Intra-septal injections of glucose and glibenclamide attenuate galanin-induced spontaneous alternation performance deficits in the rat

Brain Research 813 Ž1998. 50–56

Research report

Intra-septal injections of glucose and glibenclamide attenuate galanin-induced spontaneous alternation performance deficits in the rat Mark R. Stefani, Paul E. Gold

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Neuroscience Graduate Program and Department of Psychology, UniÕersity of Virginia, CharlottesÕille, VA 22903, USA Accepted 18 August 1998

Abstract Injection of the neuroactive peptide galanin into the rat hippocampus and medial septal area impairs spatial memory and cholinergic system activity. Conversely, injection of glucose into these same brain regions enhances spatial memory and cholinergic system activity. Glucose and galanin may both modulate neuronal activity via opposing actions at ATP-sensitive Kq ŽK-ATP. channels. The experiments described in this report tested the ability of glucose and the direct K-ATP channel blocker glibenclamide to attenuate galanin-induced impairments in spontaneous alternation performance in the rat. Intra-septal injection of galanin Ž2.5 mg., 30 min prior to plus-maze spontaneous alternation performance, significantly decreased alternation scores compared to those of rats receiving injections of vehicle solution. Co-injection of glucose Ž20 nmol. or the K-ATP channel blocker glibenclamide Ž5 nmol. attenuated the galanin-induced performance deficits. Glibenclamide produced an inverted-U dose–response curve in its interaction with galanin, with doses of 0.5 and 10 nmol having no effect on galanin-induced spontaneous alternation deficits. Drug treatments did not alter motor activity, as measured by overall number of arm entries during spontaneous alternation testing, relative to vehicle injected controls. These findings support the hypothesis that, in the septal region, galanin and glucose act via K-ATP channels to modulate neural function and behavior. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Glucose; Memory; Galanin; K-ATP; Rat; Spontaneous alternation

1. Introduction Galanin is a peptide found in the central nervous system of mammals, including rats, monkeys and humans w3,25,26,52,57x. In the rat brain, galanin co-localizes with acetylcholine ŽACh. in a sub-population of neurons in the medial septum and diagonal band of Broca w25,51x. These combined cholinergic–galaninergic neurons project to the hippocampal region w26x. Galanin receptors are richly expressed throughout the basal forebrain, including the septal nuclei, the ventral hippocampus and entorhinal cortex, ventral pallidum and the amygdala w53x. The functionŽs. of endogenous galanin in these areas is not yet known, but galanin is hypothesized to inhibit the release and effects of ACh w14,15,30x. Intra-cranial administration of galanin impairs memory and decreases cholinergic system activity in rats w14,17, 19,24,30,45,48x. Intra-ventricular infusions of galanin im-

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pair learning and memory on inhibitory avoidance and spatial delayed non-match-to-sample tasks w19,24,45x, and decrease hippocampal and frontal cortex ACh output w19x. Infusions of galanin into the ventral hippocampus impair memory on delayed non-match-to-position and water maze tasks w24,30x. Intra-septal injections of galanin potentiate the impairing effect of peripherally administered scopolamine on a spatial delayed non-match-to-sample task w45x, and simultaneously decrease hippocampal theta rhythm and impair spatial memory, as measured by T-maze choice accuracy w17x. Central administration of glucose has effects on memory and cholinergic system function opposite those of galanin. Intra-ventricular glucose injections enhance inhibitory avoidance w23x and attenuate deficits in spontaneous alternation scores produced by peripheral injections of the acetylcholine receptor antagonist scopolamine w34x. Intra-hippocampal glucose infusions simultaneously enhance spontaneous alternation scores and increase the release of ACh in the hippocampus w44x. Intra-septal glucose injections also enhance spontaneous alternation scores w54x, and attenuate morphine-induced decreases in spontaneous

0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 7 6 - 2

M.R. Stefani, P.E. Gold r Brain Research 813 (1998) 50–56

alternation performance and hippocampal ACh levels w38,39x. While the mechanismŽs. mediating the effects of galanin and glucose in the central nervous system are not yet known, a common locus of action may be the ATP-sensitive Kq ŽK-ATP. channel. K-ATP channels are hypothesized to couple neuronal glucose metabolism to neurotransmitter release via fluctuations in intracellular ATP levels w2x. Glucose, via ATP, and sulfonylurea class drugs such as glibenclamide decrease K-ATP channel conductance w4,33x. Galanin receptor activation increases K-ATP channel conductance in pancreatic b cells via an inhibitory G-protein w10,11x. Decreased K-ATP channel conductance renders the cell more sensitive to depolarizing stimuli, and increases the likelihood of stimulus-evoked neurotransmitter release. Conversely, increased channel conductance, by hyperpolarizing the cell, decreases the likelihood of stimulus-evoked neurosecretion. Indirect evidence supports the interaction of galanin and K-ATP channels in the central nervous system w6,35x. The experiments described in this report tested the hypotheses that: Ž1. intra-septal injection of galanin would impair spontaneous alternation performance, and Ž2. co-administration of glucose or the direct K-ATP channel blocker glibenclamide would attenuate galanin-induced spontaneous alternation performance deficits. Spontaneous alternation is a non-rewarded exploratory task considered to assess spatial working memory w39,50x. The septal area was chosen as the site of drug administration because of its involvement in spatial working memory w16x. The septal area is sensitive to glucose, GABAergic, cholinergic and opioidergic modulation of learning and memory w7,8,16,38,39,54x, and is rich in both galanin receptors and K-ATP channels w29,53x. In this report we show that intraseptal administration of galanin produces spontaneous alternation deficits which in turn can be blocked by co-administration of either glucose or glibenclamide.

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ropine sulfate Ž108 mgrrat i.p... Stainless steel guide cannulae Ž22 gauge, Plastics One, Roanoke, VA. were implanted, directed at the dorsal medial septal area, using stereotaxic coordinates Ž0.5 mm anterior to bregma, 0.0 mm lateral of midline, 5.2 mm ventral from skull surface. derived from the atlas of Paxinos and Watson w36x. Rats were permitted a one-week recovery period, during which they were monitored for infection and handled daily. 2.3. Intracranial injections Intra-septal injections were made via a 28-gauge injection cannula ŽPlastics One, Roanoke, VA. which extended 1 mm beyond the end of the guide cannula. The injection cannula was connected to a 25 ml Hamilton syringe via a length of polyethylene tubing. Syringes were driven by an injection pump ŽHarvard Apparatus, South Natick, MA. at a rate of 0.5 mlrmin. The injection cannula was left in place for 2 min following the end of the injection period to permit diffusion. Drugs were suspended in a vehicle solution composed of Žin mM.: NaCl, 154; KCl, 3; CaCl 2 , 1.5; MgCl 2 , 1.0; NaH 2 PO4 , 2.0; Na 2 HPO4 , 2.0; glucose, 2.0, pH 7.4. For Experiment 2, the vehicle solution also contained Tween-80 Ž1.5% wrv final concentration., which was added to aid in the solubilization of glibenclamide. Galanin was prepared as a 2 = Ž5 mgrml. stock solution in 1 = vehicle solution, aliquoted, and frozen at y208C until needed. Glibenclamide was prepared at 2 = final concentration in 1 = vehicle solution on the day of experiment. The glibenclamide solution was sonicated in a bath sonicator ŽBranson Instruments, Shelton, CT. to ensure even suspension. When galanin and glibenclamide were co-administered, equal 5 ml volumes of the respective 2 = galanin and glibenclamide solutions were combined immediately before injection to give a total volume of 10 ml at the desired final Ž1 = . concentrations of each drug. All chemical reagents were purchased from Sigma ŽSt. Louis, MO..

2. Materials and methods

2.4. BehaÕioral procedures

2.1. Subjects

One week post-surgery, the rats were tested for performance of a spontaneous alternation task on the 4-arm radial maze Žplus-maze.. Rats were assigned to one of the following treatment groups Ž n’s represent the number of rats in each group included in data analysis.: Experiment 1: vehicle control ŽVEH, n s 10., 2.5 mg Ž0.79 nmol. galanin ŽGALN, n s 11., galanin q20 nmol glucose ŽGALNrGLC, n s 10.. Experiment 2: vehicle control ŽVEH, n s 8., 2.5 mg galanin ŽGALN, n s 11. or galanin q0.5, 5 or 10 nmol glibenclamide Ž n s 6, 8 and 8 for 0.5, 5 and 10 nmol glibenclamide, respectively.. Each rat received a 1 ml injection of vehicle, or vehicle plus drug 30 min prior to spontaneous alternation testing. Spontaneous alternation testing was conducted by placing the rat on the center platform of the plus-maze and allowing 12 min of

Male Sprague–Dawley rats ŽHilltop Laboratory, Dublin, VA. weighing 275–300 g on arrival were used as subjects for the studies described below. A total of 121 rats were used; 54 and 67, respectively, for experiments 1 and 2. Rats were housed individually in a room maintained on a 12 h light–dark cycle Žlights on at 0700 h. and had ad libitum access to food and H 2 O. Housing and care of laboratory animals was in compliance with institutional and federal regulations. 2.2. Surgery Rats were anesthetized with sodium pentobarbital Ž50 mgrkg i.p.. 20 min following the administration of at-

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M.R. Stefani, P.E. Gold r Brain Research 813 (1998) 50–56

Fig. 1. The points plotted represent the approximate locations of acceptable intra-septal cannula placements. Animals with cannula placements outside of the septal area were excluded from analysis. The atlas plates shown are adapted from the stereotaxic atlas of Paxinos and Watson w36x.

unimpeded exploration. The number and sequence of arm entries were recorded for calculation of a ‘percent alternation score’. An alternation consisted of four different arm choices out of five consecutive arm entries. A 4r5 alternation score was computed by dividing the number of observed alternations in overlapping quintuplets by the number of possible alternations, and multiplying the quotient by 100. The plus-maze was composed of four arms joined to an central platform. Each arm was 55 cm long and 10 cm wide, with 12 cm high walls. The central platform was 25 cm across. The floor and walls of the central platform, and the floors of the arms were made of gray-painted wood. The walls of the arms were made of poster board, and were periodically changed to prevent the accumulation of odors. The maze floor was washed with ethanol Ž70% vrv. between animals.

on glass slides and stained with Cresyl violet. Stained sections were evaluated for accuracy of cannula placement. Animals with cannula placements outside of the septal area were excluded from subsequent analysis. Fig. 1 illustrates

2.5. Histology At the conclusion of behavioral testing, the rats were euthanized with an overdose of sodium pentobarbital and perfused with solutions of 0.9% NaCl and 10% formalin. Their brains were removed and stored in a 30% sucroser10% formalin solution until sectioning. Serial sections Ž40 mm thick. through the septal area were mounted

Fig. 2. Intra-septal injection of galanin ŽGALN; 2.5 mg in 1 ml. significantly impaired plus-maze spontaneous alternation scores relative to those of vehicle injected control animals ŽVEH.. Co-administration of glucose ŽGALNrGLC; 20 nmol in 1 ml. blocked the impairments produced by galanin. wl p- 0.05 vs. VEH and GALNrGLC. Numbers within bars indicate the number of rats per group included in data analysisx.

M.R. Stefani, P.E. Gold r Brain Research 813 (1998) 50–56

the approximate locations of acceptable intra-septal cannula placements.

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Differences between treatment groups were analyzed by one-way, between-subject ANOVA and Fisher post-hoc tests.

The dose–response curve for glibenclamide was in the shape of an inverted-U. There were no significant between-group differences in the number of arm entries made during the 15-min test period Ž F4,36 s 0.344, p’s ) 0.05.. The number of arm entries for each group were as follows Žmean " standard error.: VEH, 24.6 " 1.6; GALN, 28.1 " 1.7; GALNrGLB-0.5, 26.3 " 1.2; GALNrGLB-5, 26.3 " 2.6, GALNrGLB-10, 29.9 " 1.7.

3. Results

4. Discussion

3.1. Experiment 1

The impairing effect of intra-septal galanin on spontaneous alternation performance observed in the present studies is consistent with previous reports showing that injection of galanin into the septo-hippocampal system impairs performance on tasks with spatial and working memory components w17,30,45,47x. The present results are most similar to those of Givens et al. w17x, who found that intra-septal galanin injections impaired T-maze alternation performance and decreased hippocampal theta-rhythm. Robinson and Crawley w45x found that intra-septal administration of galanin potentiated scopolamine-induced performance deficits on a spatial delayed-non-match-to-sample task, though galanin alone did not impair performance. Galanin also impairs spatial delayed non-match-to-sample and water maze task performance when injected into the ventral hippocampus w30,47x. Thus, as noted by Givens et al. w17x, galanin affects spatial memory whether administered at the origin or termination of the septo-hippocampal projection. Intra-ventricular galanin injections, by contrast, did not impair spontaneous alternation scores in a study by Ukai et al. w59x, although inhibitory Žpassive. avoidance performance was impaired. Galanin did not significantly change the overall number of arm entries relative to that of control animals, implying that the behavioral effects of galanin were due to impairment of alternation performance, and not to changes in motor activity or to differences in time between arm choices Žinter-trial interval.. Simultaneous intra-septal administration of glucose with galanin fully attenuated the galanin-induced decrement in spontaneous alternation scores. These results are concordant with the body of literature describing the memory-enhancing effects of glucose administration Žfor reviews, see Refs. w18,60,61x.. In general, glucose produces effects on behavior opposite those of galanin when injected into the septo-hippocampal system, enhancing performance on tasks with spatial and working memory components. Both intraseptal and intra-hippocampal infusions of glucose enhance spontaneous alternation performance w44,54x. Intra-septal injections of glucose also attenuate spontaneous alternation performance deficits induced by intra-septal injection of the opioid agonist morphine w38x. Thus, glucose, like galanin, modulates behavior when administered at either the origin or termination of the septo-hippocampal projection.

2.6. Statistical analysis

Rats given an intra-septal injection of galanin Ž2.5 mg. had significantly lower spontaneous alternation scores than did rats injected with vehicle solution alone Ž F2,28 s 10.22; p s 0.0005. Žsee Fig. 2.. Co-administration of glucose Ž20 nmol. blocked the impairing effects of galanin Ž p - 0.05.. There were no significant between-group differences in the number of arm entries made during the 15-min test period Ž F2,28 s 0.123, p’s ) 0.05.. The number of arm entries for each group were as follows Žmean " standard error.: VEH, 24.2 " 1.1; GALN, 25.1 " 1.7; GALNrGLC, 21.3 " 1.0. 3.2. Experiment 2 Intra-septal galanin injection again produced significantly lower alternation scores compared to those of rats receiving vehicle alone Ž F4,36 s 4.68; p s 0.004. Žsee Fig. 3.. Co-administration of 5 nmol, but not 10 nmol or 0.5 nmol, of glibenclamide attenuated the effect of galanin.

Fig. 3. Intra-septal injection of galanin ŽGALN; 2.5 mg in 1 ml. significantly impaired plus maze spontaneous alternation scores relative to those of vehicle-injected control animals ŽVEH.. Co-administration of the K-ATP channel blocker glibenclamide at a dose of 5.0 nmol, but not 0.5 or 10.0 nmol, blocked the impairments produced by galanin. wl p- 0.05 vs. VEH and GALN plus glibenclamide Ž5.0 nmol.. Numbers within bars indicate the number of rats per group included in data analysisx.

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M.R. Stefani, P.E. Gold r Brain Research 813 (1998) 50–56

The precise mechanisms of action for galanin and glucose in the central nervous system are not yet known. A possible common locus of action within the central nervous system is the ATP-sensitive Kq ŽK-ATP. channel. The K-ATP channel consists of an inwardly rectifying Kq channel, Kir6.2, coupled to a sulfonylurea receptor subunit w21x. K-ATP channel conductance is blocked by increasing intracellular concentration of ATP, derived from glucose metabolism, and by sulfonylurea class drugs such as glibenclamide w4,33x. Decreased channel conductance renders the cell more sensitive to depolarizing stimuli and increases the likelihood of stimulus-evoked secretion. In pancreatic b cells, glucose and glibenclamide stimulate insulin release by blocking K-ATP channel conductance. Conversely, galanin opens K-ATP channels via a G-protein dependent signal transduction pathway, hyperpolarizing the b cell and blocking insulin secretion w10,11x. K-ATP channels have been suggested as a mechanism coupling neuronal glucose metabolism and neurotransmitter release w2x. In the brain, evidence for the functional coupling of galanin receptors and K-ATP channels is indirect. Galanin receptors and sulfonylurea receptors, a marker of K-ATP channels, co-localize in several areas of the rat brain, including the septal region and the ventral hippocampus w29,53x. Galanin attenuates the hypothermia produced by ACh receptor agonists; glibenclamide antagonizes this effect of galanin w35x. In rat hippocampal slice preparations, galanin blocks glutamate-mediated anoxic depolarization of CA3 neurons, an effect also antagonized by glibenclamide w6x. Given that galanin and glucose inversely affect behavior and cholinergic system function, and are both known to antagonistically modulate K-ATP channel function, the present studies examined the ability of the direct K-ATP channel blocker glibenclamide to antagonize galanin-induced spontaneous alternation performance deficits. The observed attenuation of galanin-induced spontaneous alternation deficits by both glibenclamide and glucose supports the hypothesis that, within at least some areas of the central nervous system, galanin receptors are functionally coupled to K-ATP channels. The inverted-U dose–response curve observed for glibenclamide in its attenuating effect on the galanin-induced performance deficit is similar to that observed by Patel and Hutson w35x in their examination of the interaction of glibenclamide and galanin on cholinergic receptor agonist mediated hypothermia. While the data reported here support the hypothesis that galanin, glucose and glibenclamide act through the K-ATP channel to influence spontaneous alternation scores, it is possible that these compounds, rather than acting in concert to modulate K-ATP channel function, act at pharmacologically distinct loci which in turn interact ‘downstream’ to affect behavior. Galanin has been observed to reduce muscarinic receptor mediated phosphoinositol ŽPI. turnover in rat and primate hippocampus w15,31,32x, possibly by

reducing Ca2q influx through voltage-gate Ca2q channels w32x. It is not clear from the Palazzi et al. w32x studies whether the galanin-induced reductions in PI turnover were due to a direct effect of galanin on Ca2q channels, or were secondary to an increase in Kq conductance. Galanin receptors have been reported to be coupled to voltage-sensitive Ca2q channels in rat insulinoma w20x and pituitary w22x cells. Glibenclamide, too, may modulate ion channels other than, or in addition to Kir6.2, as has recently been sug¨ ¨ ¨ et al. w1x. Moreover, the modulatory gested by Ammala sites of ATP and glibenclamide are located on different subunits of the K-ATP channel—the Kir6.2 Kq channel and the SUR, respectively w58x —making it possible that glucose and glibenclamide attenuate the effects of galanin via pharmacologically different but behaviorally indistinguishable mechanisms. However, as ATP sensitivity is conferred on the Kir6.2 Kq channel only in the presence of the sulfonylurea receptor subunit w58x, the present data support the K-ATP channel as a plausible common site of action for both glucose and glibenclamide in their antagonism of galanin-induced behavioral impairments. In addition to K-ATP channel modulation, glucose administration might affect neurotransmitter release and neuronal function via osmotic effects w5,13,49x, or by augmenting central ACh synthesis by increasing the availability of the ACh precursor acetyl-CoA, a glucose metabolite w12,27,28,40x. An osmotic effect is unlikely; intra-cranial injections of iso-osmotic yet metabolically inert compounds such as D-lactate and L-glucose neither enhance performance nor attenuate morphine-induced behavioral deficits w42,56x. The ACh synthesis hypothesis is founded on observations that glucose administration increases hippocampal ACh output w12,43,44x and attenuates the effects of central cholinergic antagonists w37,41,55x, results that are also fully concordant with an effect of glucose on behavior and ACh release mediated by K-ATP channel regulation. In conclusion, intra-septal injections of glucose and the direct K-ATP channel blocker glibenclamide attenuate spatial memory impairments induced by intra-septal injection of the neuroactive peptide galanin. The present data further corroborate both the memory-impairing effects of centrally administered galanin and the memory-enhancing effects of glucose Žfor reviews, see Refs. w9,18,46,61x.. As galanin, glucose and glibenclamide are all known to modulate K-ATP channel conductance, the present results support the possibility that galanin receptors in the rodent septal area are functionally coupled to K-ATP channels, and that K-ATP channels may be the mechanism by which galanin and glucose antagonistically influence behavior. Acknowledgements This work was supported by research grants from NIA ŽAG07648. and NINDS ŽNS32914.. M. Stefani was sup-

M.R. Stefani, P.E. Gold r Brain Research 813 (1998) 50–56

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