Neurothem. Int. Vol.21, No. 3, pp. 313-327, 1992 Printedin Great Britain.All fightsreserved

0197-0186/9255.00+0.00 Copyright© 1992PergamonPress Ltd

MUSCARINIC CHOLINERGIC RECEPTOR BINDING IN RAT H I N D L I M B SOMATOSENSORY CORTEX FOLLOWING P A R T I A L D E A F F E R E N T A T I O N BY SCIATIC N E R V E TRANSECTION* UWE-KARSTEN HANISCH,~ THOMAS ROTHE,~ KNUT KROHN~ and ROBERT W . DYKES2Jf ]Paul-Flechsig-lnstitut ffir Hirnforschung, Abteilung ffir Neurochemie, Universitit Leipzig, Friedrich-Ludwig-Jahn-Allee59, 07010 Leipzig, Germany 2Centre de recherche en sciences neurologiques, D~partement de physiologic, Facult~ de mSdecine, Universit8 de Montreal, C.P. 6128, Succursale A, Montreal, Qutbec, Canada H3C 3J7 (Received 18 February 1992; accepted 1 April 1992) AlmrKt--Peripheral nerve injury or amputation leads to extensive changes within the central representations of the mammalian body surface. The mechanisms responsible for post-traumatic reorganization of these maps in adults may also, at least partly, underlie a more general feature of the somatosensory system---the capacity for stimulus-dependent plasticity. Acetyicholine has been implicated in both of these processes. We studied the binding of the ligands [3I-I]QNB and [3I-I]pirenzepinein rat hindlimb somatosensory cortex from 1 to 14 days following sciatic nerve transection. Although the [3I'I]QNBbinding was not different from normal levels in tissue homogenates of the affected somatosensory cortex, differences were demonstrated when binding was measured on a layer-by-layer basis. [3H]QNB binding was changed only in certain layers, at certain times. The predominant effects appeared to be a decrease in binding in the middle layers from 4 to 14 days after the transection. Combining the [3H]QNB data with data obtained from the more M rselective ligand [~H]pirenzepine suggested that complex changes occur among several muscarinic receptors, including receptors with non-M i subtype characteristics. Moreover, unilateral nerve transection affects the hindlimb somatosensory regions in both hemispheres.

The reorganization of the somatotopically-ordered map o f the body found within the primary somatosensory cortex of mammals has become a useful tool for understanding neuronal plasticity. Neurons in adult somatosensory cortex acquire new receptive fields on adjacent body surfaces soon after the original one is lost by amputation or nerve injury (for review see Merzenich et al., 1988). A number of different hypotheses have been tendered to explain the cellular processes involved in the creation o f a new receptive

* This paper is dedicated to the memory of Professor D. Biesold who died on 29 May 1991 at the age of 65. Dr Voiker Bigl, long time colleague and friend of Dietmar Biesold acted as Executive Editor in the processing of this paper. t Author to whom all correspondence should be addressed. Abbreviations: ACh, acetylcholine; CHAT, choline acetyltransferase; HCh3, hemicholinium-3; PDBu, phorbol12,13-dibutyrate; PKC, protein kinase C; QNB. quinuclidinyl benzilate; SDHACU, sodium-dependent highaffinity choline uptake ; SNT, sciatic nerve transection.

field, but few are supported by strong empirical evidence. The types of mechanisms that might explain neuronal plasticity are constrained by the time course of the events that follow deafferentation. The rapidity of the appearance of new receptive fields and their subsequent evolution make sprouting of axonal processes during this early period less likely than an alteration in the efficacy o f existing connections (see Dykes, 1991, for a review of these issues). In the visual system o f young kittens norepinephrine has been impficated in the process o f occular dominance plasticity (Kasamatsu and Imamura, 1991), and perhaps both aoetylcholine (ACh) and norepinephrine are involved (Bear and Singer, 1986). More anteriorly on the cortical mantle the neuromodulator implicated in neuronal plasticity has more often been A C h (Gorman and Woody, 1991 ; Metherate and Dykes, 1988a, b). In the somatosensory cortex Dykes (1991) has hypothesized that ACh is a permissive agent that, when present in sufficient quantities, allows neurons depolarized by

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afferent inputs to strengthen the efficacx of the active connections. If this hypothesis is valid, then a corollary o f the hypothesis is that the s o m a t o s e n s o r y cortex s h o u l d show evidence for an altered state following deaffere n t a t i o n reflecting changes in cholinergic function. Initial tests of this corrolary did not d e m o n s t r a t e a n y change in [3H]quinuclidinyl benzilate ([3H]QNB) binding in raccoon somatosensory cortex within 1 - 1 6 wk after digit a m p u t a t i o n ( S a m p s o n e l al., 1988) a n d in rat somatosensory cortex up to 63 days following sciatic nerve injury (Hanisch e t al., 1989 ; R o t h e e t al., 1990). Nevertheless, we have pursued this q u e s t i o n with a more detailed analysis o f radioligand b i n d i n g to muscarinic receptors in rat s o m a t o s e n s o r y cortex. N o r m a l l y in the rat, the sciatic nerve provides i n p u t to a b o u t 8 5 % of the h i n d l i m b r e p r e s e n t a t i o n in g r a n u lar cortex. Within 2 days following sciatic nerve t r a n section (SNT), the remaining sensory input conveyed in the s a p h e n o u s nerve e x p a n d s nearly 3-fold to control a b o u t one third of the region h a v i n g lost its i n p u t from the transection (Wall a n d Cusick, 1984). Walt (1988a, b) has described in detail the sequence o f events t h a t occur during this time. We have e x a m i n e d the l a m i n a r distribution o f [3H]QNB a n d [3H] pirenzepine binding sites in this region while the reorganizational process was occurring. T h e c h a n g e s observed are subtle, involving more t h a n one class o f muscarinic receptors, a n d seem to be limited to certain sublayers o f the cortex.

EXPERIMENTAL PROCEDURES

Adult male Wistar rats (250-350 g) were anesthetized with i.p. injections of pentobarbital sodium (50 mg/kg). The left hindlimb was shaved and an incision was made over the posterior thigh, exposing the sciatic nerve. A 5 mm section was removed between two ligatures; the wound was closed and treated with antibiotics. The animals survived for periods up to 14 days before they were sacrificed. As controls, untreated animals were kept under identical conditions for 4 days. On the day of the sample, animals were decapitated and the brains were removed rapidly in a cold environment (4°C) and frozen in isopentane ( - 7 0 ° C ) within 2 min. Consecutive coronal sections (10/~m) were cut at - 16 to - 2 0 ° C with a Kryostat microtome (Tissue Tek, Miles, U.S.A.) and thawmounted on gelatin-coated slides. Coronal sections were taken from a range spanning the area of granular cortex representing the hindpaw (Chapin and Lin, 1984). The atlas of Paxinos and Watson (1982) was used for stereotaxic orientation. Adjacent sections were stained for Nissl substance to confirm that indeed the hindlimb representation had been sampled. Binding studies were performed as described by Schliebs et al. (1989) using [3H]QNB (1.11 TBq/mmol) and [3H] pirenzepine (2.05 TBq/mmol) from the Radiochemical

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Centre, Amcrsham (England). Briefly, incubation ol t1~c slides was carried out in humid chambers at room Jempera-. lure by flooding the sections with sodium potassium phosphate buffer (50 mM, pH 7.4) containing [3H]QNB at diflkrent concentrations (1.5, 3.9 and 11.3 nM). Alter 3 h slides were given two rinses of 5 rain each in ice-cold butter bet\~re drying in a light nitrogen stream. Nonspecific binding was determined in adjacent brain sections, co-incubated with 100 /tM atropine sulphate. For assaying the M ~receptor subtype, slides were incubated with sodium phosphate buffer ( I 0 raM. pH 7.4) containing [~H]pirenzepine at different concentrations (t0.9, 17.1 and 32.2 nM), for I h at room temperature. Following two rinses of I min each in ice-cold buffer, the slides were dried in a nitrogen stream. Nonspecific binding was assayed in adjacent sections by adding 100 I~M atropine sulphate to the incubation buffer. Tissue sections were apposed to tritium-sensitive film (Ultrofilm, LKB) at 4 C together with slides containing standards of known radioactivity level (calibration standards were prepared from a brain paste: see Unnerstall et al., 1982). After an exposure of 3 wk, the films were developed with Kodak D-19 developer for 5 rain at 20C, fixed, rinsed and dried. Quantitative analysis of the autoradiograms was done with a computer-assisted imaging device (BVS A6471 ), using a software package written at the AMBA Programming System in the Institute of Neurobiology and Brain Research, Magdeburg (Germany). Measurements of optical density were obtained at 50 successive scanning positions along an axis perpendicular to the cortical surface, and passing through the granular cortex at 2.0-3.0 mm lateral from the pial surface to the ventral margin of layer VI. Measures were calibrated by reference to the standards. No measurable nonspecific binding was observed for either of the ligands. The 50 measurements were grouped into 12 laminar subdivisions of four adjacent measurements each. These bins correspond to the divisions used by Lamour and Dykes (1988). They can be related directly to the laminar arrangement of the primary somatosensory cortex. The first bin is approximately the thickness of layer 1, and the other bins correspond to the following layers : 2-4 (layer ll/IIl), 5 (layer IV. 6-8 (layer V) and 9-12 (layer VI). Note that some imprecisions may arise from individual differences in laminar thickness. The average was calculated for each of these bins from scanning data of 2 to 4 animals. These means were used in a nonlinear curve fitting program (Rothe and Schliebs. 1988) to estimate KD and B,,,~. Note that. because up to 4 measurements were taken from each animal, and averaged with those of other animals, the SEM of these results reflects variation among the animals only indirectly. Statistical comparisons were performed using Welch's two-tailed t-test (Sachs, 1978). Binding of [ ~H]phorbolester- 12,13-dibutyrate ([ 3H]PDBu, Amersham) to activated membrane-bound protein kinase C (PKC, EC 2.7.1.37) was measured in homogenates of tissue samples from the hindlimb somatosensory cortex obtained by the method of Hanisch et al. (1989); a 2 mm diameter cylindrical block of cortex was taken from the brain at 1.5 mm posterior and 2.5 mm lateral to bregma. This position was corrected according to measurements of the brain being studied (Hanisch et al., 1989). The binding assay was performed using a modified procedure according to Gleiter e/ al. (1988). One hundred #1 of a tissue suspension (about 25 pg protein) were incubated for 30 rain in glass tubes with 4 different concentrations of [3H]PDBu (2-25 nM) and 0.1%

Receptor binding in somatosensory cortex bovine serum albumine in a total volume of 200 ~I Tris-HCl buffer (50 raM, pH 7.4). Incubations were terminated by rapid filtration through glass fibre filters (see Schliebs et al., 1983), followed by three washes with 4 ml ice-cold buffer. Filters were measured for retained radioactivity in a Philips PW 4700 liquid scintillation counter. Nonspccific binding was determined in the presence of 100/~M PDBu (Sigma, dissolved in dimethylsulfoxide). Routinely, it represented about 5% of the total binding at a iigand cohcentration of 5 nM. Protein was measured according to Lowry et al. (195 l). All chemicals used were commercial products of the highest purity available. RESULTS [ 3H]QNB binding in the deafferented cortex When studied in tissue homogcnates of the rat hindlimb cortex there were no obvious differences detectable between [aH]QNB binding of control animals and animals having undergone SNT at various times prior to the assay. In a sample of animals at each of 5 times following SNT, as described more fully by Roth¢ et al. (1990), the [aH]QNB binding was not different from control animals. N o r were there any left-right differences in the samples. Laminar measurements revealed, however, that these global values masked changes limited to specific layers of the cortex. Densitometric readings through the hindlimb granular cortex of [3H]QNB autoradiographs (Fig. I) in both control and nerve-transected

315

rats showed that the density of ligand binding varied with the laminar position, lacing least in the most superficial and deepest parts o f the cortex and having local maxima in layers lII and VI. The readings obtained from layers III, IV, and (less clearly) V in control animals were different from those obtained in animals having undergone SNT 14 days before they were sacrificed ; in the experimental group, [3H]QNB binding was decreased (Fig. 2). In an effort to measure these local changes in muscarinic receptors (i) more precisely and (ii) as a function of time following SNT, the Bm,~ and Ko values were calculated for laminar subdivisions from autoradiographic material available at l, 2, 4, 7 and 14 days after SNT. To display the results, the readings were grouped into 12 bins representing 48 out of the 50 individual positions measured throughout the cortical thickness (see scales in Fig. 2). After scaling for the cortical thickness, these bins correspond approximately to the cortical layers indicated in the figure. Figure 3 illustrates the mean values obtained from control animals for each of the 12 bins. The overall average for this cortical region was 2493_+171 fmoles/mg for Bm,~ and 1.28 + 0.13 nM for KD. In Fig. 4 the data are expressed for the experimental animals as a function of time following deafferentation as a percentage change over controls. Bmax

Fig. 1. [3H]QNB autoradiograph indicating the axis of scanning throughout the layers within the rat somatosensory cortex.

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did not change significantly in any bin in the contralateral cortex until 4 days after nerve transection [Fig. 4(A)]. On the fourth day significant changes were limited to a small change in layer II or upper layer III (bin 3) and an approximately 20% reduction in layers IV and V(a) (bins 5 and 6). The changes in layers IV and V(a) were still present 14 days after SNT. The KD values were much more variable throughout the experiment, especially on day 2 [Fig. 4(B)]. Nevertheless, a significant increase appeared throughout layer II/III (bins 2, 3 and 4) on the first day after deafferentation. On days 4 and 14 the KD values were reduced throughout all layers, but reached significance only in layers IV and V(a).

[ SH]pirenzepine binding in the deafferented cortex The experiment summarized in Fig. 4 was also performed with [3H]pirenzepine. The optical density measurements from the autoradiographic material were digitized in the same manner providing 50 measures of binding through the depth o f the hindlimb somatosensory cortex. Figure 5 illustrates one o f these profiles obtained 14 days after deafferentation and compares it to the control values obtained with the same ligand concentration. It is immediately apparent that the differences between the two curves are found predominantly in layers V and VI, rather than m layers III and IV as was the case with [3H]QNB binding (Fig. 2). The Bin,, and KD values for each of 12 bins through the contralateral hindlimb cortex were

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plotted in a manner analogous to that done for [~H]QNB. These results for 1, 2, 4, 7 and 14 days following SNT are presented in Fig. 6. The overall average and SEM of the Bm~ for the cortex in control animals was 984 5=31 fmoles/mg protein. The KD was 10.26+ 1.44 nm (without bin 12). There were immediate changes following deafferentation. On the first day, the B , ~ for [3H]pirenzepine increased by about 20% in layers V and VI (bins 6 11). This change was transient and only two bins showed significant changes in B , ~ at day 2. Day 4 gave variable results, but by day 7, Bin,, was clearly reduced in a compact portion spanning lower layer III, layer IV, and the upper part of layer V(b). This reduction was also transient, being absent at 14 days.

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The changes in KD for [3H]pirenzepine seemed to lag behind the changes seen for Bm~. There was little change on the first day, but day 2 showed an elevation in layers III, IV and upper V. Seven days after SNT nearly all bins showed a reduction in Ko. A comparison of Figs 4 and 6 shows that the changes observed for [3H]pirenzepine did not strictly follow the changes seen with [3H]QNB. Since pirenzepine is a relatively selective antagonist for M. receptors, while QNB can bind to several classes of muscarinic receptors, and since the data demonstrated that M~ receptors behaved differently than the overall population of muscarinic receptors labeled by [3H]QNB, the effects observed with [3H]QNB, especially at day 14, imply that alterations must also have occurred in non-M ~ receptors. N o n - M t receptors in the deafferented cortex

By subtracting the absolute values of B ~ for [3H]pirenzepine from those determined for [3H]QNB, the resulting difference was used as a proxy for nonpH]pirenzepine binding sites. This was done bin-bybin in control animals [the overall mean and SEM for the control animals was 1631 + I 12 fmoles/mg protein (without bin 12)]. Then the data for the experimental groups were expressed as a percentage of the control values for each bin. The results were a measure of changes in non-[3H]pirenzepine binding sites in the experimental animals. The histograms in Fig. 7(A) suggest that there were decreases in Bmax in layer V(a) which did not reach significance until about 4 days after deafferentation. Although not reflected by the data set from day 7, a reduction is clearly present

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Unilateral injury to a peripheral nerve or unilateral deafferentation provokes changes in the electrophysiological characteristics of neurons in the primary somatosensory cortex on the side ipsilateral to the injury as well as on the contralateral side (Calford and Tweedale, 1990). In our study, measurements were obtained from the [3H]QNB and [3H]pirenzepine autoradiographs on both the ipsilateral as well as the contralateral sides. The data from the ipsilateral side. expressed as a percentage of the results on the ipsilateral side of the control animals for each of the experimental groups, are summarized in Figs. 7(B), 8 and 9. [3H]QNB receptors [Fig. 8(A)] decreased in the ipsilateral hindlimb region as they had in the contralateral region. The reduction occurred in layer V not before day 4 and was present at day 7 as well. By day 14 the bin representing layer V(a) was still reduced ; additionally layer IV and one bin within layer VI(a) were significantly reduced. On the contrary, the number of receptors binding [3H]pirenzepine [Fig. 9(A)] showed a reduction throughout layers II/IIl, IV, V, and VI(a) at day 14 that was not seen in the contralateral hemisphere. This reduction, starting with day 4, seemed to parallel the changes in [3H]QNB. As a result, the receptors with non-M ~characteristics [Fig. 7(B)] were significantly affected only in two bins at day 7. The time courses of the Ko values are represented by the histograms of Figures 8(B) and 9(B). Although not true for all bins, generally the changes in these values were of the same direction and laminar localization as their corresponding Bm,~ deviations. Most obvious, the KD of [3H]pirenzepine binding was decreased throughout most of the cortex on day 14. In summary, receptor changes occurred in both the ipsilateral and contralateral hindlimb representations. Changes in [3H]QNB binding followed a similar time course within a similar portion of the cortical depth on both sides up to day 14. At this time, however, the alterations of [XH]pirenzepine and (as a consequence) of non-[3H]pirenzepine binding sites seemed to differ in the two hemispheres; M~ receptors were mostly changed ipsilaterally, non-M, receptors were changed more contralaterally. Phorbolester binding in hindlimb somatosensorv cortex following sciatic nerve transection

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Fig. 9. Changes in Bmo~(A) and Ko (B) values for [~H]pirenzepine binding in the ipsilateral hindtimb area of the primary somatosensory cortex at selected days after SNT. All data given as mean + SEM, *P < 0.05.

Receptor binding in somatosensorycortex deafferented cortex might be a first step to an understanding of post-traumatic reorganization within primary somatosensory cortex, the understanding of the biochemical mechanisms that control plasticity requires, however, more data about the effector systems at the post-receptor level. Because changes in the activity of the cholinergic system have been shown to lead to changes in second messengers (Woody and Grucn, 1988), [3H]PDBu binding was studied in homogenized tissue samples of the hindlimb regions at various times after SNT. The binding of the labeled ligand appeared to be unaffected at all times tested, as shown in Fig. 10. The increase in Kn values did not reach the level of significance. However, homogenates of hindlimb cortex did not show the changes in B=,x and KD of cholincrgic receptors that are shown here A D IPSfLATERAL

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by laminar analysis of receptor binding, and in the same way, this analysis remains to be done on a laminar basis before effects of deafferentation on this marker for protein kinase C can be excluded from consideration. DISCUSSION We observed complex alterations in the binding parameters of the muscarinic cholinergic antagonists [3H]QNB and [aH]pirenzepine that were restricted to specific laminae and restricted to specific times following deafferentation. The results differed for those two ligands allowing inferences about the muscarinic receptor subtypes involved and the schedules they followed. The restricted laminar distribution of these changes and the fact that the changes were often in opposite directions in different layers provide an explanation for why previous efforts to study muscarinic receptors have given equivocal information (Sampson et al., 1988; Hanisch et al., 1989; Rothe et al., 1990). Muscarinic receptors in the deafferented hindlimb cortex

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Fig. 10. Binding of [3H]phorbol-12,13-dibutyrate to membranes prepared from the hindpaw cortex of rats with sciatic nerve transection. B=, (A) and Kv (B) values are given as pcrocntage of control m¢aaurom¢ntsin untreated animals. The binding was studied in homogenates of the ipsilateral and contralateral areas taken from animals of the stated survival times.

The changes described for the binding of [3H]QNB and [aH]pirenzcpine were expressed by comparison to a common control group of untreated animals. Thus, it was important to confirm thor significance in several ways. First, the changes developed with different time courses. For example, the maximal number of [3H]QNB receptors were not different from control values 1 and 2 days after injury, but changed afterward, whereas those for [3H]pirenz¢pin¢ appeared immediately. Second, the changes for each ligand were restricted to a subset of neighboring bins, whereas other portions of the laminar profile did not change. For example, the reduction of muscarinic receptors revealed by [3H]QNB at day 14 involved several adjacent bins forming a compact profile, although only the maximal bins reached the level of significance. Third, the present results obtained with [3H]QNB agree with the previous findings that did not reveal changes in homogenates. The decreases determined for the laminar Bmaxof [3H]QNB at post-SNT days 4 and 14 were quite small; they were statistically significant but the maximum decreases were only 25% and were restricted to a group of bins. Thus it is understandable that their effect in tissue homogenates would be hardly detectable. Moreover, the small decrease in the maximal number of binding sites was accompanied by a decrease in the KD of the ligand providing an additional reason why changes in the binding of

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UwE-KARSTEN HANISCHet al.

[3H]QNB in homogenates at 3 nM were not detected (Rothe et al., 1990). Muscarinic receptors do not represent a homogeneous entity, neither in terms of their molecular biology, nor in terms of their pharmacological features (Bonner, 1989). Since [3H]QNB is not a subtypeselective ligand the binding obtained with it reflects the entire population of muscarinic receptors also in terms of altered binding parameters. In contrast, [3H]pirenzepine has high affinity for binding sites of the M1 type. The decrease in [3H]QNB binding was not paralleled by similar changes in the binding of [3H]pirenzepine. Relatively large changes in [3H] pirenzepine //max were observed only at day 7 and the most pronounced changes involved the middle part of the cortex, Fourteen days after SNT, when [3H]QNB binding was still reduced in layers IV and V(a), there was no reduction in binding by [3H]pirenzepine. These differences in the Bm,x profiles for the two ligands indicated that changes must have occurred also in non-M~ binding sites. We estimated the size of these changes from a bin-by-bin subtraction of the two ligands and compared the resulting distributions for the control and the SNT animals. The greatest difference between control animals and SNT animals occurred in layers IV and V(a) where the difference reached almost 50% within 2 wk. Although a subpopulation of M 2 receptors is likely to be located at presynaptic sites (Quirion et al., 1989), the major reduction of binding sites detected with [3H]QNB in parietal cortex should be found at postsynaptic sites. Nevertheless, our conclusions about the non-Mr receptor populations have been derived indirectly and it would be desirable to have experimental confirmation of our inference that receptors with M2 and/or M 3 characteristics are affected by SNT. Ipsilateral effects of deafferentation on muscarinic receptors

In the ipsilateral cortex, Bma, was decreased for [3H]QNB in a similar way and with a similar profile to that seen on the contralateral side. Changes, however, also involved reductions in layer V(b) at day 4 and in layer VI(a) at day 14. The early changes in the ipsilateral KD for [~H]QNB were a tendency towards increased values in the superficial layers, with parallel decreases in the infragranular layers; pronounced reductions were found for the bins representing layer V(b). Recently Calford and Tweedale (1990) have documented receptive field changes in the somatosensory representations of both hemispheres in the flying fox

after unilateral pharmacological and surgical manipulation of peripheral nerves. The changes observed in the present study may be a reflection of the processes giving rise to these physiological changes. Although they are difficult to interpret, our previous work also provides evidence for an ipsilateral effect on another biochemical marker of function ; 24 h following SNT, we demonstrated a decreased [3H]hemicholinium-3 ([3H]HCh3) binding in layer VI ipsilateral to the nerve transection (Rothe et al., 1990). The fact that the M~ receptor population was affected principally on the ipsilteral side and in layers not related to the arrival of afferent information from the thalamus suggests that this class of receptors may have a preferential association with callosal pathways. From a more practical point of view, the ipsilateral changes show also that an interhemispheric comparison is not a suitable reference as an internal control. Electrophysioloyical evidence jor laminar changes in ACh receptors

Comparing the neuronal responses elicited by iontophoretically administered ACh in the granular hindlimb cortex of normal and deafferented rats 2-3 wk after SNT, Lamour and Dykes (1988) suggested that deafferentation might be accompanied by changes in ACh receptors. The proportions of neurons influenced by ACh in the deafferented cortex were not affected after SNT, but the character of the responses were altered ; the magnitude of the responses to ACh was generally smaller in SNT animals and the time course of the neuronal discharges differed. The effects were most pronounced in layers V and VI, where the majority of ACh-sensitive neurons are located (see also Lamour et al., 1982). Since the electrophysioiogical experiments delivered ACh in the vicinity of the recording electrode, those data do not reveal neuronal discharges evoked via remote dendritic receptors. For methodological reasons such as this, the radioligand binding assays may not necessarily show a receptor distribution identical to the profile of the electrophysioiogical data. Changes in [3H]pirenzepine binding sites shown in Fig. 6 cannot explain entirely either (i) the overall reduction in muscarinic receptors or (ii) the changed electrophysiological response pattern. Receptors other than those binding [3H]pirenzepine seem to account for the decrease in Bmaxrevealed by [3H]QNB. Perhaps a reduction of non-M ~muscarinic receptors located on proximal dendrites is one of the biochemical substrates of the unusually low neuronal responses to ACh.

Receptor binding in somatosensory cortex The few response enhancements seen with ACh administration in the supragranular layers after SNT arc not easily explained by a simple correlation with muscarinic receptor changes. Lamour et al. (1982) rtportcd that among the cell studied in rat somatosensory cortex, 24% responded to the administration of nicotine and the nicotinic agonist, butyrylcholine. The significantly higher proportion of neuronal responses to butyrylcholine following SNT (Lamour and Dykes, 1988) points also to a nicotinic component among the cholinergic effects of deafferentation. Whether the numbers of nicotinic receptors might be changed, or even their subunit composition(s) (Denerius et al., 1991), is not known. Changes in other cholinergic markers

In the context of biochemical evidence for a changed cholinergic system, the demonstration of altered muscarinic receptors provides a first indication for the proposed postsynaptic effects (Lamour and Dykes, 1988). Previous work has shown so far only decreases in presynaptic markers, i.e. the activity of the ACh-synthesizing enzyme choline acetyltransferase (CHAT), the sodium-dependent high-affinity choline uptake (SDHACU), and the [3H]HCh3 binding; a 15% reduction was observed for ChAT activity in the contralateral hindlimb cortex of rats 3 days after SNT (Hanisch et al., 1989), but not at other survival times (Rothe et aL, 1990). Subsequently, while investigating ChAT activity in individual layers of the hindlimb cortex, we found the reduction in the activity of this enzyme to he restricted to layer V, where it was dramatically decreased (Rothe et al., 1990). In the same study, we also obtained evidence for a clear reduction of another cholinergic marker in layer V within the denervated hindlimb representation; SDHACU was decreased 12 and 24 h following SNT. The binding of [3H]HCh3 in this region was lower by 57% in layer V, whereas other layers maintained control levels. By calculating the proportional contribution that layer V makes to the entire somatosensory cortex it becomes apparent that the pronounced loss of ChAT activity (45% of corresponding control) in layer V accounts for the "average" reduction of 15% detected in the experiments on homogenates of whole cortex. This illustrates again the disadvantages associated with assay conditions lacking anatomical resolution at the level of cortical (sub)layers ; minute or contrary changes of a marker within different laminae are not well represented by a signal obtained from the whole tissue thickness.

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Limitations of biochemical studies within defined somatosensory subdivisions

The laminar analysis of binding data cannot avoid a certain variation, especially in the marginal bins at the upper and lower edges of the cortex where the slope of the profile is rather steep (Fig. 2). The division of the cortical depth into 12 bins represents, therefore, a compromise between anatomical resolution and sample size within one class. Variation among the samples taken at different cortical depths of individual animals is reduced by pooling several adjacent measures but the rapid transitions at boundaries still influence the average. As well as the problems associated with transitions between laminae, the investigation of biochemical parameters in the rat hindlimb somatosensory cortex after partial deafferentation is made more difficult by the variable stereotaxic location of this minute region (Hanisch et al., 1989 ; Rothe et al., 1990; Webster et al., 1991). Further, the sciatic and the saphenous contributions to the hindlimb representation vary among individuals (Wall and Cusick, 1984; Webster et aL, 1991). Consequently, the precise location and the layout of the idealized body map as derived from comprehensive electrophysiological studies (Chapin and Lin, 1984) cannot be used on an individual without electrophysiological confirmation. Additional sources of variation arise from the fact that the magnitude of reorganizational events may vary with the animal examined (Wall and Cusick, 1984). In spite of these limitations, this study obtained biochemical evidence for changes in muscarinic receptor binding following deafferentation. As a general result, comparisons between survival times, between hemispheres, and between ligands suggest trends of laminar-specific changes in receptors that must play an important role in cortical reorganization.

CONCLUSIONS

Electrophysiological studies of iontophoretically administered ACh in deafferented rat hindlimb cortex led to the hypothesis that changes occur in ACh receptors (Lamour and Dykes, 1988). The present work supports this hypothesis. We obtained evidence for changes in the binding characteristics of muscarinic receptors within the hindlimb representation following SNT. These changes were (i) restricted to specific laminae within primary somatosensory cortex and were (ii) observed in somewhat different patterns in both hemispheres. Changes were observed (iii) as early as a few days after transection, hence (iv) they

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accompany the initial r e o r g a n i z a t i o n a l events observed by electrophysiological techniques. The altered binding characteristics o f [3H]QNB are a first indication of changes a m o n g m u s c a r i n i c receptors. Analyses with more specific ligands are required to clarify which population(s) o f muscarinic receptors are p r e d o m i n a n t l y influenced by c h a n g e d sensory experience, and are involved in the early phases o f reorganization. Those studies m u s t be d o n e on a laminar basis in circumscribed regions o f sensory cortex. Acknowledgements--Supported by funds from the Scottish Rite Charitable Foundation of Canada, the Medical Research Council of Canada and the German Democratic Republic. REFERENCES

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