350
Brauz Research, 590 (1992) 350-355 @ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25361
Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat A d a m A. B o o k ", R o n a l d G. Wiley ~ a n d J o h n B. S c h w e i t z e r a,b " Department of Anatomy and Ne.robiolog~,, r, Department of Pathology, Uniz'ersity of Tennessee, Memphis, TN 38163 (USA) and ' DVAMC and Vanderbih Unit'ersity, Nashrilh,, TN 37232 (USA) (Accepted 23 June 1992)
Kt,v words: Nerve growth factor receptor; lmmunotoxin; Choline acetyltransferase; Tyrosine hydroxylase; Immunohistochemistry; Radioenzymatic assay
A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 lgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 lgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (CHAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 lgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKTl-saporin-injetted controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 lgG-saporin. ChAT immunohi;~tochemistry was confirmative of major c~ll loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the s~l~ctivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.
The magnocellular neurons of the medial septal nucleus (msn), nucleus of the diagonal band of Broca (.ndbB), and nucleus basalis magnocellularis (nbm) are often collectively termed the cholinergic neurons of the basal forebrain (CBF) t~. The msn and vertical limb of ndbB (vdB) project predominantly to the hippocam. pus 17, the horizontal limb of ndbB (hdB) projects primarily to the olfactory bulb "~1, and the nbm provides the cholinerg; afferent fibers to the neocortex H~. The CBF is the most consistently and severely affected system in Alpheimer's disease .~2, A considerable body of evidence documents a selective loss of cholinergic neurons in the nbm in patients with Alzheimer's disease "~'3~'. For this reason, some attempts to model Alzheimer's disease in animals have focused on producing cholinergic hypofunction by manipulating CBF nucleiS.').14. Methods used to produce forebrain cholinergic deficit in animals include the local injection of kainic or ibotenic acid into the nbm 5, electrolytical lesioning of CBF nuclei 14 and the local or intraventricular ad-
ministration of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A) ''~,'. However, none of these methods produces an entirely satisfactory model of forebrain cholinergic deficit, CBF kainic or ibotenic acid injections and electrolytic lesions destroy cholinergic neurons, but also affect many noncholinergic cells 14,21. Local administration of AF64A into various brain regions produces considerable nonspecific tissue destruction 16. lntraventricular AF64A results in a selective loss of only septohippocampal neurons, sparing cholinergic neurons in the nbm 2,u. Selectively destroying cholinergic neurons, while sparing other cell types, would provide a model of forebrain cholinergic deficit superior to the models produced by the methods described above. Neurons of the CBF possess NGF receptors while other neurotransmitter-containing neurons in the region, and even the nearby striatal cholinergic interneurons, do not express detectable levels of NGF receptors 7.~,3s. 192 IgG, a well-characterized monocional antibody to the low-affinity rat nerve growth factor (NGF)receptor t,
Correspomlence: J.B. Schweitzer, Department of Pathology, College of Medicine, The University of Tennessee, 800 Madison Avenue, Room 568M, Memphis, TN 38163, USA. Fax: (I)(901) 528-6979.
351 has been shown by autoradiography and immunohistochemistry to accumulate specifically and bilaterally in cholinergic neurons of the basal forebrain following intraventricular administration :'6'27'3°. Recently, an immunotoxin composed of 192 lgG chemically linked via a disulfide bond to the ribosome inactivating protein saporin (192 IgG-saporin) was shown immunohistochemically to destroy cholinergic neurons of the CBF following intraventricular injection 33. In order to further characterize the cholinergic deficit produced by 192 lgG-saporin, we now report the levels of ChAT activity in the terminal fields of CBF neurons following intraventricular administration of the immunotoxin. The cells that produce 192 IgG were generously supplied by E.M. Johnson, Jr. The antibody was purified from a 50% ammonium sulfate cut of ascitic fluid using standard protein A affinity methods ~. Purity was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 192 lgG was chemically coupled to saporin by modifying both 192 lgG and saporin with a ten-fold molar excess of N-succinimidyi3-(2-pyridyldithio)propionate (SPDP) (Pierce). Both reagents were dialyzed against phosphate-buffered saline (PBS). Saporin was then reduced with 5 mM dithiothreitol, desalted on a Sephadex G25 column, and added to 192 IgG in a 2.5-fold molar excess. The 192 lgG-saporin conjugate was allowed to react overnight and was subsequently purified by ion exchange and affinity chromatography using CM-52 (Whatman) and MAPS li (Bio-Rad), in order to remove excess 192 lgG and saporin, respectively. 192 igG-saporin was loaded onto the CM-52 column in 10 mM sodium phosphate buffer, containing l0 mM sodium chloride (pH 6.5), and was eluted from the column with 1.0 M sodium chloride in the same buffer. Purification using MAPS II was performed according to the manufacturer's instructions, except a 200 mM sodium citrate buffer (pH 5.0) was used to elute the sample. The purified immunotoxin was dialyzed into PBS, concentrated using miniconcentrators (Amicon), and its purity checked by SDS-PAGE. A control immunotoxin composed of the antihuman pan-Tlymphocyte (CD5) OKT1 monoclonal antibody chemically linked to saporin, referred to as OKTl-saporin, was synthesized and purified as described previously 29. Adult female Sprague-Dawley rats (Harlan)weighing 200-300 g were placed in a stereotaxic apparatus (Kopf) following the induction of deep anesthesia with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) given intramuscularly. Stereotaxic injection of 24 ~! of PBS or 8 ~g (in 24 ~1 of PBS) of 192 IgG-saporin was made into the right lateral ventricles
using standard coordinates 23 and a 10 #1 syringe with a 31-gauge needle (Hamilton). For this procedure, a burr hole was placed at bregma 1.2 mm lateral to the longitudinal suture. The needle was inserted to a depth of 3.4 mm below the cortical surface and the injection was performed at the rate of 2/zl per min. Coordinates were adjusted depending on the size of the animal. In a separate experiment, 4 animals were injected with 192 IgG-saporin (5 p,g) and 2 animals with OKT1saporin (5 /zg) by pressure microinjection into the lateral ventricle (coordinates: 1.2 mm anterior to bregma, 1.0 mm lateral to midline, 5.0 mm deep to the cortical surface 29) using glass micropipettes of 25-50 ~m tip diameter. Animals were allowed to survive for varying times following injection, with the majority of animals (n = 7) analyzed 2 weeks after injection. PBSinjected animals that were allowed to survive 2 weeks and unmanipulated animals which did not differ in ChAT activity from PBS-injected animals were used as controls. For radioenzymatic ChAT and tyrosine hydroxylase (TOH) assays, brains were rapidly removed following sacrifice of the animals, frozen on dry ice, and stored at -80°C. Standardized dissections of the olfactory bulbs, hippocampus, striatum, and neocortex (fi'ontai/ parietal) were performed. Brain tissue was homogenized in 20 vols. of PBS, centrifuged, and the supernatants were frozen at -80°C for use in the biochemical assays. ChAT activity was measured in the olfactory bulb, hippocampus, cortex and striatum, while TOH activity was measured in the striatum, using the methods of Schrier and Schuster 2"~ and Nagatsu 2°, respectively. ChAT activity is reported as counts per minute per microgram (cpm//zg) of ~4C-labeled acetyicholine produced, whereas TOH activity is reported as cpm//u,g of 3H-labeled 3,4-dihydroxyphenylacetic acid produced. The Student's t-test was used to assess differences in ChAT and TOH activity between animals that received intraventricular injections of 192 IgG-saporin and PBS and were allowed to survive 2 weeks. For immunohistochemical analysis, 2 weeks following the intraventricular injection of either 8 p,g of 192 IgG-saporin (n = 3) or PBS (n = 2), animals were transcardially perfused with 100 ml of normal saline, followed by 250 ml of 4% buffered paraformaldehyde containing 15% picric acid and 0.1% glutaraldehyde. The brains were removed, equilibrated with 30% buffered sucrose, and 40-p,m coronal sections of forebrain and brainstem were cut using a vibratome (Lancer). Sections were processed for immunohistochemistry using a monoclonal antibody against ChAT (Boehringer Mannheim) and the localization of the primary was carried out by the avidin-biotin horse-
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Fig. 1. ChAT activity expressed as percent of control (n = 3) in homogenates of CBF terminal fields and striatum of animals that received intraventricular injections of 192 lgG-saporin (8 v,g in 24 V,I of PBS). Animals injected with immunotoxin were allowed to survive for I (n = I), 4 (n = I). 7 (n = 2), or 14 (n = 2) days.
Fig. 2. ChAT activity expressed as percent of control in homogenates of CBF terminal fields of animals that received intraventricular injections of either 192 IgG-saporin (5/~g) or OKTl-saporin (5/zg) 2 weeks previously. Control animals received intraventricular injections of PBS. All data points represent the means of duplicate animals.
radish peroxidase method using an anti-rat immunoglobulin kit (Vector Laboratories). Sections were developed using diaminobenzidine and hydrogen peroxide, mounted, dried, and coverslipped. Radioenzymatic ChAT assays showed time-dependent decreases in ChAT activity in the terminal field regions of CBF neurons following intraventricular administration of 192 lgG-saporin (Fig. 1). Compared to controls, ChAT activity was unaltered in all brain regions I day following injection of the immunotoxin. By 4 days after injection, ChAT activity was clearly reduced in the cortex and olfactory bulb, marginally reduced in the hippocampus, and still unaffected in the striatum, as compared to controls, ChAT activity was nearly maximally reduced in the cortex and olfactory bulb by 14 days after injection, and showed approxi. mately a 30% decrease in hippocampus, as compared to controls. The level of ChAT activity in the striatum was unaffected by 192 lgG-saporin at all time periods examined. Animals injected with OKTl-saporin (5/zg) showed levels of ChAT activity in the cortex, olfactory bulb, and hippocampus comparable to those obtained from PBS-injected animals (Fig. 2), ChAT activity in these regions from animals given equimolar doses of 192 igG-saporin were greatly reduced. Furthermore, ChAT activity remained at the levels shown in Fig, 2 in animals which survived for a period of 60 days (mean ChAT activity of duplicate animals, expressed as percentage of PBS-injected controls: cortex 36.9%, olfactory bulb 1.3%, hippocampus 10.2%). ChAT activity measurements from animals that received 8 v.g of 192 lgG-saporin with a 2-week post-operative survival time are summarized in Fig. 3. Data in Fig. 3 do not precisely match the points shown at 14
days in Fig. 1, because additional animals were treated with a separate preparation of immunotoxin which appears to have been slightly less effective in the olfactory bulbs. Nevertheless, all 3 CBF terminal field regions show statistically significant decreases in ChAT activity in immunotoxin-treated animals, as compared to PBS-injected controls. There was no difference in TOH activity in the striatum between animals that received up to 8/~g of 192 lgG-saporin (44.6:1:9.6 cpm//zg protein: n ,= 4) and those that received PBS (44.7 + 9.8 cpm/p.g protein', n = 3 ) . ChAT immunohistochemistry of CBF nuclei from animals that received 8 tzg of 192 IgG.saporin with a
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Fig, 3, ChAT activity expressed as percent of control (n = 5) in homogenates of CBF terminal fields and striatum of animals that received 8 Izg of 192 IgG-saporin (n = 7) and were allowed to survive 2 weeks following injections. Control animals received intraventricufar injections of PBS and were allowed to survive 2 weeks. Error bars are $.E,M, ** P < 0,01, * P < 0.05, student's t-test.
353 2-week survival time revealed a nearly complete loss of ChAT positivity in neurons of the hdB (Fig. 4B; compare with Fig. 4A) and the nbm. Variable, but at least moderate, loss of ChAT-positive neurons occurred in the msn and the vdB as compared to PBS-injected controls (data not shown). These results are in qualitative agreement with our biochemical findings, in which the most significant decrements in ChAT activity occurred in the terminal field regions of hdB and nbm (the olfactory bulb and cortex, respectively). ChAT immunohistochemistry further showed that cholinergic neurons in the striatum were unaffected by 8 ~ g of 192 IgG-saporin
This study provides further evidence that 192 IgGsaporin can selectively abolish the cholinergic parameters in the CBF, a system in which NGF receptor positivity is highly c o l o c a l i z e d with C h A T positivity ~'34
ChAT activity was significantly reduced in the cortex, o l f a c t o r y bulbs, and h i p p o c a m p u s but w a s u n a f f e c t e d
in the striatum of animals that received intraventricular 192 IgG-saporin. Striatal cholinergic neurons are NGF receptor-negative in normal adult rats 7'8'as (see below). ChAT immunohistochemistry revealed a corresponding loss o f C h A T - p o s i t i v e n e u r o n s in the h d B a n d in the
nbm, with no loss of ChAT-positive neurons in the striatum, following intraventricular 192 IgG-saporin. At equimolar doses, OKTl-saporin, an immunotoxin targeted to an antigen not present in the rat central
(Fig. 4C). O t h e r c h o l i n e r g i c cell g r o u p s
(e.g., motor cranial nerve nuclei) likewise appeared unremarkable (Fig. 4D).
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Fig. 4. Photomicrographs of coronal rat brain sections processed for ChAT immunohistochemistry. All sections were processed concurrently under identical conditions. A: unremarkable distribution of cholinergic (ChAT-positive) somata in the hdB of an animal that received an intraventricular injection of PBS (24 ,¢1) and was subsequently allowed to survive for 2 weeks. B: section through the same level of the hdB of an animal that received an intraventricular injection of 192 IgO-saporin (8 ~g in 24 ~l of PBS) and was subsequently allowed to survive for 2 weeks. Positively stained neurons are completely absent. C: section through the striatum of the same animal shown in B demonstrating robust staining of striatal cholinergic interneurons. D: section through the level of the third cranial nerve nucleus of the same animal shown in B demonstrating well-stained cholinergic neurons. Bar in A, B, D = 100/+m. Bar in C -- 50 ~m. ;
354 nervous system, did not affect the cholinergic system while 192 !gG.saporin significantly reduced ChAT activity. The main advantage of using 192 IgG-saporin to produce a model of CNS cholinergic deficit therefore resides in the ability of this immunotoxin to selectively target NGF receptor-positive cholinergic neurons in the basal forebrain and concomitantly spare neighboring cholinergic and other neurons, such as the cholinergic striatal interneurons. Previous studies have shown that 30% of the cholinergic neurotransmission in the cortex is due to intrinsic, NGF receptor-negative cholinergic neurons I°. Therefore, the observed 70% reduction of ChAT activity in the neocortex seen with 8 Izg of 192 IgG-saporin represents a maximal loss of CBF input. Since the olfactory bulb has no intrinsic cholinergic neurons 28 and gets all of its input from the CBF ~5, a 100% loss is theoretically possible in this region and was nearly achieved. A less than maximal effect was achieved in the hippocampus, which correlates with the sparing in the msn that was seen with immunohistochemical methods. We are currently investigating whether higher doses, the site of injection (animals injected more anteriorly by pressure microinjection showed greater decrements in ChAT activity in the hippocampus and olfactory bulb}, or other factors may affect this immunotoxin's ability to abolish cholinergic parameters in this area of the brain. The cholinergic neurons of the striatum were consis. tently unaffected by 192 lgG-saporin at all time and dosage points investigated. Although many studies have failed to report the immunohistochemical presence of NGF receptors on adult rat striatai cholinergic neurons ~x'~5, others have observed varying amounts of weakly NGF receptor-positive neurons in adult stria. turn TM. In any case, we have previously shown that cholinergic neurons in the adult rat striatum do not accumulate 192 IgG following intraventricular administration '~''27'3°. The resting (unmanipulated) cholinergic striatal neurons, whether weakly NGF receptor.positive or not, would therefore not be expected to accumulate, nor be intoxicated by, 192 lgG-saporin. This was the observed result. The unaltered level of TOH activity in the striatum following intraventricular 192 IgG-saporin further demonstrates the specificity of this immunotoxin towards NGF receptor-positive CBF neurons. CNS care:. cholaminergic neurons are NGF receptor.negative 7. TOH, the rate-limiting enzyme in the biosynthesis of catecholamines, is a marker for both dopaminergic and adrenergic systems ~9. The assay employed 20 was not sensitive enough to detect the levels of TOH present in most basal forebrain regions (data not shown), but
TOH activity is measurable in the striatum, which has considerably more dopaminergic input than the other regions of forebrain. The observed lack of effect of 192 IgG-saporin on TOH levels in our study is therefore mainly a lack of effect on the nigrostriatal dopaminergic system. The determination of specificity of ilesioning is a complicated issue. While we have sh6wn selectivity within different cholinergic systems and with a marker for an unrelated but similar long projection system, numerous other systems remain to be investigated. Even if alterations of other systems are found, the possibility of second order (transsynaptic) alterations need to be considered. For example, the cholinergic deficit induced by intraventricular AF64A was recently shown to be associated with a transient decrease in the level of somatostatin and a transient increase in the level of neuropeptide Y in the hippocampus and parietal cortex ~. In another study, lesioning of thalamostriatal neurons by kainic acid produced a decrease in ChAT activity in the striatum that was accompanied by an increase in glutamate decarboxylase activity, highaffinity glutamate uptake, and apparent dopamine turnover 21. In both of these examples, the alterations were ascribed not to direct effects of the lesioning tool on these systems, but were interpreted as compensatory or transsynaptic effects. Though 192 IgG-saporin significantly reduced cholinergic parameters following intraventricular administration, further experimental work is needed to determine if this reduction in ChAT activity and ChAT immunopositivity is permanent and represents the actual death of CBF neurons, or if it just reflects a down-regulation in enzyme synthesis in these neurons. We have found that the decrease in ChAT activity is relatively permanent in that it persists for at least 60 days. Whether the death of cholinergic neurons has occurred is an interesting issue and several paradigms exist to assess it. There are conflicting reports in the literature concerning whether the massive loss of cholinergic neurons in the msn of the adult rat brain produced by a lesioning of the fimbria-fornix is cell death ~'2z or simple atrophy ~x24. Specific studies assessing actual cell loss in CBF nuclei following intraventricular 192 lgG-saporin are therefore indicated and include standard pathological studies on the brain and labeling neurons with a retrograde marker and subsequently assessing the effect of the immunotoxin. These studies are currently being performed. Elucidation of the specific functions of cholinergic neurons in the basal forebrain has been hampered by the previous lack of a good selective lesioning tool 4. The ability of the antibody 192 IgG to specifically
355
target saporin to CBF neurons and significantly reduce cholinergic parameters suggests 192 IgG-saporin is capable of producing a much more discrete CBF lesion than that produced by classical chemical and electrical lesioning techniques. The result is an animal model of forebrain cholinergic deficit that initially appears to be selective and may prove to be very useful in the future as a tool to probe the biochemical, physiological, and behavioral functions of the CBF. The authors gratefully acknowledge the skillful technical assistance of Ellen B. Looney. We also thank Douglas Lappi for providing the OKTl-saporin. This research has been supported in part by Grants NS25122, NS10230 and MH10121 and by the Department of Veterans Affairs Medical Center, Nashville, TN.
REFERENCES 1 Chandler, C.E., Parsons, L.M., Hosany, M. and Shooter, E., A monoclonal antibody modulates the interaction of nerve growth factor with PC 12 cells, J. Biol. Chem., 259 (1984) 6882-6889. 2 Chrobak, J.J., Spates, M.J., Stackman, R.W. and Walsh, T.J., Hemicholinium-3 prevents the working memory impairments and the cholinergic hypofunction induced by ethylcholine aziridinium ion (AF64A), Brain Res., 504 (1989) 269-275. 3 Etienne, P., Robitaille, Y, Wood, P., Gauthier, S., Nair, N.P.V. and Quirion, R., Nucleus basalis neuronal loss, neuritic plaques and choline acetyltransferase activity in advanced Alzheimer's disease, Neuroscience, 19 (1986) 1279-1291. 4 Fibiger, H.C., Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence, Trends Neurosci., 14 (1991) 220-223. 5 Flicker, C., Dean, R.L., Watkins, D.L., Fisher, S.K. and Bartus, R.T., Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat, Pharntacol. Biochem. Behat,., 18 (1983)973-981. 6 Gage, F.H., Wictorin, K., Fischer, W., Williams, L.R., Varon, S. and Bjiirklund, A., Retrograde cell changes in medial septum and diagonal band following fimbria-fornix transection: quantitative temporal analysis, Neuroscience, 19 (1986) 241-255. 7 Gage, F,H., Batchelor, P,, Chen, K.S., Chin, D., Deputy, S., Rosenberg, M,B., Higgins, G.A., Koh, S., Fischer, W. and Bj6rklund, A., NGF-receptor reexpression and NGF-mediated cholinergie neuronal hypertrophy in the damaged adult neostriaturn, Neuron, 2 (1989) 1177-1184. 8 Hefti, F., Hartikka, J., Salvatierra, A., Weiner, W.J. and Mash, D.C., Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain, Neurosci. Lett., 69 (1986) 37-41. 9 Hortnagl, H., Sperk, G., Sobal, G. and Maas, D., Cholinergic deficit induced by ethylcholine aziridinium (AF64A) transiently affects somatostatin and neuropeptide Y levels in rat brain, J. Neurochem., 54 (1990) 1608-1613. 10 Johnston, M.V., McKinney, M. and Coyle, J.T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. 11 Kiss, J. and Patei, A.J., Characterization of neurons containing nerve growth factor receptors in the rat neostriatum, Neurosci. Lett., 105 (1989) 251-256. 12 Koh, S., Oyler, G.A. and Higgins, G.A., Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain, Exp. Neurol., 106 (1989) 209-221. 13 Kromer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. 14 Lo Conte, G., Bartolini, L., Casamenti, F., Marconcini-Pepeu, I. and Pepeu, G., Lesions of the cholinergic forebrain nuclei: changes in avoidance behavior and scopolamine actions, Pharmacol. Biochem. Behav., 17 (1982)933-937.
15 Macrides, F., Davis, B.J., Youngs, W.M., Nadi, N.S. and Margolis, F.L., Cholinergic and catecholaminergic afferents to the olfactory bulb in the hamster: a neuroanatomical, biochemical, and histochemical investigation, J. Cornp. Neuroi., 203 ( 19g i ) 495-514. 16 McGurk, S.R., Hartgraves, S.L., Kelly, P.H., Gordon, M.N. and Butcher, L.L., Is ethylcholine mustard aziridinium ion a specific cholinergic neurotoxin?, Neuroscience, 22 (1987) 215-224. 17 McKinney, M., Coyle, J.T. and Hedreen, J.C., Topographic analysis of the innervation of the rat neocortex and hippocampus by the basal forebrain cholinergic system, J. Comp. Neurol., 217 (1983) 103-121. 18 Mesulam, M.-M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. 19 Molinoff, P.B. and Axelrod, J., Biochemistry of catecholamines, Annu. Rev. Biochem., 40 (1971) 465-500. 20 Nagatsu, T., Levitt, M. and Undenfried S., Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis, J. Biol. Chem., 239 (1964) 2910-2917. 21 Nieoullon, A., Scarfone, E., Kerkerian, L., Errami, M. and Dusticier, N., Changes in choline acetyltransferase, glutamic acid decarboxylase, high-affinity glutamate uptake and dopaminergic activity induced by kainic acid lesion of the thalamostriatal neurons, Neurosci. Lett., 58 (1985) 299-304. 22 O'Brien, T.S., Svendsen, C.N., Isacson, O. and Sofroniew, M.V., Loss of true blue labelling from the medial septum following transection of the fimbria-fornix: evidence for the death of cholinergic and non-cholinergic neurons, Brain Res., 508 (1990) 249-256. 23 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. 24 Peterson, G.M., Lanford, G.W and Pnwell, E.W., Fate of septohippocampal neurons following fimbria-fornix transection: a time course analysis, Brain Res. Bull., 25 (1990) 129-137. 25 Schrier, B.K. and Schuster, L., A simplified radiochemical assay for choline acetyltransferase, J. Neurochem., 14 (1967) 977-985. 26 Schweitzer, J.B., Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebram neurons, Brain Res., 423 (1987) 309-317. 27 Schweitzer, J.B., Nerve growth factor receptor-mediated transport from CSF labels cholinergic neurons: direct demonstration by a double-labeling study, Brain Res., 490 (1989)390-396. 28 Sharif, N.A., Chemical and surgical lesions of rat olfactory bulb: changes in thyrotropin-releasing hormone and other systems, J. Neurochem., 50 (I 988) 388-394. 29 Siena, S., Lappi, D.A., Bregni, M., Formosa, A., Villa, S., Soria, M., Bonadonna, G. and Oianni, A.M., Synthesis and characterization of an antihuman T.lymphocyte saporin immunotoxin (OKTI-SAP) with in vivo stability into non-human primates, Blood, 72 (1988) 756-765. 30 Thomas, L.B., Book, A.A. and Schweitzer, J.B., Immunohistochemical detection of a monoclonal antibody directed against the NGF receptor in basal forebrain neurons following intraventricular injection, .l. Neurosci. Methods, 37 (1991) 37.-45. 31 Wenk, H., Meyer, U. and Bigl, V., Centrifugal cholinergic connections in the olfactory system of rats, Neuroscience, 2 (1977) 797-800. 32 Whitehouse, P.J., Price, DJ., Clark, A.W., Coyle, J.T. and DeLong, M.R., Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol., 10 (1981) 122-126. 33 Wiley, R.G., Oeitmann, T.N. and Lappi, D.A., Immunolesioning: selective destruction of neurons using immunotoxin to rat NGF receptor, Brain Res., 562 (1991) 149-153. 34 Woolf, N.J., Gould, E. and Butcher, L.L., Nerve growth factor receptor is associated with cholinergic neurons of the basal forebrain but not the pontomesencephalon, Neuroscience, 30 (1989) 143-152. 35 Yan, Q. and Johnson Jr. E.M., lmmunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain, J. Comp. Neurol., 290 (1989) 585-598.
Brauz Research, 590 (1992) 350-355 @ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25361
Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat A d a m A. B o o k ", R o n a l d G. Wiley ~ a n d J o h n B. S c h w e i t z e r a,b " Department of Anatomy and Ne.robiolog~,, r, Department of Pathology, Uniz'ersity of Tennessee, Memphis, TN 38163 (USA) and ' DVAMC and Vanderbih Unit'ersity, Nashrilh,, TN 37232 (USA) (Accepted 23 June 1992)
Kt,v words: Nerve growth factor receptor; lmmunotoxin; Choline acetyltransferase; Tyrosine hydroxylase; Immunohistochemistry; Radioenzymatic assay
A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 lgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 lgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (CHAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 lgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKTl-saporin-injetted controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 lgG-saporin. ChAT immunohi;~tochemistry was confirmative of major c~ll loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the s~l~ctivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.
The magnocellular neurons of the medial septal nucleus (msn), nucleus of the diagonal band of Broca (.ndbB), and nucleus basalis magnocellularis (nbm) are often collectively termed the cholinergic neurons of the basal forebrain (CBF) t~. The msn and vertical limb of ndbB (vdB) project predominantly to the hippocam. pus 17, the horizontal limb of ndbB (hdB) projects primarily to the olfactory bulb "~1, and the nbm provides the cholinerg; afferent fibers to the neocortex H~. The CBF is the most consistently and severely affected system in Alpheimer's disease .~2, A considerable body of evidence documents a selective loss of cholinergic neurons in the nbm in patients with Alzheimer's disease "~'3~'. For this reason, some attempts to model Alzheimer's disease in animals have focused on producing cholinergic hypofunction by manipulating CBF nucleiS.').14. Methods used to produce forebrain cholinergic deficit in animals include the local injection of kainic or ibotenic acid into the nbm 5, electrolytical lesioning of CBF nuclei 14 and the local or intraventricular ad-
ministration of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A) ''~,'. However, none of these methods produces an entirely satisfactory model of forebrain cholinergic deficit, CBF kainic or ibotenic acid injections and electrolytic lesions destroy cholinergic neurons, but also affect many noncholinergic cells 14,21. Local administration of AF64A into various brain regions produces considerable nonspecific tissue destruction 16. lntraventricular AF64A results in a selective loss of only septohippocampal neurons, sparing cholinergic neurons in the nbm 2,u. Selectively destroying cholinergic neurons, while sparing other cell types, would provide a model of forebrain cholinergic deficit superior to the models produced by the methods described above. Neurons of the CBF possess NGF receptors while other neurotransmitter-containing neurons in the region, and even the nearby striatal cholinergic interneurons, do not express detectable levels of NGF receptors 7.~,3s. 192 IgG, a well-characterized monocional antibody to the low-affinity rat nerve growth factor (NGF)receptor t,
Correspomlence: J.B. Schweitzer, Department of Pathology, College of Medicine, The University of Tennessee, 800 Madison Avenue, Room 568M, Memphis, TN 38163, USA. Fax: (I)(901) 528-6979.
351 has been shown by autoradiography and immunohistochemistry to accumulate specifically and bilaterally in cholinergic neurons of the basal forebrain following intraventricular administration :'6'27'3°. Recently, an immunotoxin composed of 192 lgG chemically linked via a disulfide bond to the ribosome inactivating protein saporin (192 IgG-saporin) was shown immunohistochemically to destroy cholinergic neurons of the CBF following intraventricular injection 33. In order to further characterize the cholinergic deficit produced by 192 lgG-saporin, we now report the levels of ChAT activity in the terminal fields of CBF neurons following intraventricular administration of the immunotoxin. The cells that produce 192 IgG were generously supplied by E.M. Johnson, Jr. The antibody was purified from a 50% ammonium sulfate cut of ascitic fluid using standard protein A affinity methods ~. Purity was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 192 lgG was chemically coupled to saporin by modifying both 192 lgG and saporin with a ten-fold molar excess of N-succinimidyi3-(2-pyridyldithio)propionate (SPDP) (Pierce). Both reagents were dialyzed against phosphate-buffered saline (PBS). Saporin was then reduced with 5 mM dithiothreitol, desalted on a Sephadex G25 column, and added to 192 IgG in a 2.5-fold molar excess. The 192 lgG-saporin conjugate was allowed to react overnight and was subsequently purified by ion exchange and affinity chromatography using CM-52 (Whatman) and MAPS li (Bio-Rad), in order to remove excess 192 lgG and saporin, respectively. 192 igG-saporin was loaded onto the CM-52 column in 10 mM sodium phosphate buffer, containing l0 mM sodium chloride (pH 6.5), and was eluted from the column with 1.0 M sodium chloride in the same buffer. Purification using MAPS II was performed according to the manufacturer's instructions, except a 200 mM sodium citrate buffer (pH 5.0) was used to elute the sample. The purified immunotoxin was dialyzed into PBS, concentrated using miniconcentrators (Amicon), and its purity checked by SDS-PAGE. A control immunotoxin composed of the antihuman pan-Tlymphocyte (CD5) OKT1 monoclonal antibody chemically linked to saporin, referred to as OKTl-saporin, was synthesized and purified as described previously 29. Adult female Sprague-Dawley rats (Harlan)weighing 200-300 g were placed in a stereotaxic apparatus (Kopf) following the induction of deep anesthesia with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) given intramuscularly. Stereotaxic injection of 24 ~! of PBS or 8 ~g (in 24 ~1 of PBS) of 192 IgG-saporin was made into the right lateral ventricles
using standard coordinates 23 and a 10 #1 syringe with a 31-gauge needle (Hamilton). For this procedure, a burr hole was placed at bregma 1.2 mm lateral to the longitudinal suture. The needle was inserted to a depth of 3.4 mm below the cortical surface and the injection was performed at the rate of 2/zl per min. Coordinates were adjusted depending on the size of the animal. In a separate experiment, 4 animals were injected with 192 IgG-saporin (5 p,g) and 2 animals with OKT1saporin (5 /zg) by pressure microinjection into the lateral ventricle (coordinates: 1.2 mm anterior to bregma, 1.0 mm lateral to midline, 5.0 mm deep to the cortical surface 29) using glass micropipettes of 25-50 ~m tip diameter. Animals were allowed to survive for varying times following injection, with the majority of animals (n = 7) analyzed 2 weeks after injection. PBSinjected animals that were allowed to survive 2 weeks and unmanipulated animals which did not differ in ChAT activity from PBS-injected animals were used as controls. For radioenzymatic ChAT and tyrosine hydroxylase (TOH) assays, brains were rapidly removed following sacrifice of the animals, frozen on dry ice, and stored at -80°C. Standardized dissections of the olfactory bulbs, hippocampus, striatum, and neocortex (fi'ontai/ parietal) were performed. Brain tissue was homogenized in 20 vols. of PBS, centrifuged, and the supernatants were frozen at -80°C for use in the biochemical assays. ChAT activity was measured in the olfactory bulb, hippocampus, cortex and striatum, while TOH activity was measured in the striatum, using the methods of Schrier and Schuster 2"~ and Nagatsu 2°, respectively. ChAT activity is reported as counts per minute per microgram (cpm//zg) of ~4C-labeled acetyicholine produced, whereas TOH activity is reported as cpm//u,g of 3H-labeled 3,4-dihydroxyphenylacetic acid produced. The Student's t-test was used to assess differences in ChAT and TOH activity between animals that received intraventricular injections of 192 IgG-saporin and PBS and were allowed to survive 2 weeks. For immunohistochemical analysis, 2 weeks following the intraventricular injection of either 8 p,g of 192 IgG-saporin (n = 3) or PBS (n = 2), animals were transcardially perfused with 100 ml of normal saline, followed by 250 ml of 4% buffered paraformaldehyde containing 15% picric acid and 0.1% glutaraldehyde. The brains were removed, equilibrated with 30% buffered sucrose, and 40-p,m coronal sections of forebrain and brainstem were cut using a vibratome (Lancer). Sections were processed for immunohistochemistry using a monoclonal antibody against ChAT (Boehringer Mannheim) and the localization of the primary was carried out by the avidin-biotin horse-
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Fig. 1. ChAT activity expressed as percent of control (n = 3) in homogenates of CBF terminal fields and striatum of animals that received intraventricular injections of 192 lgG-saporin (8 v,g in 24 V,I of PBS). Animals injected with immunotoxin were allowed to survive for I (n = I), 4 (n = I). 7 (n = 2), or 14 (n = 2) days.
Fig. 2. ChAT activity expressed as percent of control in homogenates of CBF terminal fields of animals that received intraventricular injections of either 192 IgG-saporin (5/~g) or OKTl-saporin (5/zg) 2 weeks previously. Control animals received intraventricular injections of PBS. All data points represent the means of duplicate animals.
radish peroxidase method using an anti-rat immunoglobulin kit (Vector Laboratories). Sections were developed using diaminobenzidine and hydrogen peroxide, mounted, dried, and coverslipped. Radioenzymatic ChAT assays showed time-dependent decreases in ChAT activity in the terminal field regions of CBF neurons following intraventricular administration of 192 lgG-saporin (Fig. 1). Compared to controls, ChAT activity was unaltered in all brain regions I day following injection of the immunotoxin. By 4 days after injection, ChAT activity was clearly reduced in the cortex and olfactory bulb, marginally reduced in the hippocampus, and still unaffected in the striatum, as compared to controls, ChAT activity was nearly maximally reduced in the cortex and olfactory bulb by 14 days after injection, and showed approxi. mately a 30% decrease in hippocampus, as compared to controls. The level of ChAT activity in the striatum was unaffected by 192 lgG-saporin at all time periods examined. Animals injected with OKTl-saporin (5/zg) showed levels of ChAT activity in the cortex, olfactory bulb, and hippocampus comparable to those obtained from PBS-injected animals (Fig. 2), ChAT activity in these regions from animals given equimolar doses of 192 igG-saporin were greatly reduced. Furthermore, ChAT activity remained at the levels shown in Fig, 2 in animals which survived for a period of 60 days (mean ChAT activity of duplicate animals, expressed as percentage of PBS-injected controls: cortex 36.9%, olfactory bulb 1.3%, hippocampus 10.2%). ChAT activity measurements from animals that received 8 v.g of 192 lgG-saporin with a 2-week post-operative survival time are summarized in Fig. 3. Data in Fig. 3 do not precisely match the points shown at 14
days in Fig. 1, because additional animals were treated with a separate preparation of immunotoxin which appears to have been slightly less effective in the olfactory bulbs. Nevertheless, all 3 CBF terminal field regions show statistically significant decreases in ChAT activity in immunotoxin-treated animals, as compared to PBS-injected controls. There was no difference in TOH activity in the striatum between animals that received up to 8/~g of 192 lgG-saporin (44.6:1:9.6 cpm//zg protein: n ,= 4) and those that received PBS (44.7 + 9.8 cpm/p.g protein', n = 3 ) . ChAT immunohistochemistry of CBF nuclei from animals that received 8 tzg of 192 IgG.saporin with a
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Fig, 3, ChAT activity expressed as percent of control (n = 5) in homogenates of CBF terminal fields and striatum of animals that received 8 Izg of 192 IgG-saporin (n = 7) and were allowed to survive 2 weeks following injections. Control animals received intraventricufar injections of PBS and were allowed to survive 2 weeks. Error bars are $.E,M, ** P < 0,01, * P < 0.05, student's t-test.
353 2-week survival time revealed a nearly complete loss of ChAT positivity in neurons of the hdB (Fig. 4B; compare with Fig. 4A) and the nbm. Variable, but at least moderate, loss of ChAT-positive neurons occurred in the msn and the vdB as compared to PBS-injected controls (data not shown). These results are in qualitative agreement with our biochemical findings, in which the most significant decrements in ChAT activity occurred in the terminal field regions of hdB and nbm (the olfactory bulb and cortex, respectively). ChAT immunohistochemistry further showed that cholinergic neurons in the striatum were unaffected by 8 ~ g of 192 IgG-saporin
This study provides further evidence that 192 IgGsaporin can selectively abolish the cholinergic parameters in the CBF, a system in which NGF receptor positivity is highly c o l o c a l i z e d with C h A T positivity ~'34
ChAT activity was significantly reduced in the cortex, o l f a c t o r y bulbs, and h i p p o c a m p u s but w a s u n a f f e c t e d
in the striatum of animals that received intraventricular 192 IgG-saporin. Striatal cholinergic neurons are NGF receptor-negative in normal adult rats 7'8'as (see below). ChAT immunohistochemistry revealed a corresponding loss o f C h A T - p o s i t i v e n e u r o n s in the h d B a n d in the
nbm, with no loss of ChAT-positive neurons in the striatum, following intraventricular 192 IgG-saporin. At equimolar doses, OKTl-saporin, an immunotoxin targeted to an antigen not present in the rat central
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Fig. 4. Photomicrographs of coronal rat brain sections processed for ChAT immunohistochemistry. All sections were processed concurrently under identical conditions. A: unremarkable distribution of cholinergic (ChAT-positive) somata in the hdB of an animal that received an intraventricular injection of PBS (24 ,¢1) and was subsequently allowed to survive for 2 weeks. B: section through the same level of the hdB of an animal that received an intraventricular injection of 192 IgO-saporin (8 ~g in 24 ~l of PBS) and was subsequently allowed to survive for 2 weeks. Positively stained neurons are completely absent. C: section through the striatum of the same animal shown in B demonstrating robust staining of striatal cholinergic interneurons. D: section through the level of the third cranial nerve nucleus of the same animal shown in B demonstrating well-stained cholinergic neurons. Bar in A, B, D = 100/+m. Bar in C -- 50 ~m. ;
354 nervous system, did not affect the cholinergic system while 192 !gG.saporin significantly reduced ChAT activity. The main advantage of using 192 IgG-saporin to produce a model of CNS cholinergic deficit therefore resides in the ability of this immunotoxin to selectively target NGF receptor-positive cholinergic neurons in the basal forebrain and concomitantly spare neighboring cholinergic and other neurons, such as the cholinergic striatal interneurons. Previous studies have shown that 30% of the cholinergic neurotransmission in the cortex is due to intrinsic, NGF receptor-negative cholinergic neurons I°. Therefore, the observed 70% reduction of ChAT activity in the neocortex seen with 8 Izg of 192 IgG-saporin represents a maximal loss of CBF input. Since the olfactory bulb has no intrinsic cholinergic neurons 28 and gets all of its input from the CBF ~5, a 100% loss is theoretically possible in this region and was nearly achieved. A less than maximal effect was achieved in the hippocampus, which correlates with the sparing in the msn that was seen with immunohistochemical methods. We are currently investigating whether higher doses, the site of injection (animals injected more anteriorly by pressure microinjection showed greater decrements in ChAT activity in the hippocampus and olfactory bulb}, or other factors may affect this immunotoxin's ability to abolish cholinergic parameters in this area of the brain. The cholinergic neurons of the striatum were consis. tently unaffected by 192 lgG-saporin at all time and dosage points investigated. Although many studies have failed to report the immunohistochemical presence of NGF receptors on adult rat striatai cholinergic neurons ~x'~5, others have observed varying amounts of weakly NGF receptor-positive neurons in adult stria. turn TM. In any case, we have previously shown that cholinergic neurons in the adult rat striatum do not accumulate 192 IgG following intraventricular administration '~''27'3°. The resting (unmanipulated) cholinergic striatal neurons, whether weakly NGF receptor.positive or not, would therefore not be expected to accumulate, nor be intoxicated by, 192 lgG-saporin. This was the observed result. The unaltered level of TOH activity in the striatum following intraventricular 192 IgG-saporin further demonstrates the specificity of this immunotoxin towards NGF receptor-positive CBF neurons. CNS care:. cholaminergic neurons are NGF receptor.negative 7. TOH, the rate-limiting enzyme in the biosynthesis of catecholamines, is a marker for both dopaminergic and adrenergic systems ~9. The assay employed 20 was not sensitive enough to detect the levels of TOH present in most basal forebrain regions (data not shown), but
TOH activity is measurable in the striatum, which has considerably more dopaminergic input than the other regions of forebrain. The observed lack of effect of 192 IgG-saporin on TOH levels in our study is therefore mainly a lack of effect on the nigrostriatal dopaminergic system. The determination of specificity of ilesioning is a complicated issue. While we have sh6wn selectivity within different cholinergic systems and with a marker for an unrelated but similar long projection system, numerous other systems remain to be investigated. Even if alterations of other systems are found, the possibility of second order (transsynaptic) alterations need to be considered. For example, the cholinergic deficit induced by intraventricular AF64A was recently shown to be associated with a transient decrease in the level of somatostatin and a transient increase in the level of neuropeptide Y in the hippocampus and parietal cortex ~. In another study, lesioning of thalamostriatal neurons by kainic acid produced a decrease in ChAT activity in the striatum that was accompanied by an increase in glutamate decarboxylase activity, highaffinity glutamate uptake, and apparent dopamine turnover 21. In both of these examples, the alterations were ascribed not to direct effects of the lesioning tool on these systems, but were interpreted as compensatory or transsynaptic effects. Though 192 IgG-saporin significantly reduced cholinergic parameters following intraventricular administration, further experimental work is needed to determine if this reduction in ChAT activity and ChAT immunopositivity is permanent and represents the actual death of CBF neurons, or if it just reflects a down-regulation in enzyme synthesis in these neurons. We have found that the decrease in ChAT activity is relatively permanent in that it persists for at least 60 days. Whether the death of cholinergic neurons has occurred is an interesting issue and several paradigms exist to assess it. There are conflicting reports in the literature concerning whether the massive loss of cholinergic neurons in the msn of the adult rat brain produced by a lesioning of the fimbria-fornix is cell death ~'2z or simple atrophy ~x24. Specific studies assessing actual cell loss in CBF nuclei following intraventricular 192 lgG-saporin are therefore indicated and include standard pathological studies on the brain and labeling neurons with a retrograde marker and subsequently assessing the effect of the immunotoxin. These studies are currently being performed. Elucidation of the specific functions of cholinergic neurons in the basal forebrain has been hampered by the previous lack of a good selective lesioning tool 4. The ability of the antibody 192 IgG to specifically
355
target saporin to CBF neurons and significantly reduce cholinergic parameters suggests 192 IgG-saporin is capable of producing a much more discrete CBF lesion than that produced by classical chemical and electrical lesioning techniques. The result is an animal model of forebrain cholinergic deficit that initially appears to be selective and may prove to be very useful in the future as a tool to probe the biochemical, physiological, and behavioral functions of the CBF. The authors gratefully acknowledge the skillful technical assistance of Ellen B. Looney. We also thank Douglas Lappi for providing the OKTl-saporin. This research has been supported in part by Grants NS25122, NS10230 and MH10121 and by the Department of Veterans Affairs Medical Center, Nashville, TN.
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