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Galanin microinjected into the medial septum inhibits scopolamine-induced acetylcholine overflow in the rat ventral hippocampus
BRAIN RESEARCH ELSEVIER
Brain Research 709 (1996) 81-87
Research report
Galanin microinjected into the medial septum inhibits scopolamine-induced acetylcholine overflow in the rat ventral hippocampus John K. Robinson a, *, Alessandro Zocchi b, Agu Pert b, Jacqueline N. Crawley a a Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Bethesda, MD 20892, USA b Unit on Behavioral Pharmacology, Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892, USA Accepted 11 October 1995
Abstract
Galanin-like immunoreactive terminals hyperinnervate the basal forebrain cholinergic neurons in Alzheimer's disease. To investigate the hypothesis that galanin acts directly on basal forebrain cell bodies, in vivo microdialysis studies were conducted in awake rats which analyzed the actions of galanin on acetylcholine release. Microinjection of galanin into the cholinergic cell body region of the medial septum-diagonal band (MS-I)BB) inhibited acetylcholine release in the ventral hippocampus. These results are consistent with an interpretation that galanin teiminals synapsing on cholinergic cell bodies of the basal forebrain may serve to inhibit the release of acetylcholine in the terminal fields of the cholinergic neurons. Keywords: Activity; Receptor antagonist; Behavior; Microdialysis; Neuropeptide
1. Introduction
The cholinergic septohippocampal pathway is thought to play an important modulatory role in hippocampal activity [8], especially activity related to learning and memory [7]. Lesions of the septo-hippocampal pathway have been shown to disrupt performance in many learning and memory paradigms 1120]. Release of acetylcholine (ACh) in the hippocampus is temporally associated with increased power of the hippocampal theta rhythm [18], thought to be critical for the phenomenon of long-term potentiation in hippocampus [12]. A number of neuropeptiides are localized in the medial septum-diagonal band of Broca (MS-DBB), including somatostatin, calcitonin gene-related peptide, cholecystokinin, dynorphin-A, Met-enkephalin, and galanin [11]. Galanin (GAL) is the most: abundant of the neuropeptides in this region [11], and is the only neuropeptide that coexists with ACh in the rat. GAL-like immunoreactivity
* Corresponding author, at Present address: Department of Psychology, State University of New York at :Stony Brook, Stony Brook, NY 117942500, USA. Fax: (1) ( 5 1 6 ) 632-7876; e-mail: [email protected] Elsevier Science B.V. SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 3 0 7 - 5
is seen in about 30% of the medial septum-diagonal band cell bodies and projections to the ventral hippocampus [151. The coexistence of GAL with ACh in the MS-DBB, projecting to the ventral hippocampus is the best available model system in rodents for studying the functional interactions between galanin and acetylcholine. GAL administered intraventricularly to awake rats has been shown to block ACh release stimulated by muscarinic receptor antagonist, scopolamine, in ventral hippocampus [3,5]. GAL had no significant effect on baseline ACh release. In the dorsal hippocampus, which contains a much lower expression of galanin receptors, intraventricularly administered galanin had no effect on scopolamine-stimulated ACh release [5]. The site of action at which GAL inhibits ACh release has not been identified. GAL administered into the MSDBB of rats has been shown to simultaneously disrupt T-maze delayed alternation performance and the hippocampal theta rhythm [6]. Further, GAL administered into the MS-DBB potentiated scopolamine-induced disruption of an operant delayed non-matching-to-sample task [24]. Taken together, these results suggest that galanin may act on medial septum cell bodies to exert its inhibitory role in memory processes.
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J.K. Robinsonet al. /Brain Research 709 (1996)81-87
The present experiments were conducted to determine whether galanin microinjected into the MS-DBB influences hippocampal ACh release. The previously published experiments [3,5] that showed that intraventricularly injected galanin reduces ventral hippocampal ACh release, were replicated, and then compared with the effects of galanin microinjected into the MS-DBB of awake, behaving rats.
2. Materials and methods
2.1. Subjects and surgery The subjects were male Sprague-Dawley rats, 300-350 g at time of surgery. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and approved by the NIMH Animal Care and Use Committee. Rats were anesthetized with chloral hydrate anesthesia, and under aseptic conditions, implanted with a C M A / 1 2 microdialysis probe guide with a stainless-steel stylet (Carnegie Medicin, Stockholm, Sweden) into the ventral hippocampus (5.5 mm posterior to Bregma, 5.1 mm ventral to surface of the skull, and 5.0 mm lateral to the midline). In addition, rats were implanted with a guide cannula made of stainless steel hypodermic tubing either into the lateral ventricle (0.5 mm posterior to Bregma, 3.5 mm ventral to surface of the skull, 1.0 mm lateral to the midline) or into the MS-DBB (0.7 mm anterior to Bregma, 5.6 mm ventral to surface of the skull, 1.5 mm lateral to the midline at a 15 degree angle from vertical), after Paxinos and Watson [22]. A dental cement cap held the cannulae in place, and four screws anchored the cap into the skull. The subjects were allowed one week of recovery before the start of testing.
apparatus and handling during the sampling procedures. Three baseline samples were then collected, prior to the MS-DBB injection. Typically, eight post-injection samples were collected. Initially, a pilot study was conducted in which three doses of scopolamine (0.1, 0.5, and 1.0 m g / k g i.p.) were tested, to determine an optimal intermediate dose for the subsequent experiments. Galanin (Rat GAL 1-29, Bachem Bioscience, Philadelphia, PA) was dissolved in Ringer's solution, and injected in a volume of 5.0 4xl over 25 s for intraventricular injection, and injected in a 0.5/xl volume over 57 s for the MS-DBB microinjections, through a 31-gauge, 1.9 cm stainless steel injector, extending 0.2 cm ventral to the implanted guide cannula, using a Sage microinfusion pump (Orion Instruments, Cambridge, MA). For both injection routes, the injector was left in place for an additional one min before removal and insertion of the stylet. Vehicle control subjects received only Ringer's solution. Scopolamine hydrobromide was dissolved in saline and administered i.p. immediately following the MS-DBB microinjection. 2.3. Quantitation of acetylcholine concentrations The analytical system consisted of a Bioanalytical Systems (West Lafayette, IN) high pressure liquid chromatograph, equipped with an LC-4B detector. The potential was set at +500 mV on a platinum working electrode versus a Ag/AgC1 reference electrode. BAS MF-6150 analytical column cartridge, and a BAS MF-6151 immobilized enzyme reactor cartridge were used. Mobile phase (35 mM Na2PO4, pH adjusted to 8.5 with H3PO4, 0.05% antibacterial (Kathon, Bioanalytical Systems, West Lafayette, IN), prepared fresh weekly, was pumped at a flow rate of 0.6 ml/min.
2.2. Experimental procedures 2.4. Locomotor activity At approximately 18 h prior to the start of the experiment, the stylet from the hippocampal microdialysis cannula was removed and a C M A / 1 2 microdialysis probe with a 3 mm membrane that extended 3 mm from the implanted cannula. On the day of the experiment, the animal was placed in a plexiglass cylinder inside a Digiscan activity monitor (Omnitech Electronics, Columbus, OH). Tubing was attached by a counterbalance arm and swivel (Harvard Scientific, Cambridge, MA) to permit free movement. The probe was perfused with artificial cerebrospinal fluid (NaCI 147 mM, KCI 4.0 mM, CaCI 2 1.3 mM, and 1 /xM neostigmine bromide (Sigma, St. Louis, MO)) at a rate of 0.5/xl/min. 10-/xl samples were collected every 20 min and injected into the HPLC immediately for ACh analysis. The first four samples taken from the subject were discarded. This time period represented habituation to the
Three Digiscan locomotor activity parameters were quantitated at 20 min intervals. The measure of horizontal movements incorporated both exploration and static repetitive motion such as grooming. The vertical measure primarily assessed rearings. The ambulatory parameter recorded successive interruptions of two photobeams, and was most representative of spontaneous exploratory activity. The measurements were recorded immediately following collection of the perfusate sample. 2.5. Data analysis ANOVAs and posthoc testing was conducted using SuperanovaT M (v.l.1) statistical software. The calculation of percent of baseline ACh release from peak height was determined by comparison to the mean peak heights of three pre-injection samples of ACh standards (1.0 /zM)
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
83
within each experimental day. ANOVAs were conducted on post-injection samples of ACh and Digiscan activity parameters.
from subjects with incorrect placements were removed from further analysis.
2.6. Histology
3. Results
At the completion of the experiment, subjects were decapitated under chloral hydrate anesthesia. The brains were fixed in 10% formalin, sectioned, and stained with thionin. The microinjection cannula placements and the microdialysis probe placements were then evaluated. Data
Locations of microinjection and microdialysis sites from the subjects included in the data analyses are shown in Fig. 1. Fig. 1A shows a representative MS-DBB placement. Fig. 1C summarizes the locations of placements at the MS-DBB region. Fig. 1B shows a representative ventral
A
Fig. 1. Microinjectioncannula and microdialysisprobe placements.Photomicrograph(A) and reconstructionof injectionsites (C) in medial septum/diagonal band of Broca. Photomicrograph(B) and reconstructionof tip of microdialysisprobe sites (D) in ventral hippocampus.
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
84
Table 1 Effects of scopolamine (0.1-1.0 m g / k g i.p.) on acetylcholine release Treatment
Rat
Acetylcholine (pmol/sample)
no.
Baseline 1
No-injection
1 2 3 4 5 6 7 8
Scop 0.1 Scop 0.5 Scop 1.0
0.26 0.15 0.07 0.63 0.11 0.37 0.62 0.23
Post-injection 2
3
0.23 0.11 0.07 0.63 0.09 0.37 0.62 0.13
0.23 0.07 0.04 0.47 0.19 0.30 0.62 0.17
1
0.23 0.11 0.07 0.58 0.09 0.37 1.65 0.30
2
0.15 0.07 0.07 0.84 0.60 0.52 5.70 0.67
3
4
5
6
7
8
0.23 nd 0.04 0.63 0.91 3.50 5.10 0.67
0.23 nd 0.07 1.32 0.67 2.70 3.00 0.63
nd nd 0.04 0.32 0.45 1.60 2.20 0.23
nd nd 0.08 0.26 0.48 1.30 1.60 0.23
nd nd 0.08 0.21 0.17 0.86 2.00 0.27
nd nd 0.04 0.21 0.13 0.34 1.50 0.13
Data from individual animals at each dose are shown, nd, not determined.
hippocampus cannula placement. Fig. 1D summarizes the locations of placements at the ventral hippocampus site. Eleven rats were excluded because a substantial part of the dialysis membrane was outside the region of the ventral hippocampus. An additional three rats showed extensive tissue damage in the region surrounding the dialysis membrane and were excluded. MS-DBB microinjection cannula placements were determined to be correct in all subjects with correct microdialysis probe placement. Table 1 shows the effects of scopolamine administered i.p. on ACh overflow in the ventral hippocampus. Data from two subjects at each dose were obtained. These pilot data suggest that the 0.5 m g / k g and 1.0 m g / k g doses produce comparable responses, while in contrast, the 0.1 m g / k g dose did not produce a consistent increase in ACh
GALANIN (ICV) + SCOPOLAMINE (LP.)
overflow. Based upon these pilot subjects, an intermediate 0.3 m g / k g dose of scopolamine was chosen for subsequent experiments. Fig. 2 shows the effects of galanin (1.6 nmol) administered i.c.v, on ACh overflow in the ventral hippocampus induced by scopolamine (0.3 m g / k g i.p.). ANOVA of the post-injection samples revealed a significant main effect of t r e a t m e n t (F1, 4 -- 8.3, P < 0.05), sample (F7,28 = 7.5, P < 0.0001), and a significant treatment × sample interaction (F7,28 = 4.4, P < 0.002). Because this interaction was significant, individual sample numbers were analyzed by paired contrasts, revealing that the galanin group differed from the Ringer's solution group at the f o u r t h (F1, 4 = 10.2, P < 0.04) and fifth (F1, 4 = 7.8, P < 0 . 0 5 ) post-injection samples. Fig. 3 shows the effects of galanin (0.025, 0.4, and 1.6 nmol) administered into the MS-DBB on ACh overflow in the ventral hippocampus induced by scopolamine (0.3
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Fig. 2. Intraventricular galanin (1.6 nmol) blocked scopolamine (0.3 m g / k g i.p.)-induced acetylcholine overflow in the ventral hippocampus of awake rats. Data in Fig. 2Fig. 3Fig. 4 are shown as mean + S.E.M, Number of subjects per treatment group is shown in parentheses. GAL, rat galanin 1-29; RING, Ringer's solution vehicle controls. * P < 0.05, comparison of Ringer's solution controls and GAL.
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Fig. 3. Galanin (0.025-1.6 nmol) microinjected into medial septum/diagonal band of Broca blocked scopolamine-induced acetylcholine overflow in the ventral hippocampus of awake rats. * P < 0.05, comparison of Ringer's solution controls and GAL doses.
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
Ambulation 600"
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revealed no significant effects of dose of galanin administered into the medial septum, but did show significant dose × sample interactions for horizontal activity (F3,30 = 1.7, P < 0.03), ambulations (F3,30 = 2.2, P < 0.002), and approached significance for vertical rearings (/73,30 = 1.5, P < 0.08). Because these dose × sample interactions were significant, Dunnett's posthoc comparisons were conducted at each post-injection sample. Significant effects ( P < 0.05) were detected at the first (0.4 nmol dose), second (0.4, 1.6 nmol doses) and fourth (0.4, 1.6 nmol doses) post-injection sample for the ambulation measure, the fifth (0.025, 0.4 nmol doses) post-injection sample for the vertical measure, and the fourth (0.4, 1.6 nmol doses) and fifth (0.025, 0.4 nmol doses) post-injection sample for the horizontal measure.
100"
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4. Discussion
60"
0
20
40
60
80
100
120
140
160
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Horizontal 3000'
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Fig. 4. Galanin (0.025-1.6 nmol), microinjected into the medial septum/diagonal band of Broca, reduced scopolamine-inducedexploratory activityduring microdialysisas shown on three measures of behavioral activityin a Digiscanopen field. m g / k g i.p.). ANOVA of the post-injection samples showed a significant main effect of treatment (F3,11 = 3.6, P < 0.05), a significant main effect of sample (F7,77 = 3.4, P < 0.003), and a significant treatment × sample interaction (F21,77 = 2.9, P < 0.0004). Dunnett's posthoc comparisons conducted at each post-injection sample were significant (P < 0.05) for all doses of GAL at the second and third post-injection samples. Fig. 4 shows the effects of GAL microinjected into the MS-DBB on exploratory locomotor activity induced by scopolamine (0.3 m g / k g i.p.). ANOVA of the post-injection periods for horizontal activity (F3.11 = 2.9), vertical rearings (F3,11 = 1.1), and ambulatory activity (F3,11 = 2.5)
The present experiments showed that GAL administered into the MS-DBB cell body region potently blocked scopolamine-stimulated ACh overflow in the ventral hippocampus. Galanin (1.6 nmol) administered i.c.v, attenuated scopolamine-stimulated ACh overflow in ventral hippocampus, demonstrating that data from the present experiments are consistent with those reported by other laboratories [3,5]. Behavioral studies have shown that GAL is disruptive of memory-task performance, supporting an inhibitory functional role of GAL in the septohippocampal pathway. When microinjected i.c.v., GAL disrupted the acquisition of the Morris water maze [26] and the radial maze [13]. GAL given i.c.v, or intrahippocampally also attenuated improvement in T-maze delayed alternation performance in basal-forebrain lesioned rats produced by acetylcholine [14]. GAL administered into the MS-DBB disrupted Tmaze delayed alternation performance and the hippocampal theta rhythm [6]. GAL given i.c.v. [23] or into the ventral hippocampus [25], impaired an operant delayed non-matching-to-sample task (DNMTS), and GAL potentiated scopolamine disruption of DNMTS when administered into the MS-DBB [24]. The behavioral component of the present study indicates that microinjection of GAL into the MS-DBB produces significant inhibition of spontaneous exploratory behavior induced by scopolamine. However, it is important to note that this observation may not indicate a general role of GAL in mediating exploratory behavior or anxiety, because GAL alone administered i.c.v, has previously been shown to have no effect upon spontaneous exploratory activity measured in a Digiscan apparatus [9]. The present results are consistent with an interpretation that the MS-DBB is one site at which GAL may act to inhibit hippocampal cholinergic transmission. Galanin has inhibitory effects in the rat hippocampus on (a) ACh
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J.K. Robinson et al. / Brain Research 709 (1996) 81-87
release in vitro and in vivo [1,3,5], (b) carbachol-stimulated phosphoinositide turnover in vitro [21], and (c) the slow cholinergic excitatory post-synaptic potential in CA1 pyramidal neurons in vitro [4]. This latter effect has been interpreted as resulting from presynaptic inhibition of ACh release [4]. It is possible that galanin can inhibit ACh release by acting at both the cell body and terminal regions. One important question to be determined by future studies is whether galanin blocks ACh release when administered into the ventral hippocampus. This question could be answered, using in vivo microdialysis, by administering GAL by reverse perfusion through the dialysis probe, while dialyzing for ACh from the same probe, in the ventral hippocampus. It is interesting to speculate that endogenous GAL could serve as an inhibitory mechanism normally regulating ACh release. In the rat, several populations of galanin-containing neurons are potential sources of endogenous galanin which could mediate the inhibition of scopolamine-induced hippocampal ACh release. First, the MS-DBB contains many galanin-immunoreactive interneurons, some of which may synapse on adjacent cholinergic cell bodies [15,16]. Second, the galanin-immunoreactive projections from the paraventricular and perifornical regions of the hypothalamus to the lateral septum [17] may synapse on cells, perhaps GABAergic, that project to the MS-DBB and thereby modify the activity of cholinergic projections to the hippocampus. Third, some noradrenergic projections from locus coeruleus may also release coexisting galanin. Fourth, the cholinergic neurons in which galanin coexists with acetylcholine may also return collaterals to the MSDBB, and may release galanin normally as part of an inhibitory, negative feedback loop. In the human brain, GAL does not coexist with ACh, but GAL-like immunoreactivity is localized in interneurons and projections to the basal forebrain [10]. In Alzheimer's disease, GALimmunoreactive fibers and terminals hyperinnervate cholinergic neurons of the nucleus basalis and diagonal band [2,19]. If GAL inhibits the activity of ACh neurons in these regions, acting at the cell body, the overexpression of GAL in Alzheimer's disease may be further reducing the activity of remaining cholinergic neurons.
References [1] Bartfai, T., Langel, 0, Bedecs, K. Andell, S., Land, T., Gregersen, S., Ahr6n, B., Girotti, P., Consolo, S., Corwin, R., Crawley, J., Xu, X., Wiesenfeld-Hallin, Z. and Hfkfelt, T., Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes, Proc. Natl. Acad. Sci. USA, 90 (1993) 11287-11291. [2] Chan-Palay, V., Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer's and Parkinson's disease: a hypothesis for the role of galanin accentuating cholinergic dysfunction in dementia, J. Comp. Neurol., 272 (1988) 543-557. [3] Consolo, S., Bertorelli, R., Girotti, P., La Porta, C., Bartfai, T., Parenti, M. and Zambelli, M., Pertussis toxin-sensitive G-protein
mediates galanin's inhibition of scopolamine-evoked acetylcholine release in vivo and carbachol-stimulated phosphoinositide turnover in rat ventral hippocampus, Neurosci. Lett., 126 (1991) 29-32. [4] Dutar, P., Lamour, Y. and Nicoll, R.A., Galanin blocks the slow EPSP in CA1 pyramidal neurons from ventral hippocampus, Eur. J. PharmacoL, 164 (1989) 355-360. [5] Fisone, G., Wu, C.F., Consolo, S., Nordstrom, O., Brynne, N., Bartfai, T., Melander, T. and HSkfelt, T., Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo and in vitro studies, Proc. Natl. Acad. Sci. USA, 84 (1987) 7339-7343. [6] Givens, B., Olton, D.S. and Crawley, J.N., Galanin in the medial septal area impairs working memory, Brain Res., 582 (1992) 71-77. [7] Hagan, J.J. and Morris, R.G.M The cholinergic hypothesis of memory: a review of animal experiments. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 20, plenum Press, New York, 1988, pp. 217-323. [8] Halliwell, J.V., Physiological mechanisms of cholinergic action in the hippocampus. In S.-M. Aquilonius and P.-G. Gillberg (Eds.), Progress in Brain Research, VoL 84, Elsevier Science Publishers, 1990, pp. 255-272. [9] Holmes, P.V., Koprivica, V., Chough, E. and Crawley, J.N., Intraventricular galanin does not affect behaviors associated with locus coeruleus activation in rats, Peptides, 7 (1994) 1303-1308. [10] Kordower, J.H. and Mufson, E.J., Galanin-like immunoreactivity within the primate basal forebrain: differential staining patterns between humans and monkeys, J. Comp. Neurol., 294 (1990) 281292. [11] Lamour, Y., Senut, M.C., Dutar, P. and Bassant, M.H., Neuropeptides and septo-hippocampal neurons: electrophysiological effects and distributions of immunoreactivity, Peptides, 9 (1988) 13511359. [12] Larson, J., Wong, D. and Lynch, G., Patterned stimulation at the theta frequency is optimal for induction of long-term potentiation, Brain Res., 368 (1986) 347-350. [13] Malin, D.H., Novy, B.J., Lett-Brown, A., Plotner, R.E., May, B.T., Radulescu, S.J., Crothers, M.K., Osgood, L.D. and Lake, J.R., Galanin attenuates retention of one-trial reward learning, Life 8ci., 50 (1992) 939-944. [14] Mastropaolo, J., Nadi, N., Ostrowski, N.L. and Crawley, LN., Galanin antagonizes acetylcholine on a memory task in basal-forebrain lesioned rats, Proc. Natl. Acad. Sci. USA, 85 (1988) 98419845. [15] 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 Res., 360 (1985) 130-138. [16] Melander, T., Staines, W.A. and Rokaeus, A., Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs, Neuroscience, 19 (1986) 223-240. [17] Merchenthaler, I., Lopez, F.J. and Negro-Vilar, A., Anatomy and physiology of central galanin-containing pathways, Prog. Neurobiol., 40 (1993) 711-769. [18] Mizumori, S.J.Y., McNaughton, B.L. and Barnes, C.E., A comparison of supramammillary and medial septum influences on hippocampal field potentials and single-unit activity, J. Neurophysiol., 61 (1989) 15-31. [19] Mufson, E.J., Cochran, E., Benzing, W. and Kordower, J.H., Galaninergic innervation of the cholinergic vertical limb of the diagonal band (Ch2) and bed nucleus of the stria terminalis in aging, Alzheimer's disease and Down's syndrome, Dementia, 4 (1993) 237-250. [20] Olton, D.S. and Wenk, G.L., Dementia: Animal models of the cognitive impairments produced by degeneration of the basal forebrain cholinergic system. In H.Y. Meltzer (Ed.), Psychopharmacol-
J.K. Robinson et al. / Brain Research 709 (1996) 81-87 ogy: The Third Generation of Progress, Raven Press, New York,
1987, pp. 941-953. [21] Pallazzi, E., Fisone, G., Iq[6kfelt, T., Barffai, T. and Consolo, S., Galanin inhibits the muscarinic stimulation of phosphoinositide turnover in rat ventral hippocampus, Eur. J. Pharmacol., 148 (1988) 479-480. [22] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates (2nd Edn.), Academic Press, San Diego, CA, 1986. [23] Robinson, J.K. and Crawley, J.N., Intraventricular galanin impairs delayed non-matching to sample performance in rats, Behav. Neurosci., 107 (1993) 458-467.
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[24] Robinson, J.K. and Crawley, J.N., Intraseptal galanin potentiates scopolamine disruption of an operant, delayed non-matching-to-sample memory task, J. Neurosci., 13 (1993) 5119-5125. [25] Robinson, J.K. and Crawley, J.N., Analysis of anatomical sites at which galanin impairs delayed non-matching-to-sample in rats, Behay. Neurosci., 108 (1994) 941-950. [26] Sundstrom, E., Archer, T., Melander, T. and H6kfelt, T., Galanin impairs aquisition but not retrieval of spatial memory in rats studied in the Morris swim maze, Neurosci. Lett., 88 (1988) 331-335.
Brain Research 709 (1996) 81-87
Research report
Galanin microinjected into the medial septum inhibits scopolamine-induced acetylcholine overflow in the rat ventral hippocampus John K. Robinson a, *, Alessandro Zocchi b, Agu Pert b, Jacqueline N. Crawley a a Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Bethesda, MD 20892, USA b Unit on Behavioral Pharmacology, Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD 20892, USA Accepted 11 October 1995
Abstract
Galanin-like immunoreactive terminals hyperinnervate the basal forebrain cholinergic neurons in Alzheimer's disease. To investigate the hypothesis that galanin acts directly on basal forebrain cell bodies, in vivo microdialysis studies were conducted in awake rats which analyzed the actions of galanin on acetylcholine release. Microinjection of galanin into the cholinergic cell body region of the medial septum-diagonal band (MS-I)BB) inhibited acetylcholine release in the ventral hippocampus. These results are consistent with an interpretation that galanin teiminals synapsing on cholinergic cell bodies of the basal forebrain may serve to inhibit the release of acetylcholine in the terminal fields of the cholinergic neurons. Keywords: Activity; Receptor antagonist; Behavior; Microdialysis; Neuropeptide
1. Introduction
The cholinergic septohippocampal pathway is thought to play an important modulatory role in hippocampal activity [8], especially activity related to learning and memory [7]. Lesions of the septo-hippocampal pathway have been shown to disrupt performance in many learning and memory paradigms 1120]. Release of acetylcholine (ACh) in the hippocampus is temporally associated with increased power of the hippocampal theta rhythm [18], thought to be critical for the phenomenon of long-term potentiation in hippocampus [12]. A number of neuropeptiides are localized in the medial septum-diagonal band of Broca (MS-DBB), including somatostatin, calcitonin gene-related peptide, cholecystokinin, dynorphin-A, Met-enkephalin, and galanin [11]. Galanin (GAL) is the most: abundant of the neuropeptides in this region [11], and is the only neuropeptide that coexists with ACh in the rat. GAL-like immunoreactivity
* Corresponding author, at Present address: Department of Psychology, State University of New York at :Stony Brook, Stony Brook, NY 117942500, USA. Fax: (1) ( 5 1 6 ) 632-7876; e-mail: [email protected] Elsevier Science B.V. SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 3 0 7 - 5
is seen in about 30% of the medial septum-diagonal band cell bodies and projections to the ventral hippocampus [151. The coexistence of GAL with ACh in the MS-DBB, projecting to the ventral hippocampus is the best available model system in rodents for studying the functional interactions between galanin and acetylcholine. GAL administered intraventricularly to awake rats has been shown to block ACh release stimulated by muscarinic receptor antagonist, scopolamine, in ventral hippocampus [3,5]. GAL had no significant effect on baseline ACh release. In the dorsal hippocampus, which contains a much lower expression of galanin receptors, intraventricularly administered galanin had no effect on scopolamine-stimulated ACh release [5]. The site of action at which GAL inhibits ACh release has not been identified. GAL administered into the MSDBB of rats has been shown to simultaneously disrupt T-maze delayed alternation performance and the hippocampal theta rhythm [6]. Further, GAL administered into the MS-DBB potentiated scopolamine-induced disruption of an operant delayed non-matching-to-sample task [24]. Taken together, these results suggest that galanin may act on medial septum cell bodies to exert its inhibitory role in memory processes.
82
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The present experiments were conducted to determine whether galanin microinjected into the MS-DBB influences hippocampal ACh release. The previously published experiments [3,5] that showed that intraventricularly injected galanin reduces ventral hippocampal ACh release, were replicated, and then compared with the effects of galanin microinjected into the MS-DBB of awake, behaving rats.
2. Materials and methods
2.1. Subjects and surgery The subjects were male Sprague-Dawley rats, 300-350 g at time of surgery. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and approved by the NIMH Animal Care and Use Committee. Rats were anesthetized with chloral hydrate anesthesia, and under aseptic conditions, implanted with a C M A / 1 2 microdialysis probe guide with a stainless-steel stylet (Carnegie Medicin, Stockholm, Sweden) into the ventral hippocampus (5.5 mm posterior to Bregma, 5.1 mm ventral to surface of the skull, and 5.0 mm lateral to the midline). In addition, rats were implanted with a guide cannula made of stainless steel hypodermic tubing either into the lateral ventricle (0.5 mm posterior to Bregma, 3.5 mm ventral to surface of the skull, 1.0 mm lateral to the midline) or into the MS-DBB (0.7 mm anterior to Bregma, 5.6 mm ventral to surface of the skull, 1.5 mm lateral to the midline at a 15 degree angle from vertical), after Paxinos and Watson [22]. A dental cement cap held the cannulae in place, and four screws anchored the cap into the skull. The subjects were allowed one week of recovery before the start of testing.
apparatus and handling during the sampling procedures. Three baseline samples were then collected, prior to the MS-DBB injection. Typically, eight post-injection samples were collected. Initially, a pilot study was conducted in which three doses of scopolamine (0.1, 0.5, and 1.0 m g / k g i.p.) were tested, to determine an optimal intermediate dose for the subsequent experiments. Galanin (Rat GAL 1-29, Bachem Bioscience, Philadelphia, PA) was dissolved in Ringer's solution, and injected in a volume of 5.0 4xl over 25 s for intraventricular injection, and injected in a 0.5/xl volume over 57 s for the MS-DBB microinjections, through a 31-gauge, 1.9 cm stainless steel injector, extending 0.2 cm ventral to the implanted guide cannula, using a Sage microinfusion pump (Orion Instruments, Cambridge, MA). For both injection routes, the injector was left in place for an additional one min before removal and insertion of the stylet. Vehicle control subjects received only Ringer's solution. Scopolamine hydrobromide was dissolved in saline and administered i.p. immediately following the MS-DBB microinjection. 2.3. Quantitation of acetylcholine concentrations The analytical system consisted of a Bioanalytical Systems (West Lafayette, IN) high pressure liquid chromatograph, equipped with an LC-4B detector. The potential was set at +500 mV on a platinum working electrode versus a Ag/AgC1 reference electrode. BAS MF-6150 analytical column cartridge, and a BAS MF-6151 immobilized enzyme reactor cartridge were used. Mobile phase (35 mM Na2PO4, pH adjusted to 8.5 with H3PO4, 0.05% antibacterial (Kathon, Bioanalytical Systems, West Lafayette, IN), prepared fresh weekly, was pumped at a flow rate of 0.6 ml/min.
2.2. Experimental procedures 2.4. Locomotor activity At approximately 18 h prior to the start of the experiment, the stylet from the hippocampal microdialysis cannula was removed and a C M A / 1 2 microdialysis probe with a 3 mm membrane that extended 3 mm from the implanted cannula. On the day of the experiment, the animal was placed in a plexiglass cylinder inside a Digiscan activity monitor (Omnitech Electronics, Columbus, OH). Tubing was attached by a counterbalance arm and swivel (Harvard Scientific, Cambridge, MA) to permit free movement. The probe was perfused with artificial cerebrospinal fluid (NaCI 147 mM, KCI 4.0 mM, CaCI 2 1.3 mM, and 1 /xM neostigmine bromide (Sigma, St. Louis, MO)) at a rate of 0.5/xl/min. 10-/xl samples were collected every 20 min and injected into the HPLC immediately for ACh analysis. The first four samples taken from the subject were discarded. This time period represented habituation to the
Three Digiscan locomotor activity parameters were quantitated at 20 min intervals. The measure of horizontal movements incorporated both exploration and static repetitive motion such as grooming. The vertical measure primarily assessed rearings. The ambulatory parameter recorded successive interruptions of two photobeams, and was most representative of spontaneous exploratory activity. The measurements were recorded immediately following collection of the perfusate sample. 2.5. Data analysis ANOVAs and posthoc testing was conducted using SuperanovaT M (v.l.1) statistical software. The calculation of percent of baseline ACh release from peak height was determined by comparison to the mean peak heights of three pre-injection samples of ACh standards (1.0 /zM)
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
83
within each experimental day. ANOVAs were conducted on post-injection samples of ACh and Digiscan activity parameters.
from subjects with incorrect placements were removed from further analysis.
2.6. Histology
3. Results
At the completion of the experiment, subjects were decapitated under chloral hydrate anesthesia. The brains were fixed in 10% formalin, sectioned, and stained with thionin. The microinjection cannula placements and the microdialysis probe placements were then evaluated. Data
Locations of microinjection and microdialysis sites from the subjects included in the data analyses are shown in Fig. 1. Fig. 1A shows a representative MS-DBB placement. Fig. 1C summarizes the locations of placements at the MS-DBB region. Fig. 1B shows a representative ventral
A
Fig. 1. Microinjectioncannula and microdialysisprobe placements.Photomicrograph(A) and reconstructionof injectionsites (C) in medial septum/diagonal band of Broca. Photomicrograph(B) and reconstructionof tip of microdialysisprobe sites (D) in ventral hippocampus.
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
84
Table 1 Effects of scopolamine (0.1-1.0 m g / k g i.p.) on acetylcholine release Treatment
Rat
Acetylcholine (pmol/sample)
no.
Baseline 1
No-injection
1 2 3 4 5 6 7 8
Scop 0.1 Scop 0.5 Scop 1.0
0.26 0.15 0.07 0.63 0.11 0.37 0.62 0.23
Post-injection 2
3
0.23 0.11 0.07 0.63 0.09 0.37 0.62 0.13
0.23 0.07 0.04 0.47 0.19 0.30 0.62 0.17
1
0.23 0.11 0.07 0.58 0.09 0.37 1.65 0.30
2
0.15 0.07 0.07 0.84 0.60 0.52 5.70 0.67
3
4
5
6
7
8
0.23 nd 0.04 0.63 0.91 3.50 5.10 0.67
0.23 nd 0.07 1.32 0.67 2.70 3.00 0.63
nd nd 0.04 0.32 0.45 1.60 2.20 0.23
nd nd 0.08 0.26 0.48 1.30 1.60 0.23
nd nd 0.08 0.21 0.17 0.86 2.00 0.27
nd nd 0.04 0.21 0.13 0.34 1.50 0.13
Data from individual animals at each dose are shown, nd, not determined.
hippocampus cannula placement. Fig. 1D summarizes the locations of placements at the ventral hippocampus site. Eleven rats were excluded because a substantial part of the dialysis membrane was outside the region of the ventral hippocampus. An additional three rats showed extensive tissue damage in the region surrounding the dialysis membrane and were excluded. MS-DBB microinjection cannula placements were determined to be correct in all subjects with correct microdialysis probe placement. Table 1 shows the effects of scopolamine administered i.p. on ACh overflow in the ventral hippocampus. Data from two subjects at each dose were obtained. These pilot data suggest that the 0.5 m g / k g and 1.0 m g / k g doses produce comparable responses, while in contrast, the 0.1 m g / k g dose did not produce a consistent increase in ACh
GALANIN (ICV) + SCOPOLAMINE (LP.)
overflow. Based upon these pilot subjects, an intermediate 0.3 m g / k g dose of scopolamine was chosen for subsequent experiments. Fig. 2 shows the effects of galanin (1.6 nmol) administered i.c.v, on ACh overflow in the ventral hippocampus induced by scopolamine (0.3 m g / k g i.p.). ANOVA of the post-injection samples revealed a significant main effect of t r e a t m e n t (F1, 4 -- 8.3, P < 0.05), sample (F7,28 = 7.5, P < 0.0001), and a significant treatment × sample interaction (F7,28 = 4.4, P < 0.002). Because this interaction was significant, individual sample numbers were analyzed by paired contrasts, revealing that the galanin group differed from the Ringer's solution group at the f o u r t h (F1, 4 = 10.2, P < 0.04) and fifth (F1, 4 = 7.8, P < 0 . 0 5 ) post-injection samples. Fig. 3 shows the effects of galanin (0.025, 0.4, and 1.6 nmol) administered into the MS-DBB on ACh overflow in the ventral hippocampus induced by scopolamine (0.3
RING + SCOP(N-~I~) .6 nmoles GAL+SCOP(N--3)
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20
40
60
80 100 120 140 160 180 200 TIME (rain)
Fig. 2. Intraventricular galanin (1.6 nmol) blocked scopolamine (0.3 m g / k g i.p.)-induced acetylcholine overflow in the ventral hippocampus of awake rats. Data in Fig. 2Fig. 3Fig. 4 are shown as mean + S.E.M, Number of subjects per treatment group is shown in parentheses. GAL, rat galanin 1-29; RING, Ringer's solution vehicle controls. * P < 0.05, comparison of Ringer's solution controls and GAL.
6
100! 0
•
,
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-
,
-
,
-
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,
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,
-
,
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,
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20 41/ 60 80 100 120 MO 160 180 200 TIME Iminl
Fig. 3. Galanin (0.025-1.6 nmol) microinjected into medial septum/diagonal band of Broca blocked scopolamine-induced acetylcholine overflow in the ventral hippocampus of awake rats. * P < 0.05, comparison of Ringer's solution controls and GAL doses.
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
Ambulation 600"
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500"
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0.025nmoles G A L + S C O P (N=4) 0 . 4 n m o l e s G A L + S C O P (N-,3)
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40
60
80
100
120
140
160
180
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Vertical
120-
85
revealed no significant effects of dose of galanin administered into the medial septum, but did show significant dose × sample interactions for horizontal activity (F3,30 = 1.7, P < 0.03), ambulations (F3,30 = 2.2, P < 0.002), and approached significance for vertical rearings (/73,30 = 1.5, P < 0.08). Because these dose × sample interactions were significant, Dunnett's posthoc comparisons were conducted at each post-injection sample. Significant effects ( P < 0.05) were detected at the first (0.4 nmol dose), second (0.4, 1.6 nmol doses) and fourth (0.4, 1.6 nmol doses) post-injection sample for the ambulation measure, the fifth (0.025, 0.4 nmol doses) post-injection sample for the vertical measure, and the fourth (0.4, 1.6 nmol doses) and fifth (0.025, 0.4 nmol doses) post-injection sample for the horizontal measure.
100"
80-
4. Discussion
60"
0
20
40
60
80
100
120
140
160
180
200
Horizontal 3000'
g
\ 1000'
0,
•
• 20
-
• 40
-
, 60
-
, - , - , - T - - 8D 100 120 140 160 TIME
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Fig. 4. Galanin (0.025-1.6 nmol), microinjected into the medial septum/diagonal band of Broca, reduced scopolamine-inducedexploratory activityduring microdialysisas shown on three measures of behavioral activityin a Digiscanopen field. m g / k g i.p.). ANOVA of the post-injection samples showed a significant main effect of treatment (F3,11 = 3.6, P < 0.05), a significant main effect of sample (F7,77 = 3.4, P < 0.003), and a significant treatment × sample interaction (F21,77 = 2.9, P < 0.0004). Dunnett's posthoc comparisons conducted at each post-injection sample were significant (P < 0.05) for all doses of GAL at the second and third post-injection samples. Fig. 4 shows the effects of GAL microinjected into the MS-DBB on exploratory locomotor activity induced by scopolamine (0.3 m g / k g i.p.). ANOVA of the post-injection periods for horizontal activity (F3.11 = 2.9), vertical rearings (F3,11 = 1.1), and ambulatory activity (F3,11 = 2.5)
The present experiments showed that GAL administered into the MS-DBB cell body region potently blocked scopolamine-stimulated ACh overflow in the ventral hippocampus. Galanin (1.6 nmol) administered i.c.v, attenuated scopolamine-stimulated ACh overflow in ventral hippocampus, demonstrating that data from the present experiments are consistent with those reported by other laboratories [3,5]. Behavioral studies have shown that GAL is disruptive of memory-task performance, supporting an inhibitory functional role of GAL in the septohippocampal pathway. When microinjected i.c.v., GAL disrupted the acquisition of the Morris water maze [26] and the radial maze [13]. GAL given i.c.v, or intrahippocampally also attenuated improvement in T-maze delayed alternation performance in basal-forebrain lesioned rats produced by acetylcholine [14]. GAL administered into the MS-DBB disrupted Tmaze delayed alternation performance and the hippocampal theta rhythm [6]. GAL given i.c.v. [23] or into the ventral hippocampus [25], impaired an operant delayed non-matching-to-sample task (DNMTS), and GAL potentiated scopolamine disruption of DNMTS when administered into the MS-DBB [24]. The behavioral component of the present study indicates that microinjection of GAL into the MS-DBB produces significant inhibition of spontaneous exploratory behavior induced by scopolamine. However, it is important to note that this observation may not indicate a general role of GAL in mediating exploratory behavior or anxiety, because GAL alone administered i.c.v, has previously been shown to have no effect upon spontaneous exploratory activity measured in a Digiscan apparatus [9]. The present results are consistent with an interpretation that the MS-DBB is one site at which GAL may act to inhibit hippocampal cholinergic transmission. Galanin has inhibitory effects in the rat hippocampus on (a) ACh
86
J.K. Robinson et al. / Brain Research 709 (1996) 81-87
release in vitro and in vivo [1,3,5], (b) carbachol-stimulated phosphoinositide turnover in vitro [21], and (c) the slow cholinergic excitatory post-synaptic potential in CA1 pyramidal neurons in vitro [4]. This latter effect has been interpreted as resulting from presynaptic inhibition of ACh release [4]. It is possible that galanin can inhibit ACh release by acting at both the cell body and terminal regions. One important question to be determined by future studies is whether galanin blocks ACh release when administered into the ventral hippocampus. This question could be answered, using in vivo microdialysis, by administering GAL by reverse perfusion through the dialysis probe, while dialyzing for ACh from the same probe, in the ventral hippocampus. It is interesting to speculate that endogenous GAL could serve as an inhibitory mechanism normally regulating ACh release. In the rat, several populations of galanin-containing neurons are potential sources of endogenous galanin which could mediate the inhibition of scopolamine-induced hippocampal ACh release. First, the MS-DBB contains many galanin-immunoreactive interneurons, some of which may synapse on adjacent cholinergic cell bodies [15,16]. Second, the galanin-immunoreactive projections from the paraventricular and perifornical regions of the hypothalamus to the lateral septum [17] may synapse on cells, perhaps GABAergic, that project to the MS-DBB and thereby modify the activity of cholinergic projections to the hippocampus. Third, some noradrenergic projections from locus coeruleus may also release coexisting galanin. Fourth, the cholinergic neurons in which galanin coexists with acetylcholine may also return collaterals to the MSDBB, and may release galanin normally as part of an inhibitory, negative feedback loop. In the human brain, GAL does not coexist with ACh, but GAL-like immunoreactivity is localized in interneurons and projections to the basal forebrain [10]. In Alzheimer's disease, GALimmunoreactive fibers and terminals hyperinnervate cholinergic neurons of the nucleus basalis and diagonal band [2,19]. If GAL inhibits the activity of ACh neurons in these regions, acting at the cell body, the overexpression of GAL in Alzheimer's disease may be further reducing the activity of remaining cholinergic neurons.
References [1] Bartfai, T., Langel, 0, Bedecs, K. Andell, S., Land, T., Gregersen, S., Ahr6n, B., Girotti, P., Consolo, S., Corwin, R., Crawley, J., Xu, X., Wiesenfeld-Hallin, Z. and Hfkfelt, T., Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes, Proc. Natl. Acad. Sci. USA, 90 (1993) 11287-11291. [2] Chan-Palay, V., Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer's and Parkinson's disease: a hypothesis for the role of galanin accentuating cholinergic dysfunction in dementia, J. Comp. Neurol., 272 (1988) 543-557. [3] Consolo, S., Bertorelli, R., Girotti, P., La Porta, C., Bartfai, T., Parenti, M. and Zambelli, M., Pertussis toxin-sensitive G-protein
mediates galanin's inhibition of scopolamine-evoked acetylcholine release in vivo and carbachol-stimulated phosphoinositide turnover in rat ventral hippocampus, Neurosci. Lett., 126 (1991) 29-32. [4] Dutar, P., Lamour, Y. and Nicoll, R.A., Galanin blocks the slow EPSP in CA1 pyramidal neurons from ventral hippocampus, Eur. J. PharmacoL, 164 (1989) 355-360. [5] Fisone, G., Wu, C.F., Consolo, S., Nordstrom, O., Brynne, N., Bartfai, T., Melander, T. and HSkfelt, T., Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo and in vitro studies, Proc. Natl. Acad. Sci. USA, 84 (1987) 7339-7343. [6] Givens, B., Olton, D.S. and Crawley, J.N., Galanin in the medial septal area impairs working memory, Brain Res., 582 (1992) 71-77. [7] Hagan, J.J. and Morris, R.G.M The cholinergic hypothesis of memory: a review of animal experiments. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 20, plenum Press, New York, 1988, pp. 217-323. [8] Halliwell, J.V., Physiological mechanisms of cholinergic action in the hippocampus. In S.-M. Aquilonius and P.-G. Gillberg (Eds.), Progress in Brain Research, VoL 84, Elsevier Science Publishers, 1990, pp. 255-272. [9] Holmes, P.V., Koprivica, V., Chough, E. and Crawley, J.N., Intraventricular galanin does not affect behaviors associated with locus coeruleus activation in rats, Peptides, 7 (1994) 1303-1308. [10] Kordower, J.H. and Mufson, E.J., Galanin-like immunoreactivity within the primate basal forebrain: differential staining patterns between humans and monkeys, J. Comp. Neurol., 294 (1990) 281292. [11] Lamour, Y., Senut, M.C., Dutar, P. and Bassant, M.H., Neuropeptides and septo-hippocampal neurons: electrophysiological effects and distributions of immunoreactivity, Peptides, 9 (1988) 13511359. [12] Larson, J., Wong, D. and Lynch, G., Patterned stimulation at the theta frequency is optimal for induction of long-term potentiation, Brain Res., 368 (1986) 347-350. [13] Malin, D.H., Novy, B.J., Lett-Brown, A., Plotner, R.E., May, B.T., Radulescu, S.J., Crothers, M.K., Osgood, L.D. and Lake, J.R., Galanin attenuates retention of one-trial reward learning, Life 8ci., 50 (1992) 939-944. [14] Mastropaolo, J., Nadi, N., Ostrowski, N.L. and Crawley, LN., Galanin antagonizes acetylcholine on a memory task in basal-forebrain lesioned rats, Proc. Natl. Acad. Sci. USA, 85 (1988) 98419845. [15] 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 Res., 360 (1985) 130-138. [16] Melander, T., Staines, W.A. and Rokaeus, A., Galanin-like immunoreactivity in hippocampal afferents in the rat, with special reference to cholinergic and noradrenergic inputs, Neuroscience, 19 (1986) 223-240. [17] Merchenthaler, I., Lopez, F.J. and Negro-Vilar, A., Anatomy and physiology of central galanin-containing pathways, Prog. Neurobiol., 40 (1993) 711-769. [18] Mizumori, S.J.Y., McNaughton, B.L. and Barnes, C.E., A comparison of supramammillary and medial septum influences on hippocampal field potentials and single-unit activity, J. Neurophysiol., 61 (1989) 15-31. [19] Mufson, E.J., Cochran, E., Benzing, W. and Kordower, J.H., Galaninergic innervation of the cholinergic vertical limb of the diagonal band (Ch2) and bed nucleus of the stria terminalis in aging, Alzheimer's disease and Down's syndrome, Dementia, 4 (1993) 237-250. [20] Olton, D.S. and Wenk, G.L., Dementia: Animal models of the cognitive impairments produced by degeneration of the basal forebrain cholinergic system. In H.Y. Meltzer (Ed.), Psychopharmacol-
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1987, pp. 941-953. [21] Pallazzi, E., Fisone, G., Iq[6kfelt, T., Barffai, T. and Consolo, S., Galanin inhibits the muscarinic stimulation of phosphoinositide turnover in rat ventral hippocampus, Eur. J. Pharmacol., 148 (1988) 479-480. [22] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates (2nd Edn.), Academic Press, San Diego, CA, 1986. [23] Robinson, J.K. and Crawley, J.N., Intraventricular galanin impairs delayed non-matching to sample performance in rats, Behav. Neurosci., 107 (1993) 458-467.
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[24] Robinson, J.K. and Crawley, J.N., Intraseptal galanin potentiates scopolamine disruption of an operant, delayed non-matching-to-sample memory task, J. Neurosci., 13 (1993) 5119-5125. [25] Robinson, J.K. and Crawley, J.N., Analysis of anatomical sites at which galanin impairs delayed non-matching-to-sample in rats, Behay. Neurosci., 108 (1994) 941-950. [26] Sundstrom, E., Archer, T., Melander, T. and H6kfelt, T., Galanin impairs aquisition but not retrieval of spatial memory in rats studied in the Morris swim maze, Neurosci. Lett., 88 (1988) 331-335.