Basal Forebrain Cholinergic Immunolesion by 192IgG-Saporin: Evidence for a Presynaptic Location of Subpopulations of α2-and β-Adrenergic as Well as 5-HT2A Receptors on Cortical Cholinergic Terminals

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<ul><li><p>Neurochemical Research, Vol. 22, No. 8, 1997, pp. 957-966</p><p>Basal Forebrain Cholinergic Immunolesion by 192IgG-Saporin: Evidence for a Presynaptic Location ofSubpopulations of a2-and p-Adrenergic as Well as 5-HT2AReceptors on Cortical Cholinergic Terminals*</p><p>Mechthild Heider,1 Reinhard Schliebs,1,2 Steffen RoBner,1 and Volker Bigl1</p><p>(Accepted January 7, 1997)</p><p>To study whether the changes in cortical noradrenergic and serotonergic mechanisms observed inpatients with Alzheimer's disease are the consequence of reduced cortical cholinergic activity, anovel cholinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the low-affinity nerve growth factor receptor with the cytotoxic protein saporin, 1921gG-saporin) was usedto produce a specific and selective loss of cholinergic cells in rat basal forebrain nuclei. To correlatethe responses to cholinergic immunolesion in cholinoceptive cortical target regions with cholinergichypoactivity, quantitative receptor autoradiography to measure adrenoceptors and 5-hydroxytryp-tamine (5-HT) receptor subtypes, and histochemistry to estimate acetylcholinesterase activity, wereperformed in adjacent brain sections. a1-adrenoceptor and 5-HT1A receptor binding were not af-fected by cholinergic immunolesion in any of the cortical and hippocampal regions studied. How-ever, cholinergic immunolesion resulted in significantly reduced a2-and B-adrenoceptor as well as5-HT2A receptor binding in a number cortical and hippocampal regions displaying a reduced activityof acetylcholinesterase, already detectable seven days after a single injection of 192IgG-saporinand persisting up to three months post lesion without any significant recovery. The data suggestthat at least a subpopulation of a2-and (B-adrenoceptor as well 5-HT2A receptor subtype is presenton cortical and hippocampal cholinergic terminals originating in the basal forebrain. The lesion-induced receptor changes suggest that the alterations in cortical 5-HT2 receptor binding observedin patients with Alzheimer's disease might be secondary to the cholinergic deficits.</p><p>KEY WORDS: Alpha-adrenoceptor; beta-adrenoceptor; 5-hydroxytryptamine receptor; autoradiography; ace-tylcholinesterase; cerebral cortex; image analysis.</p><p>INTRODUCTION</p><p>Alzheimer's disease is known to be associated witha very early and progressive loss of cholinergic cells in</p><p>1 Paul Flechsig Institute for Brain Research, University Leipzig, Jahn-allee 59, D-04109 Leipzig, Germany.</p><p>2 Address reprint requests to: Dr. Reinhard Schliebs, University ofLeipzig, Paul Flechsig Institute for Brain Research, Department ofNeurochemistry, Jahnallee 59, D-04109 Leipzig, Germany. Tel.:+49-341-97-25766; fax +49-341-211 44 92.</p><p>* Special issue dedicated to Dr. Annica Dahlstrohm.</p><p>the nucleus basalis of Meynert. This cortical cholinergicdysfunction has been implicated for the severe cognitivedeficits in these patients. However, the changes in thecholinergic transmission are accompanied by a numberof alterations in other transmitter systems including glu-tamate, GABA, noradrenaline and serotonin (see e.g. ref.1), suggesting the importance of other neurotransmittersystems in cognition. Among these systems noradrena-line and serotonin are thought to be special significance.This is emphasized by a number of studies demonstrat-ing impairments in memory and cognitive behaviour fol-</p><p>9570364-3190/97/0800-0957$12.50/0 C 1997 Plenum Publishing Corporation</p></li><li><p>958 Heider, Schliebs, Refiner, and Bigl</p><p>lowing experimentally-induced alterations in the centralnoradrenergic and serotonergic transmission (2-7). Al-though the administration of both serotonergic and no-radrenergic drugs to animals can improve performancein various learning tasks (5,8,9), the role of the seroto-nergic system in learning and memory processes is stillcontroversial (for review, see ref. 7). It is also possiblethat serotonin affects cognitive processes by modulationof the cholinergic system and vice-versa. Affecting onetransmitter system, e.g. by drugs or by degenerative pro-cesses in the diseased brain, may alter the efficacy ofanother one. Thus, there is a big demand to examine theinteraction of transmitter systems which may play a rolein controlling cognitive function (7). This might contrib-ute to derive more validated rationales for pharmacolog-ical interventions in this process and to find newtherapeutical strategies to treat Alzheimer's disease. Toelucidate the exact nature of the interaction of acetyl-choline with other transmitter systems, adequate animalmodels are required to produce specific cholinergic def-icits in vivo. Cholinergic lesion paradims have been usedto characterize the role of the cholinergic system in cor-tical information processing, learning and memory andin cognitive behaviour. In the past, a number of studieshave applied excitotoxins or cholinotoxins to producecortical cholinergic deficits. However, the cytotoxinsused are far from being selective to cholinergic cells (seee.g. ref. 10). A recently introduced new approach byWiley et al. (11) which uses a monoclonal antibody(192IgG) to the low affinity NGF receptor p75NGFR totarget a ribosome-inactivating cytotoxic protein (saporin)selectively to neurotrophin receptor bearing cholinergicneurons, circumvents these limitations. Intraventricularadministration of this 192IgG-saporin conjugate resultedin a complete disappearance of choline acetyltransferase(ChAT) and NGF receptor immunoreactive neuronsthroughout the basal forebrain, but spared other neuronalsystems in the basal forebrain nuclei (12-18). The spe-cific loss of basal forebrain cholinergic cells led to astrong activation of microglia in all magnocellular basalforebrain nuclei corresponding to the topographic local-ization of degenerating cholinergic cells (14). Substantialimmunotoxin-induced reductions in both ChAT-immu-noreactivity and acetylcholinesterase (AChE) staining aswell as high-affinity choline uptake sites in a number ofcortical regions including hippocampus and olfactorybulb, but not in the striatum and cerebellum, were de-tectable already seven days after lesion (13,19). Thesehistochemical changes are complemented by specific al-terations in cortical cholinergic neurochemical param-eters including muscarinic acetylcholine receptorsubtypes, choline uptake, acetylcholine release and mus-</p><p>carinic receptor-mediated production of inositol phos-phates (20,21). Therefore, 192IgG-saporin was used toselectively lesion basal forebrain cholinergic cells and tostudy the impact of cortical cholinergic hypoactivity onnoradrenergic and serotonergic mechanisms in cholino-ceptive cortical target regions. Acetylcholine may influ-ence the neuronal activity of other transmitter systemsby acting through different postsynaptic transmitter re-ceptors in the cerebral cortex. Neurotransmitter receptorsare one of the decisive links in the chain of synapticinformation processing. They can markedly respond toalterations in neuronal activity by adaptive mechanismslike sub/supersensitivity or down-regulation (22), whichmakes them an appropriate tool to monitor for interactivechanges in the neuronal activity of a particular transmittersystem. Therefore, the effect of cholinergic immunolesionfor various periods of time on cortical adrenoceptor (a1-,, a2-, and (B-subtype) and 5-hydroxytrvptamine (5-HT)receptor subtypes (5-HT1A, 5-HT2A) was investigated byquantitative receptor autoradiography, which allows for ascreening of receptor changes through the whole brain.To correlate the responses to cholinergic immunolesionin cholinoceptive cortical target regions with cholinergichypoactivity, acetylcholinesterase (AChE) histochemistryin adjacent brain sections was performed.</p><p>EXPERIMENTAL PROCEDURE</p><p>Materials. [7-methoxy-3H]Prazosin (2886 GBq/mmol), [methyl-3H]Rauwolscine (2738 GBq/mmol), [ring, propyl-3H(N)]L-Dihydroal-prenolol (DMA, 3922 GBq/mmol), [propyl-2,3-ring-l,2,3-3H]8-Hydroxy-DPAT (4791 GBq/mmol), and [ethylene-3H]Ketanserin(3149 GBq/mmol) were purchased from New England Nuclear,DuPont, Germany.</p><p>The cholinergic immunotoxin 192IgG-saporin was obtained fromChemicon International Inc., Temecula, USA. Phentolamin (SIGMA),L-isoproterenol (SIGMA), mianserin (SIGMA), and serotonin(SIGMA) were used for assessing nonspecific binding, all other chem-icals used were commercial products of highest purity available.</p><p>Treatment of Animals and Tissue Preparation. Immunolesion ofthe forebrain cholinergic system was performed as described previ-ously (19). Briefly, adult male Wistar rats with initial weight of 235-250 g were anesthetized with 60 mg ketamine i.m. and 40 mgpentobarbital i.p./kg body weight. The rat's head was placed in a ster-eotaxic frame and the skull was opened at bregma 1.2 mm lateral tothe longitudinal suture by placing a small hole. Stereotaxic injectionof approximately 4 mg (in 10 ml of phosphate buffered saline) of192IgG-saporin was made into the left lateral ventricle (coordinates:posterior 0.8 mm and lateral 1.2 mm from bregma) using a 10 mlsyringe with a 31-gauge needle (Hamilton). The needle was insertedto a depth of 3.4 mm below the cortical surface and the injectionperformed at the rate of 1 m1 per minute. After injection the syringewas kept for additional 5 min at the injection site to allow for a com-plete diffusion. An equal number of animals received the vehicle aloneand were considered as controls. Seven, 15, 30, and 90 days following</p></li><li><p>Aminergic Receptor Binding after Cholinergic Immunolesion 959</p><p>injection the animals were sacrificed; the brains were frozen andtwelve mm thick serial coronal sections were cut with a cryostat mi-crotome (Miles, USA) and thaw-mounted on gelatin coated slides, andstored at 20C until use.</p><p>The animal experiments performed in this study have been ap-proved by the independent Ethical Committee for Animal Experimentsof the Regierungsprasidium Leipzig, licence no. TW 10/92.</p><p>Acetylcholinesterase Histochemistry. Histochemical staining foracetylcholinesterase (AChE) was performed in adjacent sections ineach cortical level according to the method of Andra and Lojda (23).Briefly, air dried cryocut sections were preincubated for 30 min at37C in 0.1 M Tris-maleate buffer (pH 5.0) containing 30 mM iso-OMPA to inhibit nonacetylcholinesterases. After preincubation sec-tions were incubated in a solution consisting of 1.7 mM acetylthioch-oline iodide, 40 mM sodium citrate, 12 mM cupric sulfate, 8 mMpotassium ferrycyanide, 30 mM iso-OMPA and 75 mM Tris-maleatebuffer (pH 5.0) for 60 min at 37C. The reaction was stopped byrinsing sections in 0.1 M Tris-maleate buffer (pH 5.0) followed by ashort dipping in distilled water. Finally, the sections were dehydratedand coversliped.</p><p>Receptor Binding Assays</p><p>Adrenoceptor Subtypes. [3H]prazosin binding to thea1-adenoceptor subtype was carried out using a slightlymodified procedure of Rainbow et al. (24): sections wereprewashed for 15 min in 170 mM Tris-HCl buffer (pH7.4) at 4C and dried under a stream of air at roomtemperature before performing the binding assay. Afterpreincubation, the dried slides were covered with 170mM Tris-HCl (pH 7.4) containing 1 nM [3H]prazosinand incubated in humid chambers for 30 min at roomtemperature. After incubation, sections were rinsed twiceof 10 min each with ice-cold buffer, followed by a quickdipping into ice-cold distilled water, and blown dry.Nonspecific binding was estimated in adjacent sectionsby adding 10 (iM phentolamine to the incubation buffer,and represented </p></li><li><p>960 Heider, Schliebs, RoBner, and Bigl</p><p>corresponding tissue region using a calibration curveplotted from the radioactivity of the tissue standards(kBq/mg tissue equivalent) and the densitometrically de-termined optical density values of the respective auto-radiograms. Receptor densities were expressed as fhiolspecifically bound radioligand per mg tissue (for detailsof quantification, see also ref. 30).</p><p>For evaluation of cortical regions all over the braincoronal sections at selected distances from the bregma:+2.7, +0.2, -3.3, -5.8 mm (according to ref. 31) wereused. Particularly, optical density readings were per-formed in the following regions: subfields of hippocam-pal formation including dentate gyrus, frontal cortices(Frl, Fr2, Fr3), parietal cortices (Parl, Par2), temporalcortices (Tel/Te2) occipital cortices (OC1, OC2), fore-limb and hindlimb area, piriform, cingulate, entorhinaland retrosplenial cortex as well as corpus striatum. Thedata obtained were corrected for non-specific binding.Measurements were made on three consecutive sectionsfrom each animal. The corresponding data obtained fromfour animals in each experimental group were averaged.</p><p>Statistical Analysis. A one-way analysis of variance(ANOVA) was used to examine differences in the neu-rochemical parameters measured between brain regionand coronal level and between brain regions of controland experimental animals, followed by t-tests usingDunnett's correction for multiple simultaneous compar-isons by means of the software package SPSS. Differ-ences between treatments were considered statisticallysignificant when P &lt; 0.05.</p><p>Fig. 1. Representative examples of autoradiograms obtained from ratbrain cryocut sections labeled for a1- ([3H]prazosin binding), a2,-([3H]rauwolscine binding) and (B- ([3H]dihydroalprenolol binding) ad-renoceptors as well as 5-HT1A- ([3H]8-OH-DPAT binding) and 5-HT2A-([3H]ketanserin binding) receptors.</p><p>based, computer assisted imaging device using theautoradiographic software package from Imaging Re-search Inc., MCID 4.0.</p><p>For calibration of grey values (optical density) 3H-microscale standards co-exposed with labeled sectionswere used. The density of the receptor binding sites wascalculated from the mean grey level determined in the</p><p>RESULTS</p><p>Seven, 15, 30, and 90 days following an intracer-ebroventricular injection of 4 mg 192IgG-saporin coronalbrain sections at selected distances from the bregmawere subjected to radioligand binding to detect a1-, a2-and 3-adrenoceptor as well as 5-HT1A- and 5-HT2A re-ceptor subtypes. Typical autoradiograms obtained label-ing adrenoceptor and serotonergic receptor subtypes areshown in Fig. 1. The levels of cryocutting were so se-lected to include for data analysis all cortical areas whichreceive a prominent cholinergic innervation from thebasal forebrain. To correlate immunotoxin-induced al-terations in cortical receptor binding with the loss ofcholinergic input, AChE staining was routinely per-formed in adjacent brain sections of both control andexperimental animals. In Fig. 2 the region-specific lossesof AChE staining seven, 15, 30, and 90 days after im-munolesion by 192IgG-saporin are graphically dis-played. In Figs. 3 to 5 the immunotoxin-induced changes</p></li><li><p>Aminergic Receptor Binding after Cholinergic Immunolesion 961</p><p>Fig. 2. Changes in acetylcholinesterase (AChE) activity in selectedbrain regions following cholinergic immunolesion for 7, 15, 30 or 90days as compared to corresponding control values. Brain cryocut sec-tions were st...</p></li></ul>