- Email: [email protected]
Basal forebrain lesions differentially alter galanin levels and acetylcholinergic receptors in the hippocampus and neocortex
Brain Research, 460 (1988) 17-21 Elsevier
17
BRE 13908
Basal forebrain lesions differentially alter galanin levels and acetylcholinergic receptors in the hippocampus and neocortex Gary L. Wenk I and/l, ke R6kaeus 2 1Department of Psychology, The Johns Hopkins University, Baltimore (U.S.A.) and 2Department of Biochemistry I, Karolinska Instituter, Stockholm (Sweden) (Accepted 29 March 1988) Key words: Galanin; Medial septal area; Nucleus basalis; Coexistence; Muscarinic receptor; Nicotinic receptor
The basal forebrain contains two subpopulations of cholinergic cells: the medial septal area (MSA) has projections to the hippocampus, while the nucleus basalis magnocellularis (NBM) has projections to the entire neocortex. In the rat, galanin-like immunoreactivity (GAL-LI) may coexist with acetylcholine (ACh) in MSA neurons but not in NBM neurons. In the monkey, GAL-LI may coexist with ACh in neurons throughout the basal forebrain. The present study investigated the differential distribution of GAL-LI within these regions by placing discrete lesions in the MSA and NBM of rats. Endogenous levels of GAL-LI were decreased in the hippocampus but not in the cortex. This differential decrease is consistent with the coexistence of GAL-LI with ACh in neurons within the MSA but not within the NBM. Markers for nicotinic and muscarinic cholinergic receptors, i.e. binding for [3H]nicotine and [3H]pirenzepine, were unchanged in the cortex and hippocampus following these lesions. This suggests that these cholinergic receptor sites do not exist upon projections originating in the NBM or MSA. These results provide new information about the similarities and differences of these two subpopulations of basal forebrain cells, which in turn may have functional ramifications.
INTRODUCTION The neuropeptide, galanin ( G A L ) , is distributed throughout the peripheral and central nervous system~9; it antagonizes the actions of acetylcholine (ACh) and substance P on smooth muscle preparations 4 and modulates the release of A C h in the hippocampus 6. GAL-like immunoreactivity (-LI) coexists with norepinephrine in the locus coeruleus, with serotonin in the raphe, and with A C h in the basal forebrain 15-17, which includes the medial septal area (MSA), vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis magnocellularis (NBM). These 3 basal forebrain regions provide cholinergic inputs to the hippocampus, cingulate cortex and entire neocortex, respectively 5, and are involved in learning and m e m o r y 24. Studies in the rat using double-labeling immunohistochemistry 17 indicate that G A L - L I coexists with a subpopulation of
cholinergic cells within the M S A and the vertical and horizontal limbs of the diagonal band of Broca but not in the NBM. In the monkey, G A L - L I is co-contained with virtually all acetylcholinesterase-reactive somata throughout the entire basal forebrain 16. The simultaneous release of G A L and A C h within the hippocampus and cortex could influence the role of ACh in mnemonic processes 16. In order to better define the role of galanin in the basal forebrain of the rat and its relationship to cholinergic projections to the hippocampus and cortex, the present study had two goals. The first goal was to confirm the differential distribution of G A L - L I within the basal forebrain by placing discrete lesions in the MSA and N B M of rats and measuring the endogenous levels of G A L - L I and C h A T activity within the hippocampus and cortex. The second goal was to determine the changes in the number of cholinergic receptors using [3H]nicotine and [3H]pirenzepine to
Correspondence: G.L. Wenk, Department of Psychology, The Johns Hopkins University, 34th & Charles St., Baltimore, MD 21218, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
18 label nicotinic and muscarinic type 1 receptors, respectively, in the cortex and hippocampus following the production of these lesions. Release of acetylcholine, and possibly galanin, from cholinergic terminals may be influenced by presynaptic cholinergic autoreceptors of the muscarinic or nicotinic type 2'9. A decrease in the density of either of these receptor types following the production of NBM and MSA lesions would be consistent with the existence of presynaptic cholinergic receptors upon afferent projections from these basal forebrain regions to the cortex and hippocampus. MATERIALS AND METHODS
Subjects Seventeen male Sprague-Dawley rats (250 g) were housed with ad libitum food and water and maintained on a 16 h:8 h light/dark cycle with lights on at 07.00 h.
Surgery Prior to surgery, each rat was given atropine methyl bromide (0.15 mg/kg, i.p., Sigma, St. Louis, MO), and pentobarbital (50 mg/kg, i.p., Sigma). When anesthetized, the rat was placed in a stereotaxic instrument with the incisor bar set so that bregma and lambda were in the same horizontal plane. The scalp was incised and retracted; holes were drilled through the skull at the appropriate locations and the lesion was made. The scalp was sutured and the rat was t h e n removed from the stereotaxic instrument and placed under a heat lamp until it awakened. NBM and MSA lesions were made with ibotenic acid (10/~g/j~l in phosphate-buffered saline, pH 7.7), injected from a Hamilton microsyringe 24. Following each injection, the syringe was left in place for 5 min to help limit diffusion. Thirteen rats received combined bilateral lesions of the NBM and MSA (NBM + MSA). NBM lesions were made by injecting 0.4/~1 of ibotenic acid at the following coordinates: 0.8 and 0.4 mm posterior to bregma, 2.8 mm lateral to the midline, and 6.9 mm ventral from the dura. MSA les i o n s w e r e m a d e by injecting 0.6/A of ibotenic acid at the following coordinates: 0.8 mm anterior to bregma, on the midline, and 5.8 mm ventral from the dura. Control rats (n = 4) received the same surgical
treatment as the lesioned rats except that the needle tip was lowered to a position 1 mm dorsal to the normal site of injection in the NBM and MSA and then withdrawn.
Biochemistry Twelve rats (4 controls, 8 NBM + MSA) were sacrificed 10 days later, and 5 rats (NBM + MSA) were sacrificed 3 months later. The brain was removed from each rat and rapidly dissected at 4 °C. Bilateral samples were taken from the entire hippocampus (150 mg) and the frontolateral cortex (25 mg), a region lateral to cingulate cortex and within the anterior sensory-motor region (cortical areas 10 and 2 according to Kriegll). The tissue samples were divided into two parts: one part was homogenized in 0.32 M sucrose and stored frozen at -80 °C until measures of ChAT activity and receptor binding were conducted; the second part was stored frozen at -80 °C and sent on dry-ice to Sweden for quantification of GAL-LI. Determinations of ChAT activity and the receptor densities were performed in triplicate; GAL-LI determinations were performed in duplicate. The protein content of each sample was assayed ~2 with bovine serum albumin as protein standard. GAL-LI levels. G A L - L I was determined as described previously 18, however, with the following modifications. Tissue samples were homogenized in 500 #1 of 0.1 N HC1 by sonication. The homogenates were centrifuged and 150/~1 of the supernatants were withdrawn and lyophilized before being assayed for GAL-LI under non-equilibrium conditions using cow G A L in standards. Choline acetyltransferase. A 40 ,ul sucrose aliquot was combined with 10 #1 of a solution containing 2% Triton X-100 and 50 mM E D T A (pH 7.4). ChAT activity was determined by standard methods s using [14C]acetyl coenzyme A (51.9 mCi/mmol, New England Nuclear, Boston, MA). [3H]Nicotine binding. [3H]Nicotinic receptors were determined according to a modified procedure of Shimohama et al. 22. An aliquot of sucrose homogenate was centrifuged at 1000 g for 10 min, and the supernatant was removed and centrifuged at 48,000 g for 15 min at 4 °C. The resultant pellet was resuspended in 0.05 M sodium phosphate buffer (pH 7.4) to provide a crude membrane preparation. [3H]Nico-
19 tine (4 nM) was incubated with the membrane preparation for 20 min at 25 °C, in the presence or absence of nicotine (100ktM). [3H]Pirenzepine binding. [3H]Pirenzepine receptors were determined according to the method of Luthin and Wolfe t3. Crude membrane preparations were incubated with [3H]pirenzepine (6 nM) for 60 min at 25 °C, in the presence or absence of atropine (I~M). RESULTS Combined NBM + MSA lesions significantly decreased ChAT activity (P < 0.01), but not levels of G A L - L I in the frontolateral cortex, and significantly decreased ChAT activity (P < 0.05) and G A L - L I (P < 0.01) in the hippocampus (Table I). The topography of cell loss and biochemical changes produced by similar lesions in the basal forebrain have been described previously 24. The lesions also decreased ChAT activity in parietal cortex (34.0 +_ 2.8 vs 23.7 _ 3.2, P < 0.05), but did not alter levels of G A L - L I significantly (29.4 _+ 2.9 vs 22.3 + 5.6). Decreased levels of hippocampal G A L - L I following combined NBM + MSA lesions were not significantly altered 3 months after the lesion (10 days, 23.6 _+ 2.4 vs 3 months, 28.9 _+ 3.1). The lesions did not significantly
TABLE
I
Effects of basal forebrain lesions upon galanin-like immunoreactivity (GAL-LI) and cholinergic markers in the cortex and hippocampus
Levels (~ + S.D.) of ChAT activity (nmol/mg prot./h) and GAL-LI (fmol/mg prot.), and the density of nicotinic and muscarinic type 1 receptors (fmol/mg prot.) in rats with combined NBM and MSA lesions. Control
N B M + MSA lesion
Cortex
ChAT GAL-LI [3H]Nicotine [3H]Pirenzepine
41.5 + 3.2 32.2 + 6.1 22.9 + 3.8
29.4 + 12.4" 25.6 + 2.7 23.9 + 2.7
663.7 + 176.8
787.4 + 119.8
46.2 35.4 18.7 737.4
31.0 25.0 15.1 744.3
Hippocampus ChAT GAL-LI [3H]Nicotine [3H]Pirenzepine
+ + + +
2.8 4.0 3.8 216.6
*P < 0.01 : **P < 0.05 vs c o n t r o l levels.
+ + + +
6.6** 3.3* 1.7 190.5
alter the number of [3H]nicotine or [3H]pirenzepine binding sites in hippocampus or cortex. DISCUSSION
The lesions destroyed cells within discrete basal forebrain areas that project to the hippocampus and cortex. The loss of cells within the MSA produced a decrease in the levels of ChAT activity and GAL-LI in the hippocampus, while the loss of cells within the NBM produced a decrease in the levels of ChAT activity, but not GAL-LI, in the cortex. The differential decrease of GAL-LI in the hippocampus, but not the cortex, indicates the coexistence of this neuropeptide with cells in the MSA but not in the NBM. These results are consistent with earlier reports in rats using double-staining immunohistochemistry that GAL-LI coexists with ACh in cells within the MSA but not with cholinergic cells within the NBM 17. However, in the monkey, where GAL-LI is also present in the NBM, a similar lesion would probably have decreased the level of GAL-L1 in the cortex 16. The functional significance of the coexistence of G A L within cholinergic projections to the hippocampus is unknown. G A L receptors have been localized to the ventral hippocampus in rats and disappear following lesions of the fimbria; stimulation of these receptors by G A L can inhibit the potassium-evoked release of ACh 6. Non-specific destruction of noradrenergic or serotonergic fibers within the MSA would have decreased the levels of G A L - L I in the hippocampus because GAL-LI is also co-localized within these neurotransmitter systems '5. However, the lesions produced here do not affect the serotonergic or noradrenergic fibers of passage m'24. The decrease in GAL-LI is therefore due entirely to the loss of cells in the MSA. NBM lesions did not alter the binding of [3H]nicotine to nicotinic receptors or [3H]pirenzepine to high affinity muscarinic type 1 receptors in the cortex or hippocampus. These data confirm earlier reports using [3H]pirenzepine14 or [3H]quinuclidinylbenzilate 23 to label muscarinic receptors in the cortex following NBM lesions produced by ibotenic acid. In contrast, NBM lesions produced by kainic acid decreased the number of [3H]pirenzepine binding sites in the frontal and parietal cortex 3. Ibotenic and kain-
20 ic acids m a y p r o d u c e different degrees of d a m a g e to a non-cholinergic p o p u l a t i o n of neurons within the N B M that project to the neocortex, and some of these neurons might possess muscarinic receptors 2. In addition, kainic acid lesions of the basal forebrain p r o d u c e d non-specific cell loss outside the injection area25; these cells might also possess [3H]pirenzepine binding sites. Presynaptic muscarinic 7 or nicotinic 2°'21 receptors may exist upon the terminals of m o n o amine projections from the m i d b r a i n or u p o n amino acid projections within the cortex to m o d u l a t e neurotransmitter release 7. F o r e x a m p l e , cholinergic projections from the diagonal b a n d of B r o c a in the basal forebrain may m o d u l a t e the activity of m e d i o d o r s a l anterior thalamic afferents to the cingulate cortex via muscarinic presynaptic receptors 1. In conclusion: (1) in the rat, the n e u r o p e p t i d e G A L is p r o b a b l y co-localized with A C h in neurons
REFERENCES 1 Andree, T.H., Gottesfeld, Z., DeFrance, J.F., Sikes, R.W. and Enna, S.J., Evidence for cholinergic muscarinic receptors on mediodorsal thalamic projections to the anterior cingulate cortex, Neurosci. Lett., 40 (1983) 99-103. 2 Briggs, C.A. and Cooper, J.R., Cholinergic modulation of the release of [3H]acetylcholine from synaptosomes of the myenteric plexus, J. Neurochem., 38 (1982) 501-508. 3 DcBelleroche, J., Gardiner, I.M., Hamilton, M.H. and Birdsall, N.J.M., Analysis of muscarinic receptor concentration and subtypes following lesion of rat substantia innominata, Brain Research, 340 (1985) 201-209. 4 Ekblad, E., H~kanson, R., Sundler, F. and Wahlstedt, C., Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations, Br. J. Pharmacol., 86 (1985) 241-246. 5 Fibiger, H.C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 6 Fisone, G., Wu, C.F., Consolo, S., NordstrOm, O., Brynne, N., Bartfai, T., Melander, T. and HOkfelt, T., Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7339-7343. 7 Flint, R.S., Murphy, J.M., Calkins, P.M. and McBride, W.J., Monoamine, amino acid and cholinergic interactions in slices of rat cerebral cortex, Brain Res. Bull., 15 (1985) 197-202. 8 Fonnum, F., Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465 -472. 9 Heydorn, W.E., Nguyen, K.Q., Creed, G.J. and Jacobowitz, D.M., Effect of reduction of cholinergic input on the concentration of specific proteins in different cortical regions of the rat brain, Brain Research, 339 (1985) 209-218. -
within the M S A but not within the N B M ; and (2) muscarinic type 1 and nicotinic cholinergic receptors are not located on the terminals of axonal projections from the M S A or N B M , but on either postsynaptic m e m b r a n e s or on projections that originate outside the basal forebrain region. ACKNOWLEDGEMENTS The authors thank D. Oiton for critically reading the manuscript, D. Pierce and B. Cribbs for excellent technical assistance and D. Harris for typing the manuscript. This research was s u p p o r t e d by funds from the U.S. Public H e a l t h Service (Grants N I H A G 05146 and NS 20471), Karolinska Institute, Magnus Bergvall's F o u n d a t i o n , Swedish Medical Research Council (Projects 13X-07906, 131-07906) and Swedish Society of Medicine.
10 Johnston, M.V., McKinney, M. and Coyle, J.T., Evidence for a cholinergic projection to neocortex from neurons in basal forebrain, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5392-5396. 11 Krieg, W.J.S., Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas, J. Comp. Neurol., 84 (1946) 221-275. 12 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 Luthin, G.R. and Wolfe, B.B., Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain, J. Pharmacol. Exp. Ther., 228 (1984) 648-655. 14 Mash, D.C., Flynn, D.D. and Potter, L.T., Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation, Science, 228 (1985) 115-117. 15 Melander, T., HOkfelt, T., ROkaeus, A., Cuello, A.C., Oertel, W.H., Verhofstad, A. and Goldstein, M., Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS, J. Neurosci., 6 (1986) 3640-3654. 16 Melander, T. and Staines, W.A., A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocampus in the owl monkey (Aotus trivirgatus), Neurosci. Leg., 68 (1986) 17-22. 17 Melander, T., Staines, W.A., HOkfelt, T., ROkaeus, A., Eckenstein, F., Salvaterra, P.M. and Wainer, B.H., Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat, Brain Research, 360 (1985) 130-138. 18 Palkovits, M., ROkaeus, A., Antoni, F.A. and Kiss, A., Galanin in the hypothalamo-hypophyseal system, Neuroendocrinology, 46 (1987) 417-423.
21 19 R6kaeus, .~., Galanin: a newly isolated biologically active neuropeptide, Trends Neurosci., 10 (1987) 158-164. 20 Rowell, P.P. and Winkler, D.L., Nicotinic stimulation of [3H]acetylcholine release from mouse cerebral cortical synaptosomes, J. Neurochem., 43 (1984) 1593-1598. 21 Schwartz, R.D., Lehmann, J. and Kellar, K.J., Presynaptic nicotinic cholinergic receptors labeled by [3H]aeetylcholine on catecholamine and serotonin axons in brain, J. Neurochem., 42 (1984) 1495-1498. 22 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia, J. Neurochem., 46 (1986) 288-293.
23 Watson, M., Vickroy, T.W., Fibiger, H.C., Roeske, W.R. and Yamamura, H.I., Effects of bilateral ibotenate-induced lesions of nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex, Brain Research, 340 (1985) 387-391. 24 Wenk, G., Hughey, D., Boundy, V., Kim, A., Walker, L. and Olton, D., Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems, Behav. Neurosci., 101 (1987) 325-332. 25 Zaczek, R., Simonton, S. and Coyle, J.T., Local and distant neuronal degeneration following intrastriatal injection of kainic acid, J. Neuropathol. Exp. Neurol., 39 (1980) 245-264.
17
BRE 13908
Basal forebrain lesions differentially alter galanin levels and acetylcholinergic receptors in the hippocampus and neocortex Gary L. Wenk I and/l, ke R6kaeus 2 1Department of Psychology, The Johns Hopkins University, Baltimore (U.S.A.) and 2Department of Biochemistry I, Karolinska Instituter, Stockholm (Sweden) (Accepted 29 March 1988) Key words: Galanin; Medial septal area; Nucleus basalis; Coexistence; Muscarinic receptor; Nicotinic receptor
The basal forebrain contains two subpopulations of cholinergic cells: the medial septal area (MSA) has projections to the hippocampus, while the nucleus basalis magnocellularis (NBM) has projections to the entire neocortex. In the rat, galanin-like immunoreactivity (GAL-LI) may coexist with acetylcholine (ACh) in MSA neurons but not in NBM neurons. In the monkey, GAL-LI may coexist with ACh in neurons throughout the basal forebrain. The present study investigated the differential distribution of GAL-LI within these regions by placing discrete lesions in the MSA and NBM of rats. Endogenous levels of GAL-LI were decreased in the hippocampus but not in the cortex. This differential decrease is consistent with the coexistence of GAL-LI with ACh in neurons within the MSA but not within the NBM. Markers for nicotinic and muscarinic cholinergic receptors, i.e. binding for [3H]nicotine and [3H]pirenzepine, were unchanged in the cortex and hippocampus following these lesions. This suggests that these cholinergic receptor sites do not exist upon projections originating in the NBM or MSA. These results provide new information about the similarities and differences of these two subpopulations of basal forebrain cells, which in turn may have functional ramifications.
INTRODUCTION The neuropeptide, galanin ( G A L ) , is distributed throughout the peripheral and central nervous system~9; it antagonizes the actions of acetylcholine (ACh) and substance P on smooth muscle preparations 4 and modulates the release of A C h in the hippocampus 6. GAL-like immunoreactivity (-LI) coexists with norepinephrine in the locus coeruleus, with serotonin in the raphe, and with A C h in the basal forebrain 15-17, which includes the medial septal area (MSA), vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis magnocellularis (NBM). These 3 basal forebrain regions provide cholinergic inputs to the hippocampus, cingulate cortex and entire neocortex, respectively 5, and are involved in learning and m e m o r y 24. Studies in the rat using double-labeling immunohistochemistry 17 indicate that G A L - L I coexists with a subpopulation of
cholinergic cells within the M S A and the vertical and horizontal limbs of the diagonal band of Broca but not in the NBM. In the monkey, G A L - L I is co-contained with virtually all acetylcholinesterase-reactive somata throughout the entire basal forebrain 16. The simultaneous release of G A L and A C h within the hippocampus and cortex could influence the role of ACh in mnemonic processes 16. In order to better define the role of galanin in the basal forebrain of the rat and its relationship to cholinergic projections to the hippocampus and cortex, the present study had two goals. The first goal was to confirm the differential distribution of G A L - L I within the basal forebrain by placing discrete lesions in the MSA and N B M of rats and measuring the endogenous levels of G A L - L I and C h A T activity within the hippocampus and cortex. The second goal was to determine the changes in the number of cholinergic receptors using [3H]nicotine and [3H]pirenzepine to
Correspondence: G.L. Wenk, Department of Psychology, The Johns Hopkins University, 34th & Charles St., Baltimore, MD 21218, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
18 label nicotinic and muscarinic type 1 receptors, respectively, in the cortex and hippocampus following the production of these lesions. Release of acetylcholine, and possibly galanin, from cholinergic terminals may be influenced by presynaptic cholinergic autoreceptors of the muscarinic or nicotinic type 2'9. A decrease in the density of either of these receptor types following the production of NBM and MSA lesions would be consistent with the existence of presynaptic cholinergic receptors upon afferent projections from these basal forebrain regions to the cortex and hippocampus. MATERIALS AND METHODS
Subjects Seventeen male Sprague-Dawley rats (250 g) were housed with ad libitum food and water and maintained on a 16 h:8 h light/dark cycle with lights on at 07.00 h.
Surgery Prior to surgery, each rat was given atropine methyl bromide (0.15 mg/kg, i.p., Sigma, St. Louis, MO), and pentobarbital (50 mg/kg, i.p., Sigma). When anesthetized, the rat was placed in a stereotaxic instrument with the incisor bar set so that bregma and lambda were in the same horizontal plane. The scalp was incised and retracted; holes were drilled through the skull at the appropriate locations and the lesion was made. The scalp was sutured and the rat was t h e n removed from the stereotaxic instrument and placed under a heat lamp until it awakened. NBM and MSA lesions were made with ibotenic acid (10/~g/j~l in phosphate-buffered saline, pH 7.7), injected from a Hamilton microsyringe 24. Following each injection, the syringe was left in place for 5 min to help limit diffusion. Thirteen rats received combined bilateral lesions of the NBM and MSA (NBM + MSA). NBM lesions were made by injecting 0.4/~1 of ibotenic acid at the following coordinates: 0.8 and 0.4 mm posterior to bregma, 2.8 mm lateral to the midline, and 6.9 mm ventral from the dura. MSA les i o n s w e r e m a d e by injecting 0.6/A of ibotenic acid at the following coordinates: 0.8 mm anterior to bregma, on the midline, and 5.8 mm ventral from the dura. Control rats (n = 4) received the same surgical
treatment as the lesioned rats except that the needle tip was lowered to a position 1 mm dorsal to the normal site of injection in the NBM and MSA and then withdrawn.
Biochemistry Twelve rats (4 controls, 8 NBM + MSA) were sacrificed 10 days later, and 5 rats (NBM + MSA) were sacrificed 3 months later. The brain was removed from each rat and rapidly dissected at 4 °C. Bilateral samples were taken from the entire hippocampus (150 mg) and the frontolateral cortex (25 mg), a region lateral to cingulate cortex and within the anterior sensory-motor region (cortical areas 10 and 2 according to Kriegll). The tissue samples were divided into two parts: one part was homogenized in 0.32 M sucrose and stored frozen at -80 °C until measures of ChAT activity and receptor binding were conducted; the second part was stored frozen at -80 °C and sent on dry-ice to Sweden for quantification of GAL-LI. Determinations of ChAT activity and the receptor densities were performed in triplicate; GAL-LI determinations were performed in duplicate. The protein content of each sample was assayed ~2 with bovine serum albumin as protein standard. GAL-LI levels. G A L - L I was determined as described previously 18, however, with the following modifications. Tissue samples were homogenized in 500 #1 of 0.1 N HC1 by sonication. The homogenates were centrifuged and 150/~1 of the supernatants were withdrawn and lyophilized before being assayed for GAL-LI under non-equilibrium conditions using cow G A L in standards. Choline acetyltransferase. A 40 ,ul sucrose aliquot was combined with 10 #1 of a solution containing 2% Triton X-100 and 50 mM E D T A (pH 7.4). ChAT activity was determined by standard methods s using [14C]acetyl coenzyme A (51.9 mCi/mmol, New England Nuclear, Boston, MA). [3H]Nicotine binding. [3H]Nicotinic receptors were determined according to a modified procedure of Shimohama et al. 22. An aliquot of sucrose homogenate was centrifuged at 1000 g for 10 min, and the supernatant was removed and centrifuged at 48,000 g for 15 min at 4 °C. The resultant pellet was resuspended in 0.05 M sodium phosphate buffer (pH 7.4) to provide a crude membrane preparation. [3H]Nico-
19 tine (4 nM) was incubated with the membrane preparation for 20 min at 25 °C, in the presence or absence of nicotine (100ktM). [3H]Pirenzepine binding. [3H]Pirenzepine receptors were determined according to the method of Luthin and Wolfe t3. Crude membrane preparations were incubated with [3H]pirenzepine (6 nM) for 60 min at 25 °C, in the presence or absence of atropine (I~M). RESULTS Combined NBM + MSA lesions significantly decreased ChAT activity (P < 0.01), but not levels of G A L - L I in the frontolateral cortex, and significantly decreased ChAT activity (P < 0.05) and G A L - L I (P < 0.01) in the hippocampus (Table I). The topography of cell loss and biochemical changes produced by similar lesions in the basal forebrain have been described previously 24. The lesions also decreased ChAT activity in parietal cortex (34.0 +_ 2.8 vs 23.7 _ 3.2, P < 0.05), but did not alter levels of G A L - L I significantly (29.4 _+ 2.9 vs 22.3 + 5.6). Decreased levels of hippocampal G A L - L I following combined NBM + MSA lesions were not significantly altered 3 months after the lesion (10 days, 23.6 _+ 2.4 vs 3 months, 28.9 _+ 3.1). The lesions did not significantly
TABLE
I
Effects of basal forebrain lesions upon galanin-like immunoreactivity (GAL-LI) and cholinergic markers in the cortex and hippocampus
Levels (~ + S.D.) of ChAT activity (nmol/mg prot./h) and GAL-LI (fmol/mg prot.), and the density of nicotinic and muscarinic type 1 receptors (fmol/mg prot.) in rats with combined NBM and MSA lesions. Control
N B M + MSA lesion
Cortex
ChAT GAL-LI [3H]Nicotine [3H]Pirenzepine
41.5 + 3.2 32.2 + 6.1 22.9 + 3.8
29.4 + 12.4" 25.6 + 2.7 23.9 + 2.7
663.7 + 176.8
787.4 + 119.8
46.2 35.4 18.7 737.4
31.0 25.0 15.1 744.3
Hippocampus ChAT GAL-LI [3H]Nicotine [3H]Pirenzepine
+ + + +
2.8 4.0 3.8 216.6
*P < 0.01 : **P < 0.05 vs c o n t r o l levels.
+ + + +
6.6** 3.3* 1.7 190.5
alter the number of [3H]nicotine or [3H]pirenzepine binding sites in hippocampus or cortex. DISCUSSION
The lesions destroyed cells within discrete basal forebrain areas that project to the hippocampus and cortex. The loss of cells within the MSA produced a decrease in the levels of ChAT activity and GAL-LI in the hippocampus, while the loss of cells within the NBM produced a decrease in the levels of ChAT activity, but not GAL-LI, in the cortex. The differential decrease of GAL-LI in the hippocampus, but not the cortex, indicates the coexistence of this neuropeptide with cells in the MSA but not in the NBM. These results are consistent with earlier reports in rats using double-staining immunohistochemistry that GAL-LI coexists with ACh in cells within the MSA but not with cholinergic cells within the NBM 17. However, in the monkey, where GAL-LI is also present in the NBM, a similar lesion would probably have decreased the level of GAL-L1 in the cortex 16. The functional significance of the coexistence of G A L within cholinergic projections to the hippocampus is unknown. G A L receptors have been localized to the ventral hippocampus in rats and disappear following lesions of the fimbria; stimulation of these receptors by G A L can inhibit the potassium-evoked release of ACh 6. Non-specific destruction of noradrenergic or serotonergic fibers within the MSA would have decreased the levels of G A L - L I in the hippocampus because GAL-LI is also co-localized within these neurotransmitter systems '5. However, the lesions produced here do not affect the serotonergic or noradrenergic fibers of passage m'24. The decrease in GAL-LI is therefore due entirely to the loss of cells in the MSA. NBM lesions did not alter the binding of [3H]nicotine to nicotinic receptors or [3H]pirenzepine to high affinity muscarinic type 1 receptors in the cortex or hippocampus. These data confirm earlier reports using [3H]pirenzepine14 or [3H]quinuclidinylbenzilate 23 to label muscarinic receptors in the cortex following NBM lesions produced by ibotenic acid. In contrast, NBM lesions produced by kainic acid decreased the number of [3H]pirenzepine binding sites in the frontal and parietal cortex 3. Ibotenic and kain-
20 ic acids m a y p r o d u c e different degrees of d a m a g e to a non-cholinergic p o p u l a t i o n of neurons within the N B M that project to the neocortex, and some of these neurons might possess muscarinic receptors 2. In addition, kainic acid lesions of the basal forebrain p r o d u c e d non-specific cell loss outside the injection area25; these cells might also possess [3H]pirenzepine binding sites. Presynaptic muscarinic 7 or nicotinic 2°'21 receptors may exist upon the terminals of m o n o amine projections from the m i d b r a i n or u p o n amino acid projections within the cortex to m o d u l a t e neurotransmitter release 7. F o r e x a m p l e , cholinergic projections from the diagonal b a n d of B r o c a in the basal forebrain may m o d u l a t e the activity of m e d i o d o r s a l anterior thalamic afferents to the cingulate cortex via muscarinic presynaptic receptors 1. In conclusion: (1) in the rat, the n e u r o p e p t i d e G A L is p r o b a b l y co-localized with A C h in neurons
REFERENCES 1 Andree, T.H., Gottesfeld, Z., DeFrance, J.F., Sikes, R.W. and Enna, S.J., Evidence for cholinergic muscarinic receptors on mediodorsal thalamic projections to the anterior cingulate cortex, Neurosci. Lett., 40 (1983) 99-103. 2 Briggs, C.A. and Cooper, J.R., Cholinergic modulation of the release of [3H]acetylcholine from synaptosomes of the myenteric plexus, J. Neurochem., 38 (1982) 501-508. 3 DcBelleroche, J., Gardiner, I.M., Hamilton, M.H. and Birdsall, N.J.M., Analysis of muscarinic receptor concentration and subtypes following lesion of rat substantia innominata, Brain Research, 340 (1985) 201-209. 4 Ekblad, E., H~kanson, R., Sundler, F. and Wahlstedt, C., Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations, Br. J. Pharmacol., 86 (1985) 241-246. 5 Fibiger, H.C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 6 Fisone, G., Wu, C.F., Consolo, S., NordstrOm, O., Brynne, N., Bartfai, T., Melander, T. and HOkfelt, T., Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7339-7343. 7 Flint, R.S., Murphy, J.M., Calkins, P.M. and McBride, W.J., Monoamine, amino acid and cholinergic interactions in slices of rat cerebral cortex, Brain Res. Bull., 15 (1985) 197-202. 8 Fonnum, F., Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465 -472. 9 Heydorn, W.E., Nguyen, K.Q., Creed, G.J. and Jacobowitz, D.M., Effect of reduction of cholinergic input on the concentration of specific proteins in different cortical regions of the rat brain, Brain Research, 339 (1985) 209-218. -
within the M S A but not within the N B M ; and (2) muscarinic type 1 and nicotinic cholinergic receptors are not located on the terminals of axonal projections from the M S A or N B M , but on either postsynaptic m e m b r a n e s or on projections that originate outside the basal forebrain region. ACKNOWLEDGEMENTS The authors thank D. Oiton for critically reading the manuscript, D. Pierce and B. Cribbs for excellent technical assistance and D. Harris for typing the manuscript. This research was s u p p o r t e d by funds from the U.S. Public H e a l t h Service (Grants N I H A G 05146 and NS 20471), Karolinska Institute, Magnus Bergvall's F o u n d a t i o n , Swedish Medical Research Council (Projects 13X-07906, 131-07906) and Swedish Society of Medicine.
10 Johnston, M.V., McKinney, M. and Coyle, J.T., Evidence for a cholinergic projection to neocortex from neurons in basal forebrain, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5392-5396. 11 Krieg, W.J.S., Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas, J. Comp. Neurol., 84 (1946) 221-275. 12 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 Luthin, G.R. and Wolfe, B.B., Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain, J. Pharmacol. Exp. Ther., 228 (1984) 648-655. 14 Mash, D.C., Flynn, D.D. and Potter, L.T., Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation, Science, 228 (1985) 115-117. 15 Melander, T., HOkfelt, T., ROkaeus, A., Cuello, A.C., Oertel, W.H., Verhofstad, A. and Goldstein, M., Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS, J. Neurosci., 6 (1986) 3640-3654. 16 Melander, T. and Staines, W.A., A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complex and in acetylcholinesterase-containing fibers and varicosities within the hippocampus in the owl monkey (Aotus trivirgatus), Neurosci. Leg., 68 (1986) 17-22. 17 Melander, T., Staines, W.A., HOkfelt, T., ROkaeus, A., Eckenstein, F., Salvaterra, P.M. and Wainer, B.H., Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat, Brain Research, 360 (1985) 130-138. 18 Palkovits, M., ROkaeus, A., Antoni, F.A. and Kiss, A., Galanin in the hypothalamo-hypophyseal system, Neuroendocrinology, 46 (1987) 417-423.
21 19 R6kaeus, .~., Galanin: a newly isolated biologically active neuropeptide, Trends Neurosci., 10 (1987) 158-164. 20 Rowell, P.P. and Winkler, D.L., Nicotinic stimulation of [3H]acetylcholine release from mouse cerebral cortical synaptosomes, J. Neurochem., 43 (1984) 1593-1598. 21 Schwartz, R.D., Lehmann, J. and Kellar, K.J., Presynaptic nicotinic cholinergic receptors labeled by [3H]aeetylcholine on catecholamine and serotonin axons in brain, J. Neurochem., 42 (1984) 1495-1498. 22 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia, J. Neurochem., 46 (1986) 288-293.
23 Watson, M., Vickroy, T.W., Fibiger, H.C., Roeske, W.R. and Yamamura, H.I., Effects of bilateral ibotenate-induced lesions of nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex, Brain Research, 340 (1985) 387-391. 24 Wenk, G., Hughey, D., Boundy, V., Kim, A., Walker, L. and Olton, D., Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems, Behav. Neurosci., 101 (1987) 325-332. 25 Zaczek, R., Simonton, S. and Coyle, J.T., Local and distant neuronal degeneration following intrastriatal injection of kainic acid, J. Neuropathol. Exp. Neurol., 39 (1980) 245-264.