Intracerebroventricularly administered galanin does not alter operant reaction time or differentially reinforced high rate schedule operant responding in rats

Neuroscience Letters 369 (2004) 245–249

Intracerebroventricularly administered galanin does not alter operant reaction time or differentially reinforced high rate schedule operant responding in rats ¨ Langelb , John K. Robinsona,∗ Ariel Brewera , Ulo a

Department of Psychology, Biopsychology Program Area, Stony Brook University, Stony Brook, NY 11794-2500, USA b Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden Received 1 June 2004; received in revised form 22 July 2004; accepted 28 July 2004

Abstract Galanin (Gal) is a 29/30 amino acid neuroendocrine peptide that impairs learning and memory processes, stimulates feeding, and modulates somatosensory, sex, and stress responses. Anatomical markers for Gal are found throughout the brain, including in the caudate-putamen and substantia nigra motor regions. Many of the behavioral tests that have been used to study the involvement of Gal in complex behavioral processes are motorically demanding, but no research has specifically investigated the involvement of Gal in response initiation or the maintenance of fine motor action. Therefore, the present study examined the effects of intraventricularly administered Gal on two highly sensitive operant tasks designed to detect alteration of these response properties. Response initiation was studied using a light–dark discrimination reaction time task that required a correct response within 2.5 s of a spatially and temporally uncertain stimulus onset. The ability to perform high local rates of responding was studied using an operant differential reinforcement of high rate (DRH) of responding task. Gal (10–20 ␮g, i.c.v.) did not alter reaction time or inter-response time distributions in either task, though did substantially reduce the total number of responses and reinforcers obtained on the DRH schedule. These results are consistent with a Gal-induced reduction of reinforcer efficacy rather than Gal-disruption of response initiation or response patterning. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Activity; Basal ganglia; Neuropeptide; Substantia nigra

Galanin (GAL) is a 29/30 amino acid neuropeptide that has been shown to modulate multiple neurotransmitter systems within the central nervous system, including acetylcholine, norepinephrine, dopamine, and serotonin [4,5,12]. Anatomical markers for Gal and Gal receptors are found in many CNS structures including the hypothalamus, amygdala, hippocampus, basal forebrain, mesencephalon, rhombencephalon, thalamus, entorhinal, piriform, and midline frontal cortices [17,18,30–32]. Additionally, some Gal binding and immunoreactivity is present in motor structures such as the dorsal caudate and globus pallidus [17,30–32]. There are three identified Gal receptors: GalR1, GalR2 and GalR3 [6].

∗

Corresponding author. Tel.: +1 631 632 7832; fax: +1 631 632 7876. E-mail address: [email protected] (J.K. Robinson).

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.097

GalR2 but not GalR1 mRNA expression was found in moderate levels in substantia nigra [20]. Evidence of function in motor structures is provided by the observations that Gal administered through a microdialysis probe enhanced acetylcholine overflow in the caudate-putamen [1,2,22,24], and decreases dopamine activity when administered i.c.v. or into the VTA [9]. Several types of behavior studies have provided some indirect inferences about the role of Gal in mediating motor processes: (1) studies in which Gal was administered centrally and gross ambulation was assessed in the open field or maze tests (e.g. [9,33]), (2) comprehensive behavioral phenotyping of transgenic and knockout mice that included neurological or motor competency tests (e.g. [11,34]), and (3) secondary response measures in operant tests of short-term memory (e.g. [15,16,25–27]). The results of these studies

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have been inconsistent. For example, reduced exploration in the open field produced by centrally administered Gal has been reported [9], but no gross changes in activity or motor competence have been observed in Gal overexpressing mice [34]. Collective interpretation of these findings is made complicated by several problems, especially that open field scores are often confounded by extraneous factors (e.g. stress induced by exposure to the novel environment of the open field). No study has directly assessed the effects of centrally administered Gal on fine motor performance, where the best possible performances of the animals are measured by making reinforcement contingent upon the latency or rate of responding. To accomplish this, the present study employed two highly sensitive operant tasks designed to assess changes in the motor response potential, and isolate this from confounding factors such as stress responses induced by open field tests. Response initiation was studied using a reaction time (RT) task which required a response within 2.5 s of a spatially and temporally uncertain cue lamp onset. Next, the ability to perform high local rates of responding was studied using an operant differential reinforcement of high rate of responding task (DRH). Both tasks allowed for the analysis of the maximal capacity of the rats to respond quickly or to sustain responding. The subjects were two groups of adult male Sprague– Dawley rats (seven in the reaction time experiment and six in the DRH experiment). Each subject was housed separately in a plastic tub cage. All animals were maintained in a humidity and temperature regulated room with a 12 h (07:00 h on and 19:00 h off) light–dark cycle. Their access to water was limited to 30 min, available 1 h following the experimental session, Monday through Friday. They had unlimited access to food in the homecage Monday through Friday. The experiments were carried out on weekdays and all subjects had unlimited access to both food and water Saturday and Sunday. All procedures were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals [37] and with the consent of the State University of New York at Stony Brook Institutional Animal Care and Use Committee. The behavioral investigation was performed in six identical test chambers (MED Associates, East Fairfield, VT), all of which were enclosed in a sound-attenuating enclosure. The stainless-steel apparatus consisted of two front levers with one cue lamp mounted above each one of them, and a rear cue lamp with a lever underneath it. The water dispenser was placed in between the two frontal levers. The experimental events and data recording were controlled by a Dell PC computer running MED Notation programming language. Each animal received a cannula made of stainless-steel hypodermic tubing (24 gauge and 1.7 cm long) into the right lateral ventricle under 100 mg/kg, i.p. Ketamine and 10 mg/kg, i.p. Xylazine anesthesia (from Bregma, 0.5 anterior/posterior, −3.5 dorsal/ventral, and +1.0 laterally from Bregma [23]). Four stainless screws and epoxy were used to secure the

small plastic tubing protecting the cannula and to hold the cannula in place. Cannulations occurred prior to the initiation of reaction time or DRH behavioral pretraining. At the completion of the experiments, the rats were sacrificed under deep sodium pentobarbital anesthesia, and placement of the cannula into the lateral ventricle was verified histologically to be correct in all 13 subjects. Rat Gal (Bachem Bioscience, King of Prussia, PA, USA) was dissolved in saline. Gal or saline was administered using a Hamilton syringe connected by plastic tubing to a 31 gauge, 1.9 cm stainless-steel injector. Each injection delivered a total 5 ␮l solution into the ventricles over 25 s. The injectors were left in place for an additional 60 s. The subjects were pretrained to press a front response lever when one of the front cue lamps was illuminated above the lever until baseline accuracy performance stabilized at greater than 90% correct for all subjects. The cue lamp was illuminated unpredictably at one of the two locations and after a variable (10 ± 3 s) ITI, and for 2.5 s in duration. A press on the signaled lever that occurred within that 2.5 s illumination window produced a 0.1 ml water reinforcer. A press on the non-signaled lever was scored as an incorrect response and produced no reinforcer. A failure to respond to either lever during the 2.5 s window was scored as an error of omission. Sessions were terminated at 40 min. The 40 min session duration was chosen to have the session be long enough to ensure that a sufficient number of trials was completed to establish the reliability of the performance measures but not be so long that a loss of pharmacological potency of Gal would diminish effects on behavior. Previous in vivo operant studies have used a comparable session duration (e.g. [14]) and in vitro studies that have shown strong Gal actions 20 min after i.c.v. administration that were not evident at a 60 min timepoint [13,19,28]. The subjects were first trained to press the left response lever on an fixed ratio 1.0 schedule to produce a 0.1 ml water reinforcer. Then, a DRH schedule was imposed with an initial rate of one response for each 10 s time period resulting in reward. The rate of responding required for reinforcement was then gradually increased over several sessions until 10 responses in a 10 s period was required to produce each reinforcer. Each 10 s block was separated by a 1.0 s ITI during which the cue lamp was turned off. Injections began after group performance stabilized at approximately 100 reinforcers per 40 min session. A single-subject design was employed where Monday–Wednesday–Friday were saline injection days. Both groups received either 10 or 20 ␮g Gal injections on Tuesday and Thursday, counterbalanced within each group. The results were analyzed by repeated measures ANOVA. Fig. 1 shows the effect of Gal on the rate of choice accuracy (Fig. 1A) and reaction time frequency distributions (proportion of total responses; Fig. 1B) for the RT test. Gal did not significantly influence choice accuracy (F [4,30] = 0.58, n.s.) nor the number of trials completed per condition (F [4,30] = 0.64, n.s., data not shown). Reaction times were sorted into

A. Brewer et al. / Neuroscience Letters 369 (2004) 245–249

Fig. 1. Gal (i.c.v.) did not significantly influence choice accuracy (A) or reaction times (B) in a light–dark discrimination task requiring a correct response within 2.5 s of stimulus onset for reward. For all figures, the means ± S.E.M. are presented, “Gal 10” = the 10 ␮g dose of Gal, “Gal 20” = the 20 ␮g dose of Gal, and three saline i.c.v. baseline injections sessions are indicated as “Saline A–C’.

bins of 500 ms intervals. While there was no significant main effect of condition (F [4,30] = 0.93, n.s.) nor a reaction time bin × condition interaction (F [20,150] = 0.58, n.s.). There was a significant main effect of time bin (F[5,150] = 65.0, p < .0001). In the DRH task, inter-response times were sorted into bins of 500 ms intervals. Analyses of the binned inter-response times (Fig. 2A) showed no significant main effect of condition (F [4,25] = 0.008, n.s.) nor IRT bin × condition interaction (F [16,100] = 0.25, n.s.), though there was a significant main effect of time bin (F[4,16] = 180.0, p < .0001). Gal significantly reduced the number of responses per condition (F [4,20] = 4.3, p < .02; Fig. 2B) and, similarly, reinforcers received per condition (F [4,20] = 3.8, p < .02; data not shown) for the DRH task. This paper reports the first studies to directly assess the effects of centrally administered Gal on reinforced reaction time and sustained high response rate. Both of these tasks encouraged the best possible performance by the rats as a means of assessing their maximum motor competence. Presently, Gal did not significantly alter the distribution of response

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Fig. 2. Gal (i.c.v) did not alter the distribution of inter-response times in the DRH (1.0 response/s) reinforcement schedule (A), but significantly reduced the number of responses per session (B). Presented are means ± S.E.M.

times, choice accuracy, or the number of trials completed per session in the RT test. Additionally, Gal did not alter the distribution of inter-response times in the DRH (1.0 response/s) reinforcement schedule, but significantly reduced the number of responses per session and reinforcers received per session. While the RT results are straightforward in their interpretation, the inherent feedback nature of the operant reinforcement process makes interpretation of the DRH results more complicated. In agreement with the reaction time test results, the capacity and tendency to respond at a high rate when the animal responds was not altered by Gal, as evidenced by DRH inter-response time distributions. However, while the rats may have had the capacity to respond at high rates, the decrease in the number of reinforcers received per session largely reflects an increase in the number of ten second DRH intervals in which the subjects did not respond at all. It is also possible that a decrease in reinforcer efficacy could have produced less responding in the DRH. The lack of a significant reduction in the rate of trial completion in the RT experiment suggests that the relative high cost (10

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responses/reinforcer) in the DRH versus low cost (one response/reinforcer) in the reaction time task may be one moderating factor. Some recent findings from our lab and others support this possibility. First, we have recently observed that Gal (i.c.v.) reduced the “break point” (the number of reinforcers received and responses emitted) in a progressive ratio schedule in which the number of operant responses required for each reward increased on each successive trial [7]. Second, Gal produced an increase in DOPA accumulation (indicating reduced firing) in dorso-lateral neostriaum and nucleus accumbens [9]. Finally, Gal, while not producing a conditioned place preference on its own, blocked morphine place preference and opiate withdrawal [38,39]. Future work may seek to further investigate Gal’s emerging role in the relationship between reinforcer efficacy and operant responding. The present lack of effects on the capacity of the animals under reinforcement incentive to emit fast responses and high response rates are generally consistent with behavioral studies that have examined rats treated with Gal or Gal analogs. For example, no change in swimming speed in the Morris water maze has been observed in rats similarly treated with Gal, i.c.v. [3,10,14,21,35,36]. The rate of pressing on a response lever mounted at the rear of the operant chamber by rats performing the delayed non-matching-to-position task has also not been reported to be affected by Gal administered i.c.v [15,16,25,26] or when injected into the amygdala, nucleus basalis magnocellularis, medial prefrontal cortex, or entorhinal cortex [27]. Gal injection i.c.v. and intraseptally failed to affect the number of arm entries in spontaneous alternation tests [33,36]. Gal overexpressing and Gal R1 knockout mice have been shown to be normal in neurological and motor test such as the rotorod, and to explore the open field in a manner comparable to wild-type mice [11,34]. Gal has generally also not produced pronounced effects on open field tests of ambulation, a test which is sensitive to many influences on exploratory behavior, including motor competence, alterations in novelty seeking, stress responsivity, and arousal level. One study reported a modest Gal-induced decrease in open field exploration at a 5.0 nmol, but not 0.5 nmol dose of Gal [9]. Interestingly, the mixed agonist–antagonist M35 (5.0 nmole) also produced a pronounced reduction in open field exploration and enhanced DOPA synthesis in this study [9]. In another study, intrastriatal injection of Gal reduced several locomotor parameters in the open field, including rearings, and the number of line crossings [29]. However, an apparent dose-dependency of this effect is suggested by other studies that reported no significant effects of i.c.v. Gal in the 1.0–1.5 nmoles (3.2–4.8 ␮g) range on open field exploration (e.g. [8,10]). When considered together with all the studies of Gal and various motor tests, the present findings suggest that when effects of Gal in the open field are observed, they may reflect changes in other non-motor factors affecting exploration (e.g. stress responsivity, novelty seeking), rather than a change in the maximal ability to initiate and maintain activity.

Acknowledgments The authors would like to thank Alice Blackshear and Georgia Bushell for assistance with the behavioral testing. This work was supported by a grant from the U.S. National Institute on Aging (1RO3 AG21295-01 to J.K.R.) and Swedish ¨ Research Council (to U.L.).

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