Muscarinic desensitization after septal lesions in rat hippocampus: Evidence for the involvement of G-proteins

Neuroscience Vol. 47, No. 1, pp. 9S103, Printed in Great Britain

1992

0306-4522/92 55.00 + 0.00 Pergamon Press plc 0 1992 IBRO

MUSCARINIC DESENSITIZATION AFTER SEPTAL LESIONS IN RAT HIPPOCAMPUS: EVIDENCE FOR THE INVOLVEMENT OF G-PROTEINS A. BAUER, J.-B. SCHULZand K. ZILLES* Brain Research Institute, University of Diisseldorf, Moorenstr. 5, D-4000 Diisseldorf, F.R.G. Abstract-Three days after bilateral septal lesions, regional and laminar densities of the muscarinic acetylcholine receptors of the dorsal rat hippocampus were studied. The concentration of [‘H]Nmethylscopolamine binding sites and muscarinic M, and M, receptor subtypes, as well as the affinity of muscarinic receptors and their sensitivity to modulation by 5guanylylimidodiphosphate were analysed by quantitative receptor autoradiography. The measurement of these parameters was performed with a computerized image-processing system allowing a spatial resolution down to the level of single hippocampal layers. No postlesional changes of the density of M, receptors were detected. M, receptors showed a remarkable decrease in concentration (~21%) in some hippocampal layers after septal lesions. In competition experiments the affinity of muscarinic receptors for the cholinergic agonist carbamylcholine chloride decreased significantly in all hippocampal subregions and layers of the lesioned animals. In contrast to controls, the sensitivity of muscarinic receptors of the lesioned animals could not be modulated by 5guanylylimidodiphosphate. These findings demonstrate a desensitization of muscarinic receptors in the rat hippocampus three days after septal lesions, which is caused by changes of the coupling of guanine nucleotide-binding proteins to muscarinic receptors. Therefore, the lesion-induced alteration of the muscarinic receptor-effecter complex is a major aspect of the hippocampal plasticity after choline@ denervation.

The lesion-induced effects in the cholinergic septohippocampal system have repeatedly been investigated. A cholinergic supersensitivity was found6*’ in microiontophoretic studies. Several reasons for this supersensitivity were reported. Changes in acetylcholinesterase (AChE) activity or in postsynaptic muscarinic acetylcholine receptors (mAChRs) were discussed. Some authors reported increases of receptor densities”*38 in the hippocampus; others found no changes24*3’in binding studies. Differences in type and completeness of lesions and in binding and measuring techniques may be responsible for these contradictory results. Since cholinoceptive structures in the hippocampus show differential regional and laminar distributions, a sufficient spatial and anatomical resolution of the measurements is required. Otherwise, changes of receptor concentrations in single subregions and layers may be obscured by measuring the total hippocampus. This present study was designed to detect local changes of mAChR concentrations and affinity states at a high spatial resolution. Therefore, quantitative

receptor

autoradiography

was performed

on sections.

In this way even subtle alterations in receptor number and/or affinity could be localized within hippocampal subregions and single layers. Many receptors are coupled to second messengers and effector systems via guanine nucleotide-binding (G)-proteins.‘7,3s Cerebral mAChRs are activating multiple effector systems including adenylyl cyclase and phospholipase C by different G-proteins.3*‘6y26It has been supposed that G-proteins are involved in denervation-induced processes on the receptor level.3’ Addition of guanosine triphosphate (GTP) or its nonhydrolysable analogues results in an uncoupling of agonist-bound receptors from G-proteins. Highaffinity receptors are consequently converted to lower affinities.’ We included an analysis of the sensitivity of agonist binding to mAChRs to the GTP analogue 5-guanylylimidodiphosphate (Gpp[NH]p) in the present study. Thus, the proportions of convertible high- and low-affinity sites in single layers of the dorsal hippocampus of lesioned animals and controls could be measured. EXPERIMENTAL

PROCEDURES

*To

whom correspondence should be addressed. Abbreviations: AChE, acetylcholinesterase; CCh, carbamylcholine chloride; DG, dentate gyrus; Gpp[NH]p, 5guanylylimidodiphosphate; G-protein, guanine nucleotide-binding protein; GTE, guanosine triphosphate; HDB, horizontal limb of the diagonal band of Broca; mAChR(s), muscarinic acetylcholine receptor(s); MS, medial septum; VDB, vertical limb of the diagonal band of Broca.

Ten male Wistar rats (Winkelmann, Borken, F.R.G.) housed under regular laboratory conditions with a natural light-dark cycle were included in this investigation. All rats were anaesthetized with Hypnorm@ (Jansen, Netherlands) and positioned in a David Kopf stereotaxic instrument with the incisor bar set 3.3 mm below the interaural line. Lesions (n = 5) were made by multiple injections of ibotenic acid into the medial septum (MS), the ventral and the dorsal part 95

of the vertical limb of the diagonal band of Brocd (VDBv and VDBd, respectively) and- the horizontal limb of the diaeonal band of Broca (HDB) of both sides. Six iniection sites were chosen according to Paxinos and Watsonr5 I’n each animal of the experimental group. Coordinates (anterior to the interaural line; lateral to the midline; above the interaural line): HDB right (8.3; 2.4; 1.2); HDB left (8.3; - 2.4: 1.2); VDBv right (9.3; 0.7; 1.3); VDBv left (9.3; --0.7; 1.3); VDBd (9.3; 0; 2.2); MS (9.3; 0; 3.8). The other five rats were sham-operated and constitute the control group. Ibotenic acid was dissolved in sterile saline to a concentration of 1 ng/ml and buffered with I mg NaHCO, (pH 7.4). The injections were made with a S-p1 syringe fitted with a 30-gauge needle, pst3 (Hamilton, Darmstadt, F.R.G.). The tip of the needle was lowered to the selected site and 2 min later, 0.6~1 of the prepared solution was injected during a period of 3 min. The needle remained in place for IOmin and was then slowly repositioned for subsequent injections. The animals were allowed to survive for 72 h. After decapitation the brains were removed immediately, frozen in N-methyl-butan (- SO’C) and stored in a freezer (- 70°C). Receptor binding was performed as previously described.m Therefore, the main steps are only briefly summarized. The brains were cut in a cryostat microtome (section thickness, 20 pm). A series of adjacent sections was Nissl stainedzO and used for cytoarchitectonal identification of hippocampal layers, In order to control the efficacy of cholinergic deafferentation, a further series of adjacent sections was stained for AChE.15 The autoradiographic binding procedure consisted of three main steps: (i) a preincubation in buffer; (ii) an incubation with (aa) tritiated ligand (total binding) and parallel (bb) with the same tritiated ligand together with an unlabelled displacer (nondisplaceable binding); and (iii) a washing procedure. The difference between aa and bb represents specific binding. The following tritiated ligands were used: [3H]N-methylscopolamine for both M, and M, receptors, [‘Hlpirenzepine for M, receptors and [3H]oxotremorine-M for Mr receptors. Details are given in Table I. Saturation experiments with the agonist [3H]oxotremorine-M were performed in order to exclude that changes in receptor density found in binding at a single ligand concentration result from an underestimation of low-affinity binding sites. Nine quadruplicates of randomized sections through the entire dorsal hippocampus were incubated with rising concentrations of [3H]oxotremorine-M (range, 0.23-7.98 nM) and in a parallel group with the addition of carbamylcholine chloride (CCh; 5 x IO- ’ M). Data from these saturation experiments were approximated with own computer programs performing a Scatchard analysis. Competition studies were performed in order to reveal changes in receptor affinity. Eight quadruplicates of randomized sections through the dorsal hippocampus were incubated with rising concentrations of CCh from low9 to

Table Receptor Ligand (concentration) Displacer (concentration) Incubation buffer

1. Incubation

M,+M,

Incubation

Washing

in incubation buffer 60 min at 25°C in incubation buffer 1 x 10min at 4°C in incubation buffer

RESULTS

The efficacy of ibotenic acid injection was controlled histologically. Nissl-stained serial sections through the MS demonstrated a severe loss of neurons together with an increased density of glial cells (Fig. I). The occurrence of differences in the packing densities of myelinated fibres in the hippocampus between control and lesioned animals cannot be excluded a priori. Such differences would introduce different degrees of /I-selfabsorption (“quenching”), which can considerably influence the data of receptor densities in autoradiographical studies with tritiated ligands.4’ Therefore, the density of intrahippocampal myelin sheets was measured. Sections of both groups were stained for myelin14 and measured densitometritally with an image analyser. There were no detectable alterations in the density. of meylin in lesioned animals compared with the control group. The efficacy of lesioning the cholinergic septohippocampal projection was determined by quantifying the density of AChE-reactive fibres in the dorsal hippocampus histochemically. Serial sections of controls and lesioned animals were incubated in identical solutions. The optical density of the reaction product in binding

experiments

M,

[3H]N-Methylscopolamine (0.2 nM) Atropinsulphate (I PM) 170 mM Tris-HCI (pH 7.7)

+lOmM MgCI, 20 min at 25°C

conditions

10 * M and a constant concentration of [‘H]1\(-methylscc) polamine (0.2 nM). In a parallel incubation, Gpp[NH]p (IOOpM) was added in order to measure the t:xtcnt i>l’ convertible high-affinity sites. Slices from routine receptor binding, saturation analysis and competition studies were further processed as detailed previously40 in order to measure receptor densities in stngle layers of the hippocampus. In brief, autoradiographs were processed with an image analyser. Grey values were transformed into receptor densities according to corresponding calibrated standards. By superimposition of adjacent Nissland AChEstained sections onto hard copies of the digittzed autoradiographs with a drawing microscope, the contours of the hippocampal subregions and layers were traced over with a cursor of a graphics tablet. The tablet was linked to a computer, in which the original data of the pixel values of the autoradiograph were stored. Thus, the regional and layer-specific values could be retrieved, and mean receptor densities of the anatomically defined structures were calculated. Since the sections of a lesioned animal were matched with the sections of a control animal in the binding procedure, differences between controls and lesioned animals were tested by Student’s r-test for matched pairs.

_I

[‘H]Pirenzepine (3 nM) Pirenzepine (10 PM) Mod. Krebs’ buffer (PH 7.4) with 7mM K+, 37mM Nat 20 min at 25°C in incubation buffer 60min at 25°C in incubation buffer 2 x 5 min at 4°C in incubation buffer

M2 [3H]Oxotremorine-M (0.8 nM) Oxotremorine

(1 PM) 20 mM Hepes-Tris (uH 7.5) +IOmM I&Cl, 20 min at 25°C in incubation buffer 60 min at 25°C in incubation buffer 2 x 2 min at 4°C in incubation buffer

Lesion-induced muscarinic desensitization

97

Fig. 1. Representative N&&stained sections through the medial septum and parts of the diagonal band of Broca of a control (B) and a lesioned animal (A) three days after stereotaxic ibotenic acid injection. An extensive loss of the large neurons in the septum can be seen in A.

was measured with an image analyser in 20-&m sections. The mean values of controls (n = 5) and lesioned animals (n = 5) differed ~~i~~ntly (P < 0.01, t-test for matched pairs). The optical densities after the lesion dectiased by more than 50% below the control level. The single values of both groups did not overlap. Therefore, a strong cholinergic deprivation in the hippocampus was achieved by the multiple lesions in the septum. The absolute densities of M, and M, receptors in the entire dorsal hippocampus did not show any significant alterations with any of the ligands used (i.e. [3H]N-methylscopolamine, [‘Hlpirenzepine, ~3H]oxot~mo~neM). The analysis of receptor densities in single layers (Table 2) demonstrated high amounts of M, receptors in the p~~dal and radiatum layers of CAl, lacunosum-moleculare layers of CA3 and molecular and granulosum layers of the dentate gyrus in controls. Intermediate values were observed in the oriens and lacunosum-moleculare layers of CA1 , lacunosum-moleculare layers of CA2 and the hilus (CA4). After denervation there were no significant changes in the distribution pattern nor in the density of M, receptors.

[3H]Oxotremorine-M binding sites (M2 receptors) showed highest densities in the pyramidal and radiaturn layers of CAI, in the pyr~dal layers of CA2 and CA3, and in CA4 of controls (Table 2). Binding was significantly reduced after denervation in the lacunosum-moleculare layers of CA1 (-21%), the pyramidal (- 17%), lucidum (- 18%) and radiatum (- 15%) layers of CA3, in CA4 (- 19%) and the multiforme layer of the dentate gyrus (DC) (- 16%). The other hippocampal layers showed non-significant decreases (< 19%) of receptor densities in comparison with controls (Table 2). In a last set of experiments the pharmacological properties of mAChRs were analysed. Saturation and inhibition experiments were performed on sections. Saturation analysis of [3H]oxotremo~ne-M binding in one iesioned and one control animal showed that specific binding was saturable (Fig. 2). In the Iesioned animal a significant reduction in maximal binding (B_), but no significant alteration in binding properties was found. For the lesioned animal, Scatchard analysis gave a dissociation constant (Kn) of 1.42 (r = 0.985) and a Hill coefficient (nH) of 0.98 indicating a single binding site; maximal binding (&J was 325 fmol/mg protein. The respective data for the

98

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BALER et al.

Table 2. Laminar distributions of M, ([3H]pirenzepine) and Mz ([‘H]oxotremorine-M) receptor densities (fmol/mg protein) [‘H]Pirenzepine

CA1 str. oriens CA1 str. pyramidale CA1 str. radiatum CA1 str. lacunosum-moleculare CA2 str. oriens CA2 str. pyramidale CA2 str. radiatum CA2 str. lacunosum-moleculare CA3 str. oriens CA3 str. pyramidale CA3 str. lucidum CA3 str. radiatum CA3 str. lacunosum-moleculare CA4 DG str. moleculare DG str. granulosum DG str. multiforme

. Percentage diff.

Control

Lesion

973 + 157 1276 k 170 1311 & 121 967 + 35 565 k 87 755 + 98 842 + 99 906k73 539 + 74 715+112 706 k 88 847 k 84 1015 f 95 878 f 127 1165 + 102 1006 + 105 794 + 90

1055 + 92 1287 z 135 1365 + 125 1020 + 69 537 + 53 698 + 79 810 It 112 911_+79 538 + 80 688 k 80 690+80 826 + 83 1008 k 52 815 & 91 1087 + 91 955 f 97 783 & 97

9

I 4 5 -5 -9 -4 1 0 -4 -2 -3 -1 -7 -7 -5 -1

____~_~

[3H]Oxotremorine-M

Control

Lesion

Percentage diff.

704 + 133 846: 159 808 k 146 596 + 104 523 f 107 776 + 141 611 k95 517 f 67 510 + 87 731 f 125 630 + 121 613 + 99 583 + 85 738 + 134 504,75 587 k 99 611 & 115

602_+ 110 722 & 140 669 + 123 468 _+84 503 + 102 6651144 497 + 105 438581 44Ok81 607 _+126 515 + 108 523 k 91 535 + 81 601 + 105 431 + 69 498 2 93 511&97

-14 ~- 15 -17 - 21* -4 -14 - 19 -15 - 14 -17* -182 -15* ._ 8 -19* -15 --I6 - 16*

The receptor densities of each layer and ligand are given as mean values f S.E.M. for the lesion (n = 5) and control group (n = 5). Asterisks indicate significant (P 5 0.05) differences between lesioned and control animals. Additionally, relative differences in per cent between both groups are calculated (percentage diff.) str., hippocampal layer.

experiment were KD = 1.50 (r = 0.947), nH = 0.95 and B,,,,, = 418 fmol/mg protein (Fig. 2). Inhibition experiments with CCh vs [‘H]Nmethylscopolamine discriminate between different affinity states of muscarinic receptors. All curves were best-fitted by a two-site model. Lesioned animals showed a remarkable shift to higher competitor concentrations in the dorsal hippocami (Fig. 3A). Non-linear least-square analysis gave Ki= 6.65 _t 2.1 x 10m4M and K,= 3.9+ 0.4 x 10~ 3M for highcontrol

Free [nM]

Fig. 2. Saturation experiment with [‘H]oxotremorine-M in the dorsal hippocampus in one lesioned (closed squares) and one control animal (closed circles). Each data point represents the mean value of a quadruplicate of four randomized sections. Saturation’ is reached at a lower absolute level in lesioned compared with control animals. The respective Scatchard plots are given in the inset indicating a lower maximal binding (B_) in lesioned animals (closed squares). The respective dissociation constants (Ko) are nearly identical in both groups. B, bound, F, free.

and low-afhnity binding sites, respectively, in lesioned animals. The percentage of receptors belonging to different affinity states was provided by the computer analysis of individual competition curves. The calculated proportion of low-affinity binding sites was 94% (inset of Fig. 3A). In controls, the respective inhibition constants were Ki= 7.2 _+3.0 x 10e6 M and K,= 4.1 + 1.2 x 10d4 M for high- and low-affinity binding sites, respectively. The calculated percentage of low-affinity receptors was 54% (inset of Fig. 3A). The effects of the GTP analogue Gpp[NH]p were also investigated in competition experiments. As expected, the addition of a constant concentration (100 PM) of Gpp[NH]p resulted in a shifting of the competition curves to the right in control animals (Fig. 3B). The proportion of low-affinity sites amounts to 90% (inset of Fig. 3B). In contrast, there were no measurable effects of Gpp[NH]p on the competition curves in lesioned animals (Fig. 3C). The proportion of high- and low-affinity binding sites (92% lowaffinity sites) are given in the inset of Fig. 3C. They nearly equal those found in sections of lesioned animals which were not treated with Gpp[NH]p. These results indicate a severe loss of convertible high-affinity binding sites in lesioned animals. The competition curves in single layers (Fig. 4) were similar to those found in the entire dorsal hippocampus (Fig. 3). Additionally, the same proportions of high- and low-affinity binding sites (Table 3) were found both in the case of higher and lower anatomical resolution. DISCUSSION

This is the first study to demonstrate receptor density and affinity in the denervated rat hippocampus on the single layer level The pattern of distribution

99

Lesion-induced muscarinic desensitization

-9

A

-6

-7

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Fig. 3. The in~bition of [3H]~-~~yl~pola~ne (0.26 nM) binding by CCh three days after Iesion. Each data point represents the mean value of the data of lesioned (n = 5) and control (n = 5) animals. The vertical bars indicate the standard deviations. A shows the specific binding of fH]~-methyl~o~~ne (ordinate) in lesioned (closed squares) and control (closed circles) hippocampi after competition with various concentrations of CCh (abscissa). The right-shifted curve of the bsioned animals indicates a lower aRinity for the agonist CCh. B shows the competition curves in control animals after adding 100~M GppmH]p (open circles) and without Gpp[NH]p (closed circles). C shows the competition curves in lesioned animals after adding 100 PM Gpp[NH]p (open squares) and without Gpp[NH]p (closed squares). In contrast to B, the addition of Gppwjp does not change the atIinity for the agonist in lesioned animals. The insets show the proportion of high- (open columns) and low (hatched columns)-affinity binding sites under the various experimental conditions. C, control animals; L, lesioned animals; C -, high- and low-affinity binding sites of controls in the absence of GppmH]p; C -+ , high- and low-atlinity binding sites of controls in the presence of Gpp[NHjp; L - , high- and low-affinity binding sites of lesioned animals in the absence of GppmH]p; L + , high- and low-affinity binding sites of lesioned animals in the presence of GPPW-UP.

and absolute densities of M, and M, receptors as well as the characteristics of [3Hl~-me~ylscopola~ne binding in controls are in accordance with former descriptions.‘“,39 The muscarinic M, receptors did not change in distribution pattern and density after denervation of the hippocampus. However, the density of Mr recepton decreased in all layers of the dorsal hippocampus. Muscarinic receptors are pre- and postsynaptically localized. In the case of the M, subtype, presynaptically situated receptors have been found to be only a small sub~pulation~2 For this reason it cannot be excluded, although it seems very unlikely, that a lesion-indu~d loss of pr~~apti~l M, receptors is obscured by a postsynaptic upregulation of the same sire.

The loss of M, receptors after septal lesions is in agreement with former observations of its partly presynaptic localization. ‘**‘,” Even the most pronounced decreases, however, do not exceed 2 1%. The large mass of unaffected receptors may be indicative of a postsynaptic localization. This has already been described for cholinergic denervations of the cortex.4*5.2’ The question of pre- and postsynaptic localization of receptors is not the only factor important in the interpretation of lesion experiments, but in addition differences in type and completeness of lesions must be regarded. For example, the ibotenic acid lesions in our study are large, but a certain amount of septohip~~~pal projections may survive. Finally, a qu&titatively important chdinergic intemeuronal

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Fig. 4. The inhibition of [“HJN-methylscopolamine binding (ordinate) by various concentrations of CCh (abscissa) three days after lesion.The concentration of the radioligand is 0.26nM. Each data point represents the actual value of one lesioned and one control animal, respectively. Data were evaluated in single hippocampal layers with widely different densities of [gH]N-methylscopolamine binding sites and

cholinergic axonal terminals.39A-F show that layers with a dense choline@ innervation [(A) oriens layer of CAl, (B) lucidum layer of CA3, (C) molecular layer of DG] do not differ in their competition curves from layers with a weaker innervation [(D) lacunosum-moleculare layer of CAl, (E) radiatum layer of CA3, (F) granular layer of DG]. system was described in the hip~mpus.13 Muscarinic receptors of this system may not be impaired by septal lesions. We confirm former observations, which were made in the septohippocampal system”,33 as well as in the basal forebrain-cortex system,“.s that non-selective antagonistic markers for both subtypes ([jH]Nmethylscopolamine, [3H]quinuclidinyl benzilate) exhibit no significant alterations several days after lesion. It has been suggested that antagonists have a IO times weaker affinity for presynaptic mAChRs

than the agonists. 33 This explains the differences between the absolute receptor masses of [3H]Nmethylscopolamine and the sum of ~3H~ire~pioe and [3H]oxotremorine-M (Tables 2, 3). It may also account for the fact that even at the single layer level no changes in distribution pattern or density could be found after septal lesions, when [3H]N-methylscopolamine binding sites were measured, whereas significant alterations were found with [%]oxotremorine-M. Moreover, [‘HfN-methylscopolamine seems to recognize only a part of the muscarinic

101

Lesion-induced muscarinic desensitization

Table 3. Density and proportions of high- and low-affinity muscarinic binding sites in the hippocampus of one lesioned and one control animal Lesion

Control

Percentage binding sites

Percentage binding sites

CA1 str. oriens CA1 str. pyramidale CA1 str. radiatum CA1 str. lacunosum-moleculare CA2 str. oriens CA2 str. pyramidale CA2 str. radiatum CA2 str. lacunosmn-moleculare CA3 str. oriens CA3 str. pyramidale CA3 str. lucidum CA3 str. radiatum CA3 str. lacumosum-moleculare CA4 DG str. moleculare DG str. granulosum DG str. multiforme

(fmol/mg protein)

High

Low

(fmol/mg protein)

High

Low

1708 1835 1642 1409 1278 1512 1433 1308 1194 1386 1309 1389 1371 1380 1507 1461 1287

57 49 44 51 50 38 44 50 45 47 45 50 50 51 54 51 57

43 51 56 49 50 62 56 50 55 53 55 50 50 49 46 49 43

1634 1737 1604 1319 1238 1465 1385 1267 1154 1368 1298 1349 1383 1391 1446 1482 1342

0 6 10 10 0 0 0 9 2 5 2 2 12 2 13 7 0

100 94 90 90 100 100 100 91 98 95 98 98 88 98 87 93 100

Data of one representative competition experiment with CCh and [3H]N-methylscopolamine (0.26 nM) are given. Brain sections from lesioned and control animals were incubated in the same incubation medium. str., hippocampal layer.

receptor population. This was shown by labelling with [3H]quinuclidinyl be&late. Due to its more lipophilic character [3H]quinuclidinyl benzilate is able to label more than exclusively surface receptors.” To what extent glial cell receptors are involved in changes after lesion experiments still has to be clarified. It has been suggested that receptors for several transmitters exist on the surface of glial cells. The incubation of astrocytes in cell culture with cholinergic agonists and antagonists results in hyperpolarizations or even depolarizations of astrocytes.1*,22,29It may be argued that the shift in receptor affinity is caused by local gliosis resulting in an increase of cholinergic high-affinity sites on the glial cell surface. However, this seems to be unlikely because the glial reaction in the target area of a lesioned projection three days after damage is caused by a microglial proliferation23 and this cell type is not known to express a significant amount of mAChRs. Recent investigations’**’ on astroglia report only small increases of glial fibrillary acidic protein as an indicator of gliosis. A significant decrease in the affinity of mAChRs for CCh in lesioned animals was found in the present investigation. Changes in the receptor-effecter system may be responsible for these alterations. The convertibility of high-affinity sites with the GTP analogue Gpp[NH]p was dramatically decreased after deafferentation. This may reflect an uncoupling of receptor and associated G-protein.L7s35 These alterations lead to changes in receptor efficacy. Alterations in muscarinic-stimulated hydrolysis of inositol phospholipids have already been described after denervation3’ Although these results cannot be compared directly with the present data because of the different types of lesions, the anatomical structures included and the postlesional time, they indicate

an effect on M, receptors. Our findings in saturation experiments with the agonist [‘H]oxotremorine-M support the hypothesis of a primary involvement of M, receptors. There were apparently no alterations in the dissociation constants of lesioned animals and controls; only the maximal binding decreased significantly in lesioned animals. Furthermore, we have found that the characteristic reduction in affinity, which is inducible in controls by adding a GTP analogue, cannot be achieved in lesioned animals. The processes of uncoupling of receptors and G-proteins seem already to be completed in the denervated regions. There are non-cholinergic projections from the medial septum to the hippocampus,12*19*36and examples of co-distributions of different receptors,*“’ which are coupled to second messengers via identical G-proteins, were described. Although the effects, which were observed in our study, can be interpreted as a result of the link between mAChRs and G-proteins, the induction of uncoupling may not be exclusively triggered by the lesion of the cholinergic septohippocampal system. This aspect needs likewise further experimental elucidation as well as the timecourse of postlesional alterations. Preliminary experiments with animals, which have survived for three weeks after the lesion, show a restitution of the affinity states in lesioned animals to the control level, resembling the results which were found 10 days after the lesion.” Changes in receptor affinity are of considerable importance for receptor efficacy. CONCLUSION

In summary, we found that ibotenic acid-induced lesions in MS, VDB and HDB lead to alterations of

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HAUEK et a/

hippocampal mAChRs. Significant decreases of M, receptors can be explained by their presynaptic localization, although the majority of M, receptors remain unaffected and thus seem to have a postsynaptic location. M, receptors exhibit no quantitative changes indicating a preferentially postsynaptic localization. All [3H]N-methylscopolamine-labelled mAChRs showed a remarkable decrease in affinity for the agonist CCh after deafferentation. Since the effect of

the GTP analogue Gpp[NH]p on the affinity status of mAChRs decreases dramatically in lesioned animals, alterations in the receptor-G-protein complex may be responsible for these observations.

Acknowledgements-The technical assistence of R. Dohm, M. Rath and H. Liihrer is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Zi 192/8-2; SFB 194).

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