Sensory deafferentation fails to modify muscarinic receptor binding in raccoon somatosensory cortex

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Brain Research Bulkritz, Vol. 20, pp. 597-601. B Pergamon Press plc, 1988,Printed in the U.S.A.

Sensory Deafferentation Fails to Modify Muscarinic Receptor Binding in Raccoon Somatosensory Cortex s.M. Received

15 October

1987

SAMPSON, S. M., C. SHAW, M. WILKINSON AND D. D. RASMUSSON. ~~~~o~ ~eff~~~renf~r~o~fails lo ~o~~y ~~~~~~~~i~ rt’ceprtrr bi~~i~i~ in rac’c’oon ~~f~~a~~~~~~,~(~~~l corf~x. BRAIN RES BULL 20(S) 597-601, 19X8.-The characteristics and distribution of muscarinic acetylcholine (mACh) receptor binding in primary somatosensory (SI) cortex and the caudate nucleus of raccoons were studied using [“HI-QNB, a muscarinic antagonist. The binding characteristics were similar to reported values in rat and cat. Autoradiographs produced from tissue sections labeled with [“HI-QNB showed the distribution of mACh receptors in the forebrain of the raccoon. [%I-QNB binding was highest in cerebral cortex, neostriatum and hippocampus. Within Sl cortex, binding was high in layers I-III and VI and relatively low in layers IV and V. Autoradiog~phs obtained from animals that had undergone peripheral deafferentation of part of the forepaw revealed no changes in [:‘H]-QNB binding in the affected cortical region during the time that physiological reorganization is known to occur. Muscarinic cholinergic receptors Deafferentation

In vitro autoradiography

THE in vitro autoradiographic receptor binding method has made it possible to examine the localization of various binding sites in different regions of the brain [22]. It has been suggested that this technique might be useful to determine if particular receptors are quantitatively modified during certain periods of plasticity. For example, Shaw t’t ui. [1.5,16] have demonstrated that the laminar distribution of various receptor populations in cat visual cortex changes during the critical period. Another type of plasticity that is particularly interesting, in that it occurs in adults, is the reorganization of primary somatosensory (SI) cortex in response to peripheral nerve deafferentation [4]. We have begun to examine this phenomenon in the raccoon, a species with a greatly enlarged representation of the forepaw [ 131. It is thus possible to identify individual digit areas within SI cortex from the sulcus patterns. The present paper includes a determination of the binding characteristics for the muscarinic acetylcholine (mACh) receptor in the raccoon in order to provide an indication of its comparability to the cat, another carnivore. Characterization experiments were carried out on two regions of the raccoon brain, SI cortex and the caudate nucleus. The optimal conditions for binding were then chosen to produce autoradiographs of the brain using the in vitro technique [22]. These showed the laminar distribution of mACh receptors in the cortex and the relative density in other forebrain structures.

Raccoon

Somatosensory

cortex

Autoradiographs were then obtained on a series of raccoons at different intervals after removal of the fifth digit to determine whether there was any change in the distribution of mACh binding sites in the region of SI cortex that has lost its peripheral input during the period when the somatosensory map is being reorganized. METHOD

Twelve adult raccoons, that had been maintained on a 12 hr on: 12 hr off light-dark cycle, were sacrificed at approximately 14:00 hr after sedation with ketamine (130 mg, IP) and sodium pentobarbital (13 mgikg, IP). Each animal was perfused via the left ventricle with phosphate buffered saline (PBS), followed by 0.1% formaldehyde in PBS. The brain was removed quickly and frozen at -70°C. Coronal sections, 16 pm thick, were cut at - 16 to -2o”C, thaw mounted onto slides and stored at -20°C. Ten animals had undergone surgical amputation of the 5th digit of the right forepaw under halothane anesthesia [ 131 one to 16 weeks prior to perfusion. Previous studies 15,131 indicate that the cortical organization is changing gradually throughout this period. The receptor binding method of Young and Kuhar [22], as modified by Shaw rt al. [15], was used to characterize the binding of [3H]-QNB (New England Nuclear, specific activity 21.4-37.2 Ci/mmol). Areas of caudate nucleus and cere-

*Requests for reprints should be addressed to D. D. Rasmussen, Department of Physiology and Biophysics, Dalhousie University, Halifax, N. S. Canada, B3H 4H7.

597

598

SAMPSON,

bra1 cortex from the control animals, or from the right, control hemisphere of animals with amputations, were selectively scraped from the whole brain tissue sections into separate scintillation vials containing Aquasol (2 ml). The cortical tissue included SI cortex and cingulate cortex. The radioactive content of these two types of tissue was determined on one of two LKB Rackbeta liquid scintillation counters with efficiencies of 38% and 50.4%. Unless stated otherwise, the incubation solution contained 5 nM [3H]-QNB in PBS, the incubation period was one hour, and 1O-1 M atropine sulfate was used for determining nonspecific binding. Association time courses were determined using incubation times ranging from five minutes to four hours. For displacement experiments, atropine sulfate or nicotine was used in concentrations ranging from lo-” M to lo-” M. IC,,,‘s were calculated using the program ALLFIT [2]. The saturability of the binding site was examined using [“HI-QNB concentrations ranging from 0.33 to 16 nM. For dissociation experiments, postincubation rinses ranging from five minutes to seven hours were used. [“HI-QNB did not dissociate within seven hours, likely due to the low temperature at which this was performed (4°C). At 35°C [3H]-QNB dissociates with a half-life of an hour [20,213. Since the dissociation rate could not be measured directly from these data, the equilibrium dissociation constant (K,,) was calculated from the saturation binding curve. The protein content of the tissue was determined using the method of Lowry pt cd. [lo] and the binding data were analyzed using the method of Zivin and Waud [23]. For autoradiographs, optimal binding conditions were chosen on the basis of the characterization results. These consisted of a three-hour incubation time, a [:‘H]-QNB concentration of 5-8 nM. two five-minute washes to terminate the binding and an atropine concentration of 0.1 mM for the determination of nonspecific binding. Slides were then apposed to tritium-sensitive LKB Ultratilm for exposure periods ranging from 6 to 21 days. The film was developed and fixed using standard techniques.

SHAW, WILKINSON

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AND RASMUSSON

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RESULTS

The results of this study are consistent with many others in showing that [“HI-QNB binding is saturable and of high affinity. The characteristics of binding in the cortex are shown in Fig. 1. An association time course determination (Fig. la) showed that equilibrium binding was reached within two hours. Nonspecific binding was less than 9% of total binding. As expected, the muscarinic antagonist, atropine, demonstrated high affinity for the [3H]-QNB binding site (Fig. lb) with an IC,,, of 8.7~ lO-‘u M. This high affinity for the mACh receptor makes atropine an excellent displacer when examining nonspecific [“HI-QNB binding. The poor displacement by nicotine (IC,, of 2.9~ 10m6M) confirms that [“HI-QNB does not bind to a nicotinic ACh receptor. A concentration of 8 nM was found to be sufficient to saturate binding in cortical tissue (Fig. lc) with half-maximal binding at approximately 1 nM. The maximum number of binding sites (B,,J was 472.0227.2 fmol/mg protein and the Ka was 0.8+0.1 x 10eg M. The standard deviation of the background error (SD(E,,d)) in the data was less than 0.1 and the Hill coefficient was 0.9, which suggests the presence of a single binding site. For the caudate tissue, the association time course also showed equilibrium binding within two hours. The IC,,,‘s for atropine and nicotine were 4.2~10-‘~ M and 2.2~ 10m6M,

0

?O

IONS~“Ml) FIG. 1. Saturation and kinetic studies of “[HI-QNB binding to thin sections (I6 pm) of raccoon cortex. (a) Time course of total, specific and nonspecific binding. (b) Competition curves for atropine and nicotine. (c) Saturation curve using different QNB concentrations incubated for 3 hr. All experiments run at room temperature (23”). Values shown are means2s.e.m. of four samples per point except in the case of nonspecific binding, which had two samples per point.

respectively. No appreciable dissociation of [3H]-QNB was seen in the caudate nucleus after seven hours at 4°C. Saturation binding was attained at QNB concentrations between 8 and 16 nM. The B,,, was found to be 648.9k53.8 fmoVmg protein and the Ka was 1.6&0.3x 10e9 M. The SD(E,,J was 0.11 and the Hill coefficient was 1.O. Figure 2 shows photomicrographs of a cresyl violet stained section through ST cortex and an autoradiograph of an adjacent section labeled with [3H]-QNB. The densest re-

MUSCARINIC

ACh RECEPTORS

IN THE RACCOON

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3mm

FIG. 2. (a) Coronal section through SI cortex stained with cresyl violet. (b) Autoradiograph of an adjacent section labeled with [WI-QNB. Abbreviations: Cd, caudate nucleus; Cg. cingulate cortex; H, hippocampus; P, putamen; T, thalamus. Area of SI cortex outlined in a is magnified in Fig. 3.

gion of [3HJ-QNB binding was the striatum (caudate nucleus and putamen). The homogeneous nature of QNB binding shown here is largely the result of the high optical density in this region of the autoradiographs. In autoradiographs in which the exposure time was reduced, the distribution of mACh receptors was patchy, presumably corresponding to the compartmentalization of intrinsic cholinergic components in the caudate [3]. In SI cortex, [3H]-QNB binding varied across the cortical layers. The area of SI outlined in Fig. 2 (the 3rd digit representation) is shown at higher magnification in Fig. 3. The left side of this montage is a photomicrograph of a cell-body stain to reveal the cytoarchitecture. The cortical layers are indicated by Roman numerals. The right side of the montage in Fig. 3 is an autoradiograph of the adjacent section. It can be seen that [3H]-QNB bound preferentially to all layers except layers IV and V, with the supragranular layers (I-III) having greater density of binding than layer VI. It can be seen in Fig. 2 that the anterior cingulate region (Cg) has a more uniform distribution throughout all layers. [3H]-QNB binding within the white matter, the hypothalamus and the thalamus was very low, with only the anterior thalamic nuclei (which project to cingulate cortex) showing

slightly higher binding than the remainder of the thalamus. Within the hippocampus, the stratum lacunae moleculare had low binding levels while the other layers had high binding. In the dentate gyrus, binding was low in the stratum polymorphe and high in the stratum moleculare. Examination of autoradiographs of the 5th digit region of SI cortex after the 5th digit was removed revealed no differences from normal cortex at any interval after digit removal. Neither the laminar distribution nor the overall density of QNB binding was different from that of the cortex of control animals or from the adjacent regions of SI cortex that had normal input. DISCUSSION

In general, the binding characteristics of [“HI-QNB in raccoon brain were very similar to those in cat and rat brain. The IC,, values obtained for atropine (4-9x lo-‘” M) and nicotine (2 x 1O-6M) are comparable to those reported for cat and rat brain [15,20]. A two-hour incubation period necessary to reach equilibrium is slightly longer than that reported for cat visual cortex [ 151 but is consistent with that found in rat brain [ 111. The measure of saturability with half-maximal binding around 1 nM is also similar to values reported in cat

SAMPSON,

SHAW, WILKINSON

AND RASMUSSON

Imm FIG. 3. A montage juxtaposing adjacent sections through a gyrus of SI cortex. Left, cortical layers are labeled on cresyl violet stained section; right, autoradiograph incubated with 8 nM [“HI-QNB and exposed to LKB Ultrafilm for 10 days.

and rat [15,20]. The present results showed a higher B,,, in the caudate nucleus than in the cortex. No comparable differences were observed in an experiment on the rat [ 1l] in which the two regions were reported separately; however, the B,,, values reported in that paper were much higher than those observed in the raccoon. In contrast, other studies on cat visual cortex [1.5] and rat striatum [19] have reported B max values similar to those observed in the raccoon (280689 fmol/mg protein). In terms of K,, values, QNB binding is similar in cat, rat and raccoon brain. In addition, this muscarinic antagonist bound to a single population of binding sites that did not demonstrate receptor cooperativity, as indicated by Hill coefficients around 1.0. The differences between cat and raccoon in the distribution of mACh receptors throughout the forebrain were relatively minor. The striatum also has the highest density of mACh receptors in the rat [I93 and monkey [18]. The distribution within the hippocampus is the same as that described in the rat [9] with high mACh levels ~o~esponding to dendritic regions. The Iaminar distribution of mACh receptors in raccoon cortex is the same as in rat SI cortex [ 1l] and is similar to cat visual cortex [15] except that there appeared to be a greater contrast between layers IV/V and the other layers in raccoon SI cortex. This might be related to the wide and

relatively acellular nature of layer V in SI cortex. The difference observed between sensory and cingulate cortex in mACh receptor distribution has been described in the rat 1171. Interestingly, the correlation between receptor binding and other biochemi~a1 indices is poor. For example, the overall level of AChE staining in SI cortex is substantially lower than in the striatum [8]. The laminar distribution of AChE in most cortical regions is relatively high in layers I, V and VI, but low in layers II-IV [7J. This may vary with cytoarchitectonic area, since in the cat visual cortex [l] and raccoon SI cortex (Rasmusson, unpublished observations) there is an additional band of greater AChE staining in layer III and little AChE staining in layer V. This would correlate better with the observed mACh receptor distribution, The paucity of cholinoceptive cells in superficial layers determined either histochemically [6] or physiologically [7] is more difficult to reconcile with the high mACh receptor density in these layers. The study of SI cortex in the raccoon offers a usefui model of plasticity since it has been shown to be modifiable in adult animals [5,13], in contrast to visual cortex which is modifiable only during a relatively short critical period early in life. The results of the present work are disappointing in

MUSCARINIC

ACh RECEPTORS

601

IN THE RACCOON

the sense that QNB binding is not modified during this period. Thus, the findings in a preliminary experiment f14] of bands of up-regulation in the 5th digit region two-weeks after deafferentation were not replicated. Attempts to produce such bands in this series of animals by manipulating the incubation or exposure conditions did not affect this conclusion. The results of the preliminary study may have resulted from an artefact introduced at the time of perfusion or to one animal in particular being abnormal in an undetermined way. Nevertheless, our results do not eliminate the possibility that other receptors undergo change. The fact that the charac-

of QNB binding in two regions of the raccoon brain are closely similar to those in cat and rat brain suggest that rapid screening of other binding sites (e.g., adrenergic, GABAergic, etc.) is possible using the parameters worked out for cat and rat brain.

teristics

ACKNOWLEDGEMENTS

We greatly appreciate the assistance and advice of Drs. M. Cynader, R. A. Leslie and H. A. Robertson. This work was supported by the Medical Research Council of Canada.

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