabbit, or goat serum. Sections were then incubated for 24^72 h at 4‡C in the same solution containing mouse monoclonal antibodies of demonstrated speci¢city for either CaMKII-K (Boehringer-Mannheim; 1:5000), parvalbumin (Sigma; 1:1000), GABA (Sigma; 1:10 000), or a⁄nity-puri¢ed polyclonal anti-

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bodies of demonstrated speci¢city for NR1 (Chemicon; made in rabbit; 1:500), GluR2/3 (Chemicon; made in rabbit; 1:250), or GluR5/6/7 (Pharmingen; made in mouse; 1:500) glutamate receptor subunits. Following three washes in 0.1 M phosphate bu¡er, sections were incubated in 0.1 M phosphate bu¡er containing biotinylated horse anti-mouse (Vector; 1:200), or goat anti-rabbit (Vector; 1:200) IgGs and 1% Triton X for 1 h, then treated for 1 h in avidin^biotin^peroxidase complex (Vector). After three 5-min washes in 0.1 M phosphate bu¡er, sections were reacted in phosphate bu¡er containing 0.5 mg/ml 3,3P-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide or with a Vector VIP Peroxidase substrate kit yielding a gray or pink reaction product. After a ¢nal rinse in 0.1 M phosphate bu¡er, sections were mounted on gelatin-coated glass slides, allowed to dry, dehydrated in ethanol, lipid-extracted with xylene and coverslipped with DPX mounting medium. Control sections were processed through the entire immunocytochemical protocol without either the primary or secondary antibody, and some with secondary only. These revealed no speci¢c staining. Counts of GABA-immunoreactive cells in various parts of area 3b, as described in the Results, were made by the same methods as Hendry et al. (1987). In making these counts, a 50 Wm wide eyepiece reticule was placed across the thickness of the relevant part of area 3b and the number of immunoreactive cells found within its borders were counted at a magni¢cation of 600 or 1250 times.

779

was withdrawn from anterior to posterior through VPL. In the cases in which excitotoxic lesions were made, a microelectrode was cemented to the 30-gauge needle of a 1-Wl Hamilton syringe ¢lled with 2 or 10 mM kainic acid, the tips being separated by a distance of less than 1 mm and with the beveled opening of the needle facing the tip of the microelectrode (Friedman and Jones, 1981). The needle/electrode assembly was returned to a set of coordinates previously determined from the microelectrode mapping, receptive ¢elds of neurons were mapped to con¢rm return of the assembly to the original microelectrode track, and then 100^300 nl of kainic acid solution was expelled in 200-Wm steps as the assembly was withdrawn from anterior to posterior over the coordinates corresponding to the anteroposterior extent of VPL. Two to 7 weeks after making the lesion(s) (Table 2), the lesioned and control animals were anesthetized and the hemispheres ipsilateral to the thalamic lesions or in the control animals were explored with microelectrodes to de¢ne the details of the somatotopic map in area 3b of the cerebral cortex. A ¢ne grain map of area 3b, concentrating on the representation of the digit upon which the thalamic lesion was centered in the lesioned animals, was produced. The distance between electrode penetrations was less than 500 Wm, except where the pattern of blood vessels prevented this. Multiunit responses to light tactile stimulation were recorded in 100^200-Wm steps as the microelectrode was advanced down the posterior bank of the central sulcus. At the conclusion of the mapping session, the brains were ¢xed by perfusion and blocks of the cortex and thalamus were subsequently sectioned and stained. The cortex was sectioned serially in the parasagittal plane and sections were stained with Thionin. Electrode tracks were identi¢ed, and the recording sites were matched to architectonic boundaries on two-dimensional ‘£attened’ maps (Manger et al., 1996a,b, 1997). Frontal sections of the thalamus were stained with Thionin, for CO activity or immunocytochemically for the same neural antigens as described below. Quanti¢cation of the volumes of lesions was carried out on camera lucida drawings of Nissl- and CO-stained sections through the anteroposterior extent of the thalamus. Using NIH Image software on the scanned drawings, volumes of VPL and the lesion within it were determined and the volume of the lesion reported as a percentage of the total volume of VPL (Tables 2 and 3). The volume of the hand representation in VPL, and the volume of this representation which was removed by the lesion, was determined by comparing measurements with those made in previous mapping studies on VPL (Jones and Friedman, 1982; Jones et al., 1982). No distinction was made between electrolytic lesions which destroy both neurons and neuropil and excitotoxic lesions which destroy only neurons. In an additional series of 12 adult M. mulatta and M. nemestrina monkeys, electrolytic or excitotoxic lesions were made in the ventral posterior nucleus of the thalamus exactly as described in the preceding section. Four animals received lesions in the thalami of both sides of the brain and three received unilateral lesions, giving a total of 11 experimental cases. Sur-

Thalamic lesions, cortical maps and cortical gene expression Studies designed to test the limits of thalamocortical divergence in maintaining somatosensory maps when the system was perturbed were carried out in nine adult macaque monkeys (Macaca mulatta and Macaca nemestrina). Lesions of varying size were placed within the upper limb representation of the ventral posterior nucleus of the thalamus and the representation mapped in area 3b of the ipsilateral somatosensory cortex. The cortex was mapped in 14 cerebral hemispheres from the nine animals. Nine of these cortices were associated with a lesion in the thalamus of the same side and ¢ve were associated with a normal thalamus. The monkeys that received thalamic lesions were anesthetized with intramuscular ketamine and maintained on intravenous Nembutal, and microelectrodes (5 M6 impedance) were advanced in horizontal Horsley^Clarke planes into the thalamus for physiological determination of the extent of the ventral posterior lateral nucleus (VPL) and its border with the ventral posterior medial nucleus (VPM). In six of the monkeys, electrolytic lesions were made in VPL, and in three an excitotoxic lesion was made by injecting kainic acid. Prior to making the lesion, the hand, foot and digit representations in VPL were identi¢ed by mapping the locations of neurons responding to gentle stimulation of the body surface. One track was then chosen along which neurons had receptive ¢elds on the glabrous skin of one digit throughout most of the anteroposterior extent of the track. Electrolytic lesions were made in 200-Wm steps as the electrode

Table 2. VPL lesions and extent of area 3b hand representation % VPL % VPL hand lesioned representation lesioned (estimate)

Lesion Survival Area of hand Area of D1 centered in time representation representation in area 3b representation (days) in area 3b (mm2 )a of digit (mm2 )a

Area of D2 representation in area 3b (mm2 )a

Area of D3 representation in area 3b (mm2 )a

Area of D4 representation in area 3b (mm2 )a

Area of D5 representation in area 3b (mm2 )a

0 (n = 5) 23.7 31.1 41.4 45.0 70.0

^ D2 D2 D1 D4 D1^D4

7.5 V 1.9 5.9 5.4s 10.5u 6.9 silent

4.6 V 1.1 5.6 4.7 4.7 8.7u silent

4.1 V 1.2 3.0 3.1 5.0 2.4s silent

2.9 V 0.8 3.5 3.7 2.9 6.2u silent

0 28 37 44 57 78

^ 14 14 37 14 14

29.1 V 5.4 32.6 30.1 31.4 32.9 silent

10.0 V 2.0 14.6u 13.2u 8.3 8.7 silent

a Areas not marked by a symbol are within 1 S.D. of the normal value. u: Expansion equivalent to more than 1 S.D. from the normal value. s: Reduction equivalent to more than 1 S.D. from the normal value.

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vival times ranged from 14 to 45 days. Five animals served as controls. In these animals, the pre- and postcentral gyri both ipsi- and contralateral to the thalamic lesion were sectioned at 25 or 30 Wm in the parasagittal plane. Sections were prepared for in situ hybridization, immunostaining, CO histochemistry or Nissl staining exactly as described above for brains with peripheral lesions. Analyses of lesion size in the thalamus and quanti¢cation of mRNA levels and patterns of immunocytochemical staining, including counts of GABA-immunoreactive cells, in area 3b were carried out as described above. All studies were carried out on animals in which the cortex had not previously been exposed for mapping procedures and the dura mater had not been opened. The upper limb representation in area 3b was thus located by the same topographic criteria, as described for animals with peripheral lesions.

intact proximal phalanx, which is normally represented posteriorly in area 3b, had expanded forwards to occupy the region normally containing the representation of the glabrous skin covering the middle and distal phalanges and, most anteriorly of all, that of the nail bed, an expansion of the representation of the remaining proximal part of the ¢nger of approximately 2 mm. There was also some rearrangement of the representation of the hairy skin on the back of the stump of D2 in regions bordering the D1 and D3 representations. Within the new representation, receptive ¢elds of neurons and response thresholds were not di¡erent from those on the una¡ected side and as the microelectrode was moved mediolaterally or anteroposteriorly in the representation of the stump, a systematic topographic map could be demonstrated. This experiment demonstrates the capacity of representations of remaining areas with intact innervation to expand into a representation that has lost its major source of peripheral input, and the capacity of the new representation to reorganize itself into a topographically coherent map of the relevant skin surface.

RESULTS

Typical pattern of reorganization after peripheral dea¡erentation Fig. 1A illustrates a typical map of the greater part of the contralateral half body surface obtained in area 3b by multiunit recording. Fig. 1C, D is illustrative of the pattern of reorganization that occurs in the long term in the map after moderate degrees of peripheral dea¡erentation. In this case, the distal and intermediate phalanges of the index ¢nger (D2) of one hand had been amputated as the result of injury 22 months previously (Manger et al., 1996a,b). In the a¡ected area 3b, there was a slight expansion of the representations of the adjacent digits (D1 and D3) into the region normally occupied by the representation of D2. Overall, the representation of the remaining stump of D2 measured 1.5 mm mediolaterally and 4.5 mm anteroposteriorly in comparison with the representation of the intact D2 on the contralateral side (which measured 2 mmU6.5 mm). The di¡erence could be accounted for by slight expansions of the representations of the adjacent D1 and D3. Internally the representation of D2 had undergone considerable changes, the representation of the proximal glabrous skin in particular having expanded to occupy the region in which the distal glabrous skin is normally represented. The representation of the glabrous skin covering the

Maintenance of gene expression in area 3b after peripheral nerve lesions and amputations The normal patterns of gene expression for GAD67 , GABAA receptor subunits, GABAB receptors, glutamate receptor subunits and CaMKII isoforms in the monkey somatosensory cortex have been documented by Huntsman et al. (1995, 1999), Jones et al. (1997), Huntsman and Jones (1998), Tighilet et al. (1998b) and Mun‹oz et al. (1999). Some of these patterns can be seen in Fig. 2. Overall, AMPA/kainate and NR1, NR2A, and NR2B receptor transcripts show higher expression than other transcripts. In area 3b in the present set of experiments (Fig. 3), mRNA levels were greatest for CaMKII-K (1500^2000 nCi/g), high for NR1, GABAA -RK1, GluR2, GluR4, CaMKII-L and GABAB receptor mRNAs (1000^1500 nCi/g), moderate for GABAA -RL2 and Q2, GluR1, GluR2, NR2A, and NR2B mRNAs (500^1000 nCi/g), and low for GABAA -RK2, K4, K5 and L1, GluR3, Glur5^7, NR2C and NR2D mRNAs

Table 3. VPL lesions and mRNA levels in area 3b hand representation % VPL lesioned

0 (n = 5) 1^10 (n = 4) 15 56

Survival % VPL time hand represen- (days) tation lesioned (estimate)

0 0^6 21 62

^ 14^45 14^45 47

mRNA

GAD

GABAA -R GABAA -R GABAA -R GABAA -R GABAB -R CaMKII-K GluR1 GluR2 GluR3 NR1 K1 L2 Q2 K5

c u u s

c u u s

c u u s

c u u u

c ^ u u

c, control ; u, levels unchanged in comparison with leg representation ; ^, not studied. u: Increased levels equivalent to v 1 S.D. in comparison with leg representation. s: Reduced levels equivalent to v 1 S.D. in comparison with leg representation.

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c ^ ^ u

c u u u

c u u u

c u u u

c u u s

c u u u

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Fig. 2. Photomicrographs of autoradiograms from adjacent sections through the hand representation, showing hybridization of cRNA probes complementary to mRNAs for 67-kDa GAD, CaMKII-K, GABAA -RL2, and GABAA -RK5 in a monkey in which the contralateral median nerve had been cut 32 days previously. Note lack of visible alterations in gene expression in area 3b. Scale bar = 1 mm.

(100^500 nCi/g). Most of the mRNAs showed laminaspeci¢c patterns of distribution and immunocytochemistry, using GABA- or receptor subunit-speci¢c antisera with double labeling for 28-kDa calbindin, parvalbumin, or CaMKII-K, revealed patterns of cell-speci¢c expression as well. These will not be described here. Preservation of gene expression in area 3b after peripheral nerve lesions Table 1 shows the types of peripheral dea¡erentations

and the survival times of animals whose somatosensory cortex was examined for evidence of alterations in gene expression comparable to those seen in the primary visual cortex after silencing or otherwise perturbing inputs from one eye. Despite the robustness of the ¢ndings in the visual cortex, we could never observe, qualitatively, the same e¡ects on gene expression in the somatosensory cortex after the deprivation paradigms shown in Table 1 (Figs. 2 and 4). Nor was there any evidence, at the survival times studied, of reductions in CO staining or parvalbumin immunostaining in the thalamic a¡erent plexus

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of deep layer III and layer IV comparable to that seen in deprived eye dominance columns of the primary visual cortex, or in area 3b after lesions of the ventral posterior nucleus of the thalamus (see below). Quantitative examination, comparing levels of gene expression in the upper and lower limb representations after sectioning the radial, median or ulnar nerves in the upper arm, and comparing the upper limb and face representations after mandibular nerve cuts, or the tail representation with that of the lower limb after partial amputations of the tail, revealed only minor di¡erences, none of which were statistically signi¢cant. The largest, although still modest, alterations were found only after multiple nerve cuts, e.g. the ulnar and radial nerves, especially when imposed on an animal that already had a longstanding amputation of several ¢ngers (Fig. 4C). In no animals could alterations in the number of cells immunoreactive for GABA or other neuroactive molecules be detected (Table 4). We are forced to conclude that alterations in gene expression that would alter the balance of intracortical excitation and inhibition, at least at this level of resolution and at the survival times studied, are unlikely to play a substantial role in the reorganization of cortical maps that occurs in area 3b after disruptions of peripheral sensory input. Representation of the hand in area 3b following thalamic lesions The representation of the hand in area 3b of the control animals occupied 29.1 V 5.4 mm2 of the surface area of area 3b (Fig. 1, Table 2), the representation of the thumb (D1) which is located laterally, occupied an area of 10 V 2 mm2 ; on progressing medially in area 3b, the representation of the index ¢nger (D2) occupied 7.5 V 1.9 mm2 , that of the third ¢nger (D3) 4.6 V 1.1 mm2 , that of the fourth ¢nger (D4) 4.1 V 1.2 mm2 , and that of the ¢fth ¢nger (D5) 2.9 V 0.8mm2 . The e¡ects of VPL lesions on somatotopic maps were compared with this set of normal data (Table 2). In all cases, although neuronal responses to stimulation of other parts of the body surface were

recorded throughout area 3b, only the relevant portions of the somatotopic maps are shown. Unless otherwise indicated, somatotopy and receptive ¢eld characteristics were normal in una¡ected parts of area 3b. Other cortical areas were not examined in detail, but neuronal responses were elicited from areas 1 and 3a at the borders of area 3b, and in all cases except those with the largest lesions, these appeared normal. Somatotopy of area 3b following VPL lesions In four cases, electrolytic lesions centered in the thalamic representation of one or other digit destroyed less than 10% of the volume of VPL (Fig. 5, Table 2), and mapping of the ipsilateral area 3b revealed no di¡erences in the extent of the representation of the hand or in its internal topography. A lesion that destroyed 23% of VPL, and an estimated 28% of the hand representation (Table 2, Fig. 6A), was centered in the representation of the glabrous tip of D2. In this case, the extent of the overall representation of the hand in area 3b was approximately the same as in normal cases except for a slight increase in the area devoted to the representation of D1 (Table 2). In a case in which 31% of total VPL volume and an estimated 37% of the volume of the hand representation were destroyed, the lesion was centered in the thalamic representation of D2 (Table 2, Fig. 6B). The representation of D1 was slightly enlarged and the representation of D2 was slightly reduced. The extent of the overall area 3b hand representation and the extents of the representations of D3^D5 were within normal limits (Table 2). In a case in which 41% of the total volume of VPL and an estimated 44% of the volume of the hand representation were destroyed (Table 2, Fig. 6C), the lesion was centered in the representation of D1. The extent of the hand representation and of the representations of all digits except D2 in area 3b were within normal limits; the representation of D2 was slightly enlarged (Table 2). In a case in which 45% of the total volume of VPL and an estimated 57% of the volume of the hand representa-

Fig. 3. Comparison of levels of mRNAs assayed in the hand and foot representations of ¢ve control monkeys. Note similarities in levels in the two representations.

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Fig. 4. Comparisons of levels of mRNAs in the hand and foot representations in monkeys in which the indicated peripheral nerves on the contralateral side had been cut approximately 1 month previously. These manipulations are without obvious e¡ects on mRNA levels in the hand representation except in the case in which both the ulnar and radial nerves had been cut in an animal in which three ¢ngers of the same hand had been amputated 2 years previously. In this case, there is a slight statistically signi¢cant increase in levels of CaMKII-K mRNA and a slight decrease in GAD mRNA (C).

tion (Table 2, Fig. 6D) were destroyed, the lesion was centered in the representation of D4. The extent of area 3b devoted to the representation of the hand and the extents of the representations of D1 and D2 were within normal limits (Table 2). However, the extent of the cortical representation of D4 was signi¢cantly diminished, and the representations of D3 and D5 were signi¢cantly increased (Table 2). Despite the major reduction in extent of the D4 representation, receptive ¢elds were normal in size, those on the distal aspect of the digit being characteristically small and, together, the receptive ¢elds covered the entire glabrous and hairy portions of the digit (Fig. 7). Neuronal responses elicited by light tactile stimulation of D4 were brisk and transient and not qualitatively di¡erent from those elicited in normal animals. The remainder of the somatotopic representation of the body in area 3b was normal. In a case in which 70% of the total volume of VPL and

an estimated 78% of the volume of the hand representation were destroyed (Table 2), the lesion was made along four recording tracks from which responses were obtained to stimulation of D1, D4, the hairy surface of the back of the hand, and the great toe (T1). In this case, the region of area 3b in which the hand is normally represented was totally silent, no neuronal responses could be elicited, and no representational map could be delineated. The parts of area 3b adjacent to the region of the silent hand representation showed little background activity and neuronal responses to stimulation of other parts of the body surface could be elicited only with di⁄culty. In these experiments, with the mapping methods used, there was no change in the representational map of the hand in area 3b until 23% of the volume of VPL (or an estimated 28% of the representation of the hand in VPL) was destroyed. Alterations were, however, relatively

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Fig. 5. Camera lucida outlines of frontal sections through the ventral posterior complex of a normal monkey in posterior (top left) to anterior (lower right) order. Numbers represent distance in Wm from the posterior pole of the complex. On these are plotted the outlines and extents of representative lesions that a¡ected varying amounts of the volume of VPL and made prior to mapping or examining mRNA levels in the ipsilateral area 3b.

(in these cases usually a ¢nger) starts to diminish in size and the representations of neighboring digits to expand. Then, when a second threshold is reached, the whole representation breaks down and the cortex is silenced. In what follows, the capacity of activity in the divergent thalamocortical projection to support a topographic map is shown to be paralleled by its capacity to maintain normal patterns of neurotransmitter-related gene expression.

modest until 45% of VPL and an estimated 57% of the hand representation were destroyed. Then, the cortical representation of the digit whose representation was primarily a¡ected in the VPL nucleus was reduced in size and the representation of neighboring digits expanded. When 70% of VPL and more than 70% of the hand representation was destroyed, the map of the hand was silenced. These results indicate that, within certain limits, divergence in the thalamocortical projection is su⁄cient to maintain a normal somatotopic map in area 3b despite loss of a large proportion of the ¢bers contributing to this projection. After a certain threshold is reached, the cortical representation of the a¡ected part

Gene expression in area 3b after thalamic lesions Experiments described in this section show that even

Table 4A. GABA-immunoreactive cells per 50Wm wide column through the thickness of area 3b (normal macaque monkeys ; Hendry et al., 1987) Monkey

1

2

3

Count

1

2

1

2

1

2

Face representation Hand/arm representation Trunk representation Foot/leg representation

38.0 V 2.1 38.9 V 2.8 36.6 V 3.0 37.4 V 2.9

38.6 V 2.9 36.0 V 2.7 37.1 V 3.1 37.4 V 3.4

35.7 V 5.4 34.7 V 6.0 32.0 V 6.1 35.8 V 5.5

33.7 V 5.7 33.1 V 6.4 33.4 V 6.1 33.4 V 5.9

39.1 V 2.9 38.7 V 2.8 39.4 V 3.3 39.6 V 4.1

39.2 V 3.8 41.4 V 2.5 38.0 V 3.4 38.7 V 2.2

Table 4B. GABA-immunoreactive cells per 50 Wm wide column through the thickness of area 3b (controls, nerve-lesioned and thalamiclesioned macaque monkeys) Control 1

Face representation Hand representation Foot/leg representation Hand representation, parvalbumin-weak zone Hand representation, parvalbumin+ zone

38.5 V 4.1 39.8 V 5.8 40.1 V 5.2 ^ ^

Control 2

37.5 V 4.5 37.9 V 3.3 38.7 V 3.9 ^ ^

Nerve-lesioned

Thalamic-lesioned

Radial N., Ulnar and radial 10% VPL 28 days Nn., 32 days

15% VPL

56% VPL

34.5 V 6.1 34.7 V 6.0 35.7 V 2.7 ^ ^

34.3 V 5.6 32.4 V 5.9 37.4 V 4.8 35.8 V 4.2 37.2 V 2.1

33.7 V 4.2 32.8 V 2.7 34.5 V 4.4 32.1 V 2.9 38.1 V 2.2

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35.7 V 3.9 37.8 V 4.7 32.1 V 4.6 ^ ^

37.2 V 4.2 37.8 V 3.7 38.2 V 2.1 ^ ^

A¡erent divergence and cortical maps

785

Fig. 6. Maps of the representations of the hand and adjacent regions, made in the same manner as Fig. 1 and taken from monkeys in which various amounts of the VPL nucleus of the thalamus had been ablated several weeks earlier. The representation of the digit whose representation was most a¡ected in the thalamus is shaded. Only after the 45% lesion is there overt shrinkage of the area 3b representation and expansion of the representations of adjacent digits.

after destruction of up to 50% of the somatosensory thalamus, only minor alterations in GABA- and glutamaterelated gene expression can be detected. In designing these experiments, it was argued that if divergence is a primary factor in supporting plasticity of representational maps in the somatosensory cortex, then not only the maps, as demonstrated above, but also gene expression for activity-dependent molecules related to the inhibitory and excitatory transmitter systems should be maintained intact until a point were reached at which continuing adaptation would be impossible and alterations would become manifest. Immunocytochemistry and mRNA levels in area 3b after thalamic lesions Lesions that destroyed less than 15% of VPL volume,

or less than an estimated 20% of the hand representation in VPL, were not associated with detectable changes in histochemical or immunocytochemical staining or in mRNA levels in area 3b. Lesions that destroyed more than 15% of VPL and more than an estimated 20% of the hand representation were associated with reductions in staining for CO, as well as in immunoreactivity for parvalbumin, in a localized part of the area 3b hand representation, and this was replicated with larger lesions (Figs. 8^11). The reduction in staining appeared as a ‘gap’ of reduced staining in the normally dense band of CO or parvalbumin staining that occupies layer IV and the deep aspect of layer III, a band that corresponds to the principal laminae of termination of thalamocortical a¡erents. The reduction in immunostaining for parvalbumin represents degeneration or loss of staining in thalamocortical a¡erent ¢bers. Despite this, no qualitative

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grams, except in the case of the largest lesions, which approached destruction of 50% of the hand representation in VPL (Figs. 8F and 11). Quantitative results

Fig. 7. Sizes and locations of receptive ¢elds of neurons recorded in the shrunken representation of the fourth digit (D4) in the case shown in Fig. 6 in which 45% of the VPL nucleus was destroyed.

alterations in immunocytochemical staining for the neural antigens or their mRNAs could be detected in the zone corresponding to the gap in the CO and parvalbumin staining in adjacent sections or in the autoradio-

Levels of mRNAs in area 3b. In cases with no thalamic lesions there was some normal variability in the levels of expression of the mRNAs studied in the upper and lower limb representations, as shown by the relatively large standard deviations (Fig. 3), but these were not statistically signi¢cant. There were also no signi¢cant di¡erences between the hand and foot representations in four cases in which up to 10% of the total volume of VPL and an estimated 0^6% of the VPL hand representation were destroyed (Fig. 12, Table 3). In a case in which 15% of the volume of VPL and an estimated 21% of volume of the hand representation were destroyed, there was a trend towards reduced levels of GAD67 and certain GABAA receptor subunit mRNAs and towards increased levels of GluR2 and CaMKII-K mRNAs (Fig. 12, Table 3). GAD67 mRNA was reduced by 5.32 V 1.85%, GABAA -RK1 mRNA was reduced by 4.70 V 2.76%, and CaMKII-K mRNA was increased by 4.76 V 2.83% in the hand representation in comparison with the foot representation of area 3b (Fig. 12),

Fig. 8. Digital micrographs of sections through the middle of the ventral posterior complex in six monkeys in which various amounts of the VPL nucleus were destroyed by electrolytic (A^D) or cytotoxic (E, F) lesions. A^E are stained for CO, F was hybridized to a cRNA complementary to the mRNAs for GABAB receptor Ia and Ib subunits. Scale bar = 500 Wm. VMb: basal ventral medial nucleus; VPI: ventral posterior inferior nucleus.

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Fig. 9. Adjacent sections through the hand representation, stained for parvalbumin (A), CO (B) or hybridized for GAD or CaMKII-K mRNAs in a monkey in which approximately 15% of the ipsilateral VPL nucleus was destroyed 1 month earlier. Note loss of parvalbumin immunostaining in the thalamic a¡erent ¢ber plexus in layers IIB and IV with corresponding weakening of CO staining. These changes are not, however, accompanied by any discernible reductions in mRNA levels in the same regions of the adjoining sections. Scale bar = 1 mm. Arrowheads in C indicate region of traverse through which GABAimmunoreactive cells were counted in adjacent sections immunostained for GABA (Table 4).

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Fig. 10. Adjacent sections through the hand representation, prepared as in Fig. 9, from a monkey in which approximately 25% of the ipsilateral VPL nucleus was destroyed 1 month earlier. Despite loss of the thalamic a¡erent plexus (between arrows in A and B), there is no discernible reduction in GAD or CaMKII-K mRNAs. Scale bar = 1 mm.

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Fig. 11. Adjacent sections through the hand representation of area 3b immunostained for parvalbumin (A) or hybridized for the mRNAs indicated, from a monkey in which more than 50% of the ipsilateral VPL nucleus was destroyed 1 month earlier, showing localized loss of the thalamic a¡erent plexus (between arrows, A) and now with accompanying reductions in mRNA levels. Scale bar = 1 mm.

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although these di¡erences did not reach statistical signi¢cance. There were no di¡erences between mRNA levels measured across the regions corresponding to the localized zone of reduced CO and parvalbumin immunostaining in comparison with the immediately adjoining regions. Apart from alterations in parvalbumin immunostaining, no qualitatively distinguishable alterations in immunostaining for any of the other antigens were evident. The number of GABA-immunoreactive cells was approximately the same when counted in 50 Wm wide traverses across the cortex in regions corresponding to the gap in the parvalbumin and CO staining and in the regions of normal intensity of staining bordering it (Table 4). Following destruction of 56% of the volume of VPL and an estimated 62% of the volume of the VPL hand representation (Table 3), there was a much larger gap in the CO-stained and parvalbumin-immunostained thalamic ¢ber plexus and signi¢cant di¡erences were seen in mRNA expression in the upper limb representation in comparison with that of the lower limb (Figs. 11 and 12). In the upper limb representation, levels of expression of GABAA -RK1 mRNAs were reduced by 17.19 V 7.76% (P 9 0.03), levels of GAD67 mRNAs were reduced by 15.65 V 7.61% (P 9 0.02), and levels of GluR2 mRNAs were increased by 11.61 V 4.32% (P 9 0.01) in comparison with levels in the lower limb or face representations (Fig. 12). Although there was a trend towards reductions in levels of GABAA -RL2 and Q2, and GABAB receptor mRNAs and towards an increase in CaMKII-K mRNA, these di¡erences did not reach statistical signi¢cance (Fig. 12, Table 3). Immunostaining for GABA and for GABAA -RK1, L2 and Q2 appeared reduced in the zone corresponding to the gap in the lamina of parvalbumin staining and there was suggestive evidence of an increase in CaMKII-K in the same zone but these were not quanti¢ed. In these experiments, quanti¢cation of mRNA levels in area 3b of the monkeys subjected to thalamic lesions, and with loss of thalamocortical ¢bers revealed by loss of parvalbumin immunostaining, there were no signi¢cant di¡erences in levels of expression in the upper limb representation until more than 15% of the total VPL volume (21% of the VPL hand representation) was destroyed (Table 3). Beyond this point, levels of GAD and GABAA -RK1 mRNAs were signi¢cantly reduced, levels of CaMKII-K mRNA were signi¢cantly increased and there was a trend towards an increase in GluR2 mRNA, in the hand representation (Table 3). These di¡erences were, nevertheless, relatively modest in comparison with the severity of the thalamic lesion. Fig. 12. Bar graphs showing mean alterations in mRNA levels in the hand representation of area 3b in comparison with those in the foot representation in animals in which up to 10% (upper), V15% (middle) and s 50% of the ipsilateral VPL nucleus were destroyed approximately 1 month earlier. Minor di¡erences after 10% and 15% lesions are mostly insigni¢cant. Alterations only start to reach statistically signi¢cant levels after the largest lesion.

DISCUSSION

The ¢rst set of experiments demonstrated that the typical changes in cortical representations that ensue in the short and long term from peripheral dea¡erentations of moderate size are unaccompanied by detectable changes in levels of gene expression for any of the molecules involved in the GABA and glutamate neurotransmitter

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systems in area 3b. These ¢ndings contrast dramatically with the robust changes that occur in deprived ocular dominance columns of the primary visual cortex in adult monkeys following loss or reduction of inputs from one eye (Hendry and Jones, 1988; Jones, 1993; Benson et al., 1994; Jones et al., 1994a,b). The lack of changes in the somatosensory cortex led to the proposal that the previously demonstrated divergence in the thalamocortical projections from the ventral posterior nucleus to area 3b may be su⁄ciently great to prevent any region of area 3b from being completely silenced by peripheral dea¡erentation (Rausell and Jones, 1995; Rausell et al., 1998). The subsequent experiments were designed to test the limits of this divergence in maintaining not only activity-dependent gene expression but also topographic maps in area 3b, by progressively reducing thalamic inputs by lesions of increasing size. It was found that the input from the thalamic representation of a ¢nger could be reduced by as much as 45% before the representation of that ¢nger began to shrink in area 3b and that more than 20% of the thalamic representation of the hand could be destroyed before modest changes could be detected in gene expression for neuroactive molecules in area 3b. From these results it can be argued that ascending divergence in the somatosensory pathways from periphery to cortex is capable of maintaining activity in the somatosensory cortex in the face of major peripheral dea¡erentations and that this divergence should be a major factor in promoting reorganization of somatotopic maps in this cortex when input from the periphery is perturbed by dea¡erentation or modi¢ed by other factors that alter patterns of sensory input from periphery to cortex. Cortical and subcortical contributions to somatosensory cortex plasticity Although cortical mechanisms have commonly been regarded as pre-eminent in reorganization of somatosensory cortical maps, there is now strong evidence for coexistent reorganization of the relays of the ascending somatosensory pathways in the brainstem and thalamus that, when projected upon the cortex, should contribute to this reorganization (see below). Divergence of lemniscal inputs to the thalamus and of thalamic projections to the cortex should serve to amplify changes occurring at subcortical centers so that a small change in the brainstem representation would be magni¢ed by divergence of dorsal column projections to the thalamus, and magni¢ed further by divergence in the thalamocortical projection. Reorganization in the brainstem and thalamus It is now clear that the same kinds of immediate and longer term plastic phenomena occur in the thalamus and brainstem as in the somatosensory cortex after identical manipulations of sensory input. In the brainstem, after partial dea¡erentation or temporary blocking of peripheral inputs to the gracile or cuneate nuclei, deafferented cells can immediately acquire larger (Millar et

791

al., 1976) or new (Pettit and Schwark, 1993) peripheral receptive ¢elds and cells can become activated by inputs from regions of the body adjoining those denervated, both immediately (Xu and Wall, 1999a,b) and in the long term (Dostrovsky et al., 1976; Kalaska and Pomeranz, 1982). Alterations in receptive ¢elds of single neurons in the reorganized nuclei are virtually identical to those occurring in the somatosensory cortex and the immediate changes occurring in the somatotopic map of the hand di¡er only in details from the contemporaneous changes recorded in the cortex (Xu and Wall, 1999a,b). Sprouting of intact a¡erents into a denervated dorsal column nuclei has also been proposed as an accompaniment of reorganization at these subcortical centers (Jain et al., 1997), although formation of new synapses has not been demonstrated and no suggestions have been advanced as to how sprouting contributes to the reorganization. In the somatosensory thalamus, expansions of receptive ¢elds of cells or of representations of body regions occur after destruction or reversible blockade of a dorsal column nucleus (Fadiga et al., 1978; McMahon and Wall, 1983; Parker et al., 1998; Wall and Egger, 1971), section of the gracile fasciculus (Pollin and Albe-Fessard, 1979), dorsal rhizotomies (Jones and Pons, 1998a), nerve section (Garraghty and Kaas, 1991b), amputation of a digit (Rasmusson, 1996), and in humans after spinal cord transections or limb amputations (Lenz et al., 1994, 1998). Injection of local anesthetic into the receptive ¢elds of ventral posterior neurons in rats leads to immediate emergence of new receptive ¢elds, akin to those reported in the dorsal column nuclei (Nakahama et al., 1996; Nicolelis et al., 1993; Shin et al., 1995). Hence, both the dorsal column nuclei and the ventral posterior nucleus of the thalamus become reorganized in the face of loss of sensory input, in a manner identical to, and along a similar time scale as, the somatosensory cortex. Divergence in ascending somatosensory projections The interpretation of the present results depends upon the extensive divergence previously demonstrated in the thalamocortical projection from the ventral posterior nucleus of the thalamus to area 3b in normal monkeys (Rausell et al., 1998). However, this divergence is at the upper end of a fountain of diverging projections that commences with the primary a¡erents entering the spinal cord. Individual dorsal column primary a¡erents ending in the dorsal column nuclei do not branch extensively and have limited domains of termination. However, the input to a dorsal column nucleus from a single body part arrives in a group of many ¢bers and these collectively terminate on large numbers of cells throughout much of the rostrocaudal extent of the nucleus. In the cuneate nucleus of monkeys, the group of primary a¡erent ¢bers from a ¢nger terminates in an elongated, column-like array, V0.2 mm thick and V3 mm long (Culberson and Brushart, 1989; Florence et al., 1988, 1989; Nyberg and Blomqvist, 1982; Rasmusson, 1988). In cats, single large diameter primary a¡erent ¢bers terminate in the cuneate nucleus in domains averaging 480 Wm in rostro-

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caudal length and 0.042 mm2 in cross sectional extent (Weinberg et al., 1990), encompassing approximately 1700 neurons (Heino and Westman, 1991). At any location, branches of approximately 300 cutaneous a¡erents overlap. Immediate expansions of receptive ¢elds of dorsal column nucleus cells after partial dea¡erentation are, therefore, likely to be indicative of divergent connections of this kind, many of which are normally silent. In monkeys, the fact that terminations of a¡erents from hairy and glabrous skin can lie in close proximity and even overlap (Florence et al., 1989) is undoubtedly a factor in promoting immediate expansion of the representation of hairy skin on the back of the hand into territory normally occupied by the representation of glabrous skin on the palm when the palm is denervated (Xu and Wall, 1999a,b). Small divergences of individual axons of cuneate nucleus cells, in projecting to the thalamus, will be magni¢ed in the projection of the group as a whole, thus serving to amplify at thalamic levels any alterations of the body map in the cuneate nucleus. Divergence in the thalamocortical projections should amplify this further in the cortex. In this way, the expansion of the representation of the hairy part of the hand into that of the palm is replicated in magni¢ed and only slightly topographically modi¢ed form in area 3b of the cortex (Xu and Wall, 1999a,b). Input^output divergence in the thalamus Individual medial lemniscal ¢bers emanating from the cuneate and gracile nuclei terminate in the ventral posterior nucleus of macaque monkeys in localized zones and in relation to 120^200 neurons (Jones, 1983). However, lemniscal ¢bers bearing information from one part of the body such as a ¢nger terminate in a shifted overlap throughout the anteroposterior extent of the nucleus, so that a ¢nger is represented in an anteroposterior lamella receiving hundreds of ¢bers and containing many hundreds of cells. A lamella representing a ¢nger extends V3 mm dorsoventrally, 3^3.5 mm anteroposteriorly, and is V0.1 mm thick (Jones and Friedman, 1982; Jones et al., 1982; Rausell et al., 1998). The axons of individual cells in this lamella each project to a cortical territory no greater than 600^800 Wm in surface extent (Garraghty and Sur, 1990; Rausell and Jones, 1995), but adjacent cells can project to patches of cortex up to 1.5 mm distant from one another (Rausell and Jones, 1995). This degree of divergence may provide a basis for the ‘cortical distance limit’ in short term plasticity. When a whole V3U3U0.1-mm lamella of thalamic cells representing a single ¢nger is considered the divergence in the projection of the thalamic representation of that ¢nger as a whole is much greater because cells at the anterior and posterior ends of the lamella can project to points many millimeters apart in the cortex. Recent studies show that 0.1 mm3 of ventral posterior nucleus can project to as much as 20 mm2 in surface extent of cortex (Rausell et al., 1998). Although the major zone of cortical terminations is concentrated at the center of this region, the projection of a group of ventral posterior cells in a lamella measuring 3U3U0.1 mm and representing a ¢n-

ger is still much greater than the representation of the ¢nger in area 3b as revealed by multiunit mapping. In most studies it measures 4^10 mm2 (Pons et al., 1987; Manger et al., 1996a,b; present results). Divergence and overlap in the projections from adjacent lamellae in the ventral posterior nucleus representing adjacent body parts is, therefore, greater than the recorded representation. Somatotopically inappropriate connections dependent on this divergence should be demonstrable at the single neuron level if the divergent branches form active synapses. This has been shown in a few cases in cats (Snow et al., 1988; Waldron et al., 1989) and raccoons (Smits et al., 1991; Zarzecki et al., 1993). The degree of divergence in the thalamocortical projection makes feasible the expansion of the representation of the radial nerve to ¢ll the former representation of the palm, denervated by section of the ulnar and median nerves (Garraghty and Kaas, 1991a,b; Schroeder et al., 1997; Churchill et al., 1998), especially if determined ¢rst in the dorsal column nuclei and then magni¢ed by the lemniscal projection to the thalamus and by the thalamic projection to the cortex. The present study showed that thalamocortical divergence in monkeys is su⁄ciently great that a substantial part of the ventral posterior representation of a single digit can be destroyed before the representation of that digit in area 3b starts to shrink and that of adjacent digits to expand (Jones et al., 1997). This divergence may be responsible for reorganization of cortical maps that exceeds the short term, 1.5-mm ‘distance limit’. In an interesting parallel to the present ¢ndings at a lower level of the system, Jain et al. (1997) found that survival of only a few primary a¡erent ¢bers after section of the cuneate fasciculus is su⁄cient to cause retention of at least part of the map of the upper limb in the somatosensory cortex, a map that is completely silenced if all the ¢bers are cut. Similarly, microlesions of the cuneate nucleus in rats fail to cause alterations in responsiveness of more than half the neurons recorded in somatotopically coupled parts of the ventral posterior nucleus (Alloway and Aaron, 1996), suggesting that remaining inputs to ventral posterior cells are capable of maintaining receptive ¢eld integrity and neuronal responsivity. The fact that such a large proportion of a VPL digit representation could be destroyed without majorly altering the projected representation in area 3b appears to provide con¢rmation of the view that ascending divergence should be a major factor in plasticity of somatosensory cortical maps. Among other a¡erent systems that might support a somatotopic map and activity-dependent gene expression in area 3b in the face of thalamic lesions that reduce input, the spinothalamic and corticocortical systems need to be considered. Although terminations of the spinothalamic system in VPL would have been destroyed by the lesions, many of the terminations of this system are outside VPL in smaller celled regions around and posterior to VPM (Rausell et al., 1992a,b), so that they and their cortical projections would have been largely spared by the lesions. Maintenance by commissural projections is also unlikely, since most parts of the hand representa-

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tion in area 3b are devoid of such projections (Jones and Powell, 1969; Jones et al., 1979; Jones and Hendry, 1980; Killackey et al., 1983). The fact that the area 3b map becomes silent once loss of inputs from VPL crosses a threshold seems to imply that corticocortical inputs to area 3b from other areas of the ipsilateral somatosensory cortex (which mostly receive their thalamic input from the ventral posterior nucleus anyway) are insu⁄cient to maintain activity in area 3b, as its thalamic input is lost. Neurotransmitter e¡ects in cortical plasticity E¡ects on fast inhibitory responses mediated by GABA and on excitatory transmission mediated by glutamate receptors are thought to be components of the cortical response to peripheral dea¡erentation. In the somatosensory cortex, dorsal column nuclei and thalamus, receptive ¢eld expansions occur when GABAA receptors are blocked (Alloway and Burton, 1986, 1991; Dykes et al., 1984; Hicks et al., 1986; Schwark et al., 1999), and removal of inhibition is thought to underlie immediate expansions of receptive ¢elds of somatosensory cortical neurons after loss of peripheral input by amputation or local anesthesia of a digit, an e¡ect that may depend on loss of tonic control by C ¢ber inputs over central inhibitory mechanisms (Calford and Tweedale, 1991c). Re-establishment of inhibition is thought responsible for shrinkage of these ¢elds back to their normal size over time. NMDA receptors also seem to play a role in the plastic response of the somatosensory cortex to deprivation of inputs (Kano et al., 1991; Garraghty and Muja, 1996). Because NMDA and nonNMDA receptors are both involved in somatosensory thalamic transmission (Salt and Eaton, 1990; Golshani et al., 1998), it would be surprising if thalamic plasticity did not involve one or both of these, as well as GABA receptors. Deprivation-dependent modulations of gene expression for molecules involved in maintaining the overall balance of excitation and inhibition have been di⁄cult to detect in the monkey somatosensory cortex. This is surprising in view of the dramatic e¡ects that perturbations of retinal input can have on expression of genes related to the GABAergic and glutamatergic neurotransmitter systems in the monkey visual cortex. Within 2 days or less following removal of an eye or blockade of action potentials in an optic nerve by tetrodotoxin injection, levels of many mRNAs and immunocytochemically detectable molecules start to change in the deprived ocular dominance columns. The neurotransmitters GABA and glutamate, GAD, the enzyme involved in GABA synthesis, and a number of GABAA receptor and glutamate receptor subunits are down-regulated, while other molecules related to these transmitter

793

systems, notably CaMKII-K, are up-regulated (Hendry and Jones, 1986, 1988; Hendry and Kennedy, 1986; Benson et al., 1991, 1994; Carder and Hendry, 1994; Huntsman et al., 1994; Tighilet and Jones, 1996; Tighilet et al., 1998b). Modest alterations in levels of immunocytochemically detectable GABA or other agents have been reported in monkey somatosensory cortex after deprivation of peripheral input (Cusick, 1991; Garraghty et al., 1991; Conti et al., 1996) but the present results show that these are very weak in comparison with the changes occurring in the visual cortex, even after the most massive and longest deprivations, and are di⁄cult to replicate at the mRNA level. The changes in gene expression at the levels detectable by the present methods are unlikely to play a major role in forms of plasticity identi¢able at the single cell level in deprived somatosensory cortex. Instead, the results clearly show that even in the presence of a substantially reduced thalamic input, su⁄cient activity can be maintained in the deprived cortex to permit gene expression at close to normal levels. This is undoubtedly another manifestation of the extensive divergence in the thalamic projection to the somatosensory cortex. The di¡erence between the modest changes in gene expression detectable in the somatosensory cortex after peripheral deafferentation and the robust changes seen in the visual cortex after monocular deprivation is a puzzling observation. The di¡erence may be due to the di¡erences in organization of the two systems: removal of an eye or complete blockade of activity in an optic nerve totally deprives all lateral geniculate neurons receiving inputs from that eye and all cortical ocular dominance domains to which they project of activity. In some preliminary studies on the monocularly-deprived cortex of adult monkeys, it was demonstrated that in layer IV, the deprived columns are physiologically silent (Jones et al., 1994a,b). In cats, focal lesions in homonymous parts of the two retinae similarly silence the visual cortex representation of the relevant part of the visual ¢eld, and also silence the representations of the a¡ected parts of the retinae in the lateral geniculate nucleus of the thalamus (Gilbert, 1998). Because of the extensive divergence at all levels in the ascending somatosensory system, it is not possible totally to silence a zone of somatosensory cortex or ventral posterior thalamus by peripheral nerve section and the cortex can only be silenced by very extensive lesions of the thalamus. It is likely that in this divergence lies one of the keys to the remarkable plasticity exhibited by the primate somatosensory cortex.

Acknowledgements2Supported by Grant NS21377 from the National Institutes of Health, United States Public Health Service. We thank Mr. Phong Nguyen for technical help.

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