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Behavioural Brain Research 153 (2004) 423–429 Research report Cognitive function in young and adult IL (interleukin)-6 deficient mice Daniela Braida a , Paola Sacerdote a , Alberto E. Panerai a , Mauro Bianchi a , Anna Maria Aloisi b , Stefania Iosuè a , Mariaelvina Sala a,∗ a Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Via Vanvitelli 32/A, 20129 Milan, Italy b Department of Physiology, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy Received 7 November 2003; received in revised form 19 December 2003; accepted 19 December 2003 Available online 1 February 2004 Abstract Interleukin-6 (IL-6) is a cytokine shown to affect brain function and to be involved in pathological neurodegenerative disorders such as Alzheimer’s disease (AD). In the present study we investigated the cognitive function in transgenic mice not expressing IL-6 (IL-6 KO) and in wild type (WT) genotype at 4 and 12 months of age, using a passive avoidance and an eight-arm radial maze tasks. Motor function was quantified using an Animex apparatus. Hippocampal choline acetyltransferase (ChAT) activity was evaluated in both genotypes. No difference was observed in both genotypes for spontaneous motor activity. The mean latency (s) to re-enter the shock box, was similar in both young mutant and WT mice. However, a decreased sensitivity (50%) to scopolamine (1 mg/kg) in mutant compared to WT mice, was obtained. IL-6 KO mice exhibited a facilitation of radial maze learning over 30 days, in terms of a lower number of working memory errors and a higher percentage of animals reaching the criterion as compared with WT genotype tested at both ages. Furthermore, mutant mice, at the age of 12 months, showed a faster acquisition (22 days versus 30 days to reach the criterion). The pattern of arm entry exhibited by IL-6 KO mice showed a robust tendency to enter an adjacent arm at both ages, while WT only at the age of 4 months. ChAT activity was inversely correlated with memory performance. These findings suggest a possible involvement of IL-6 on memory processes, even if the mechanism remains still unclear. © 2004 Elsevier B.V. All rights reserved. Keywords: Working memory; Reference memory; Knock out mice; Interleukin-6 1. Introduction Interleukin-6 (IL-6) is a plurifunctional cytokine implicated in the regulation of multiple aspects of immune response, haemopoiesis and inflammation [26,43]. In addition to its essential role in the function of immune system, it is present in the CNS, where it is constitutively expressed in different cell types and where specific binding sites have been found . Circulating IL-6 may exert neurochemical modifications in different mouse brain regions, such as the hippocampus, the hypothalamus and prefrontal cortex [7,44]. IL-6 can induce completely opposite actions on neurons, triggering either neuronal survival after injury or causing neuronal degeneration and cell death in disorders such as Alzheimer’s disease . ∗ Corresponding author. Tel.: +39-02-50317042; fax: +39-02-50317036. E-mail address: [email protected] (M. Sala). 0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2003.12.018 A pathological role for IL-6 on developing CNS neurons using a culture model  and a chronic treatment paradigm has been reported . Several studies implicate IL-6 as an important mediator in the development of neurologic disorders including AIDS dementia and Alzheimer’s disease . Furthermore, overexpression of IL-6 in the brain of transgenic mice (GFAP-IL6) has been shown to cause severe neurological disease, a progressive decline in avoidance learning [14,23] and a reduced long-term potentiation (LTP) in the dentate gyrus . In 10 months old senescence accelerated prone mice, a murine model for accelerated aging, the protein levels of IL-6 in the hippocampus and cerebral cortex is markedly increased . However, the role of IL-6 does not appear to be restricted to pathological conditions, since it is expressed in the normal brain, is developmentally regulated, has neurotrophic effects and is involved in the control of emotionality [3,13,14,28,30] and general behaviour . About the involvement of IL-6 on memory functions, little is known and the evidence obtained often contradictory. An improvement of scopolamine-induced cognitive deficit 424 D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429 was observed in mice peripherally treated with human recombinant IL-6 (0.125 and 0.5 g per mouse). This effect was not accompanied by changes in hippocampal levels of glutamine, aspartic acid, glutamate and GABA [8,11]. Furthermore, when continuously infused into the lateral ventricles of gerbils with 3-min forebrain ischemia, IL-6 prevented the occurrence of learning disability and hippocampal neuron loss . However, in another study, Clark et al.  found that the response to ischemic injury was the same in animals lacking IL-6 as in matched controls. Bilateral infusion of IL-6 into the rat hippocampus impaired retention using passive avoidance test . In order to better understand the role of IL-6 on CNS function, we investigated cognitive functions in transgenic male mice not expressing IL-6. Reference memory was studied using passive avoidance test, classically employed for the evaluation of drugs that interfere with cognitive functions in experimental animals  in absence or in presence of scopolamine. Working memory was tested in an eight-arm radial maze, a specific learning task that depends on intact hippocampus . Ultimately, because cholinergic system plays an important role in hippocampal memory  the choline acetyltransferase (ChAT) activity was analysed in this brain area of wild type (WT) and IL-6 knock out (KO) mice. 2. Materials and methods 2.1. Animals In order to obtain IL-6 deficient mice and to abolish IL-6 function, the sequence coding for the amino-terminal half of the protein was eliminated. ES cell clones (129 type, from the ES cell line CCE, see ) carrying the IL-6 mutation were injected into blastocysts of C57BL6 mice and transplanted into the uteri of F1 (CBA × C57BL6) foster mothers. Male chimeras were mated to MFI strain females and agouti offspring (representing germline transmission of the ES genome), were screened for the presence of the targeted IL-6 locus by Southern blot analysis of ECO-RI-digested tail DNA using the probe 5 . Female offspring heterozygous for the mutation were bred once with mice of the 129/SV/EV strain, from which the CCE ES cells were derived. The resulting heterozygous offspring were bred together to generate homozygous mice (IL−/−) for the mutation. These procedures have been described in greater detail [27,34]. Wild type (WT) littermates (IL-6+/+) were used as controls. The homozygous IL-6−/− mice and the WT IL-6+/+ mice used in these experiments were bred in our department. The mice were housed in a temperature-controlled room. Water was always available while food was provided ad libitum except during the radial maze experiments. All testing took place during the first half of the light period (between 9:00 and 13:00 h). Only male mice were used in the study. In the growth curves no differences were observed between IL-6 KO and wild-type mice. In each experimental group 10 animals aged 4 and 12 months were used. Experiments involving animals were performed in accordance with the NIH guidelines for care and use of laboratory animals. All behavioural studies were performed blind to the genotype of the animals. 2.2. Passive avoidance Scopolamine-induced amnesia for a passive avoidance response was assessed by a previously described method . Briefly, the apparatus consisted of two compartments, one light and one dark, connected via a sliding door. In the acquisition trial, each mouse was placed in the light compartment and allowed to enter the dark compartment; the time (in s) taken to do so was recorded. Mice having latencies greater than 40 s were eliminated. Once the mouse was in the dark compartment, the sliding door was closed and an unavoidable electric shock (1 mA for 1 s) delivered via the paws. The animal was then placed back in the home cage until the retention trial. The retention trial was carried out 24 h after the acquisition trial, by positioning the mouse in the light compartment and recording the time taken to enter the dark compartment (retention latency). An increased retention latency indicates that the animal has learned the association between the shock and the dark compartment. During the retention trial, a cut-off time of 180 s was used. To produce the amnesia for the avoidance task, mice were treated with 1.0 mg/kg i.p. of scopolamine 15 min before the acquisition trial. Latency to re-enter the shock box 24 h later was used as a measure of memory retention. Scopolamine hydrobromide (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in sterile pyrogen-free water and administered i.p. 2.3. Spontaneous activity meter In order to rule out an interference of motor activity on memory tasks, thus making difficult the interpretation of results, we evaluated spontaneous locomotor activity of young WT and IL-6 KO mice by using an Animex apparatus (L.K.B., Hagersten, Sweden) as elsewhere reported . Animals were tested after injection of either saline or scopolamine at the dose of 1 mg/kg. Upon the injection, the animals were returned to their cages. Fifteen min later, mice were introduced individually in a clean box of the same type as the home cage and the box was placed on the Animex apparatus. Activity counts were recorded for 30 min. 2.4. Radial maze Working memory was studied using a computerized wooden eight-arm radial maze according to Sala et al.  and modified to evaluate maze learning performance in mice . The modified radial maze consisted of eight arms (each 30 cm long, 7.5 cm wide, and with the enclosing walls 10 cm high), that extended radially from a central D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429 30 cm wide octagonal platform that served as a starting base. Small plastic cups mounted at the end of each arm held 15 mg food pellets as reinforcers. Access to the arms was controlled by eight pneumatically operated sheet-metal guillotine doors. The entire maze was painted black, elevated 50 cm from the floor, and placed in the center of a small room (2.5 m × 2.5 m) lit by fluorescent lights and fitted with several extramaze cues. Animal behavior was monitored by a video camera (Model CCD, Securit Alarmitalia) whose signals were digitized and interfaced by a PF6PLUSPAL apparatus 512×512 pixels (Imaging Technology, Woburn, MA), and sent to a video monitor (Trinitron KX-14CP1, Sony, Japan). Image analysis and pattern recognition were done by a Delta System computer (Addonics) using software provided by Biomedica Mangoni (Pisa, Italy). During each daily session, working memory was scored on the basis of the total number of errors (which corresponded to a re-entry into the arm just visited). At the same time, the pattern of arm entry was examined according to McCann et al. , who designed a method to analyse the angle chosen when the mouse entered two consecutive arms. In the eight-arm radial maze, five possible angles exist: from 0◦ , which corresponds to re-entry into the arm just visited, to 180◦ , corresponding to entry into the arm directly opposite the one just left. Angles of 45, 90, 135◦ may also be observed. The first eight-arm entries of a session were used in our analysis but since the first arm entry originates from the center of the maze, only seven angles were actually measured for each session. The frequency of each choice was calculated (frequency = number of observations/7 × 100). Starting 2 weeks before the experiment, the WT and IL-6 KO mices’ body weights were reduced by 10% by means of a restricted feeding schedule of standard chow (Harlan-Italy). The animals were kept at 90% of their free-feeding body weight for the duration of the experiment. After 3 days of free exploration the animals were trained to complete the maze as described by Sala et al. . Training continued, at the rate of one trial per day, until the mice reached the criterion of entering seven different arms in their first eight choices on 5 successive days, for a maximum of 30 days. The mean number of days taken to reach the criterion and the percentage of animals reaching the criterion were calculated. 425 2.6. Data analysis Data were expressed as mean (±S.E.M.) except for those concerning the percentage of animals reaching the criterion. One-way ANOVA for multiple comparisons followed by Tukey’s test was used. The total number of errors made in the radial maze were analysed by two-way ANOVA multiple comparisons followed by Bonferroni’s post hoc test. For non-parametric data Fisher’s exact test was used. Biochemical data were analysed by Student’s t-test. All data were analysed using GraphPad Prism (version 4) software. 3. Results 3.1. Passive avoidance Fig. 1 reports the passive avoidance response of 4-month-old WT and IL-6 KO mice. The passive avoidance response was different between groups [F(3, 36) = 74.58, P < 0.001]. Both genotypes did not differ in response latency when given vehicle. The scopolamine-induced cognitive deficit was significantly different between WT and IL-6 KO mice (P < 0.001). In fact, IL-6 KO mice showed a greater latency as compared with WT genotype. However, IL-6 KO mice given scopolamine had a retention latency significantly lower compared to the corresponding vehicle group. 3.2. Spontaneous motor activity ANOVA revealed a significant effect of treatment in mice tested for spontaneous motor activity [F(3, 36) = 247.82, P < 0.001]. Post hoc comparisons indicated no difference between WT and IL-6 KO genotype given vehicle (Fig. 2). Scopolamine induced an increase in locomotor activity in both genotypes when compared with corresponding vehicle group (P < 0.01). 2.5. ChAT activity At the end of the radial maze test, 4 months old WT and IL-6 KO mice were killed by decapitation, the right and left hippocampi were dissected and immediately frozen. Activity of the enzyme ChAT, which synthesizes acetylcholine, was measured by the formation of [14 C]acetylcholine from [acetyl-1-14 C]-acetylcoenzyme A (New England Nuclear, UK) and choline based on the method of Fonnum  with slight modification as previously described . The ChAT activity was expressed as nmol/100 mg of tissue protein per hour. Fig. 1. Effect of scopolamine (1.0 mg/kg i.p.), given 15 min before the acquisition trial, or vehicle on the passive avoidance response (mean ± S.E.M.) in wild type (WT) and IL-6 knock out (KO) mice aged 4 months. N = 10 for each group. ∗ P < 0.05, ∗∗ P < 0.01 as compared corresponding vehicle group; $$ P < 0.001 vs. scopolamine, KO genotype (Bonferroni post hoc test). 426 D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429 Fig. 2. Effect of scopolamine (1.0 mg/kg i.p.), given 15 min before the test, or vehicle on spontaneous motor activity evaluated in terms of cumulative counts (mean ± S.E.M.) for 30 min of wild type (WT) and IL-6 knock out (KO) mice aged 4 months. N = 10 for each group. ∗∗ P < 0.001 as compared with corresponding vehicle group (Bonferroni post hoc test). 3.3. Radial maze Learning, in terms of total number of errors in WT and IL-6 KO mice, at 4 and 12 months of age, over 30 days, is shown in Fig. 3. Two-way ANOVA revealed a major effect with genotype [F(1, 510) = 112.2, P < 0.0001] and days [F(29, 510) = 1.95, P < 0.003], and an interaction between genotype and days [F(29, 510) = 2.12, P < 0.0009] in 4-month-old naive mice. Post hoc comparison indicated a significant difference between WT and IL-6 KO starting from day 18. Similar differences were also found in 12-month-old naive groups [F(1, 510) = 17.13, P < 0.0001 for genotype; F(29, 510) = 4.18, P < 0.0001 for days and F(29, 510) = 1.76, P < 0.01 for the interaction]. IL-6 KO exhibited a greater learning starting from day 21. The percentage of mice reaching the criterion at different ages is shown in Fig. 4 (top). A percentage of about 80–90% was reached in IL-6 KO groups. Only about a 20% of WT 4 and 12-month-old mice reached the criterion within 30 days showing a significant difference as compared with the corresponding IL-6 KO group (P < 0.01 and P < 0.001, Fisher’s exact test). Considering the number of days taken to reach the criterion (Fig. 4, bottom), one-way ANOVA revealed a significant difference among groups [F(3, 36) = 10.51, P < 0.0001]. Post hoc comparison indicated no difference between WT and IL-6 KO 4-month-old group. In Fig. 4. Percentage of animals reaching the criterion (top) and number of days (mean ± S.E.M.) taken to reach the criterion (bottom) in wild type (WT) and IL-6 knock out (KO) naive mice aged 4 and 12 months. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 as compared with corresponding WT at the same age (Fisher’s exact test or Tukey’s post hoc test); $$$ P < 0.001 as compared with naive WT aged 4 months. contrast, WT 12-month-old group needed more training than IL-6 KO group tested at the same age (P < 0.05) and WT 4-month-old group (P < 0.001). The mean frequency of each of the five possible angles between consecutive arm entries in WT and IL-6 KO mice tested at different ages during the 5 days of training is shown in Fig. 5. WT mice tested at 4 months showed a tendency of 60%, to enter an arm adjacent (45◦ ) to the one they had just left [F(4, 45) = 448, ANOVA, P < 0.0001; P < 0.001, Tukey’s test]. WT 12-month-old mice exhibited an increased tendency (60%) to choice 90◦ angle frequency [F(4, 45) = 773, ANOVA, P < 0.001; P < 0.0001, Tukey’s test]. Concerning IL-6 KO mice strategy, a clear tendency (95%) to enter an arm adjacent (45◦ ) to the one they had just left was observed at both ages [F(4, 45) = 951.30, ANOVA test, P < 0.0001; P < 0.001, Tukey’s test for 4 months; F(4, 45) = 239, ANOVA test, P < 0.0001; P < 0.001, Tukey’s test] for 12 months. One-way ANOVA revealed significant dif- Fig. 3. Total number of errors (mean ± S.E.M.), made over 30 10-min daily sessions, starting from day 1 to 30 in wild type (WT) and IL-6 knock out (KO) mice aged 4 (left) and 12 months (right). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 as compared with IL-6 KO group (Tukey’s post hoc test). D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429 427 Fig. 5. Mean (±S.E.M.) pattern of arm entry, evaluated during the 5 days taken to reach the criterion, in wild type (WT) and IL-6 knock out (KO) mice aged 4 and 12 months. Abscissa: the five possible angles between consecutive arm entries. Ordinate: frequency (%) of occurrence during the first eight entries in any one session. ∗∗∗ P < 0.001 as compared with the remaining angles within the same age and genotype; $$ P < 0.01 as compared with 0, 135 and 180◦ within the same age and genotype; ## P < 0.01 as compared with the all remaining 45◦ (Tukey’s post hoc test). Table 1 Hippocampal ChAT activity of WT and IL-6 KO mice aged 4 months ChAT activity (mol Ach/h/100 mg) WT IL-6 KO a ∗ 51.56 ± 3.89a 12.30 ± 1.57∗ Values are means (±S.E.M.) of eight animals. P < 0.001 Student’s t-test. ferences among groups in the frequency to enter an adjacent arm [F(3, 36) = 6.08, ANOVA test, P < 0.003; P < 0.01, Tukey’s test] being decreased in WT 12-month-old group. 3.4. ChAT activity As reported in Table 1, a relevant significant decrease of ChAT activity was present in IL-6 KO mice in comparison with WT animals (P < 0.001). 4. Discussion The main results of the present study show that IL-6 deficiency leads to a facilitatory effect on learning and memory suggesting an important role of this cytokine in brain function. The results of the passive avoidance test suggest that IL-6 interacts with cholinergic system. In fact, even if IL-6 deficient mice did not show, under basal conditions, an increase in the step-through latency in comparison to WT, however they exhibited a reduced amnesic effect of scopolamine. This reduction seems to be selective for memory function since both genotypes showed a similar response to scopolamine-induced hypermotility. The present findings seem in contrast with the improvement of scopolamine-induced amnesia observed after IL-6 injection . However, in that experiment a single i.p. administration of IL-6 was given to animals and the responses recorded immediately after it. Other cytokines such as IL-1 and/or TNF could be involved in the acute effect of IL-6 . This experimental protocol is extremely different from knock out mice chronically deprived of IL-6. The facilitatory effect of gene IL-6 deletion on spatial learning was clearly demonstrated using radial maze task. Young and adult IL-6 mice exhibited a better and faster acquisition, in terms of a reduced number of errors and days to reach the criterion, in comparison to WT genotype. A tendency to enter an arm adjacent in both genotypes aged 4 months, was observed, but this tendency was lost in 12-month-old WT mice. These last findings suggest that IL-6 gene deletion allows animals to maintain, during aging, a strategy which is typical of young rodents . It can be argued that the facilitation of working and reference memory, found in IL-6 deficient mice, might be ascribed to the result of alterations in non-associative or motivational factors. At least for IL-6 KO mice aged 4 months, the lack of obvious performance abnormalities -IL-6 deficient mice appeared healthy, ate as WT genotype, did not show any signs of neurological abnormalities (data not shown) and exhibited a normal spontaneous motor activity—leads to interpret these findings as greater cognitive efficiency. On the contrary, for IL-6 KO mice aged 12 months, an interference of changes in motor activity, even if the behavior of these animals appeared normal (data not shown), cannot be excluded. Since IL-6 may be involved in the central mechanisms controlling the emotional response to stressful situations, our IL-6 deficient mice could have an altered emotional 428 D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429 level. Armario et al.  and Butterweck et al.  have found in IL-6 mutant mice an increased emotional behaviour evidenced by a lower level of ambulation and exploration of the open arms of plus-maze or in the open field. If this is truth, our IL-6 deficient mice should have exhibit a decreased performance in the radial maze and not a greater efficiency. In line with our data it has been reported a progressive age-related decline in avoidance learning performance in GFAP-IL-6 transgenic mice, which express IL-6 chronically from astrocytes in the CNS . In addition, the bilateral infusion of IL-6 into the hippocampus was shown to impair retention in the passive avoidance task . The mechanism by which the IL-6 deletion leads to an increased cognitive function remains still unclear. The decrease response to scopolamine-induced amnesia obtained in mutant mice corroborates the hypothesis of cholinergic involvement in memory function. The cognitive performance of both genotypes was inversely correlated to ChAT activity in the hippocampus. Contradictory results have been obtained until now between the relation of ChAT activity in the hippocampus and memory performance: positive, inverse or none: a mnemonic improvement after oxotremorine infusion in middle-aged rats was related to a decrease of ChAT activity in the dorsal hippocampus, rather than to an expected increase . On the other hand a decreased ChAT activity has been reported to correlate with impaired spatial memory in the radial arm maze . No changes in ChAT activity was observed in both young and old mice similarly performing in the radial maze [6,36]. A relevant reduction of -opioid receptors in the midbrain and an increased level of ␤-endorphin in the hypothalamus of IL-6 deficient mice has been previously reported, suggesting an alteration of endogenous opioid system . Short- and long-term memory, evaluated in a Y-maze  and in a passive avoidance task  have been reported to be impaired by treatment with endogenous or exogenus -opiate receptor agonists. The DAMGO-induced impairment of alternation performance was significantly improved by sistemic injection of physostigmine  suggesting an involvement of cholinergic system. Therefore, the obtained memory facilitation in IL-6 deficient mice may be due to the lack of inhibitory effect of -opioid receptors. The reason for the observed decrease of ChAT activity is unclear, but possibly reflects a compensatory mechanism. It must be pointed out, however, that the lack of expression of IL-6 gene might have affected brain development, and, as a consequence, some neuronal functions correlated with memory responses. Although we are aware that there are some limitations with the use of transgenic animals in behavioural studies (such as development of compensatory mechanism or the existence of redundancy mechanisms), taken together, our results suggest an involvement of IL-6 in learning and memory. Since IL-6 levels are elevated in neurodegenerative disorders such as Alzheimer’s disease , a therapeutic strategy against the progressive increase of this cytokine during aging, may be useful. Further studies on old IL-6 deficient mice will allow to define more precisely the role of IL-6 on memory function during aging. References  Alleva E, Citrulli F, Bianchi M, Bondiolotti GP, Chiarotti F, De Acetis L, et al. Behavioural characterization of interleukin-6 overexpressing or deficient mice during agonistic encounters. Eur J Neurosci 1998;10:3664–72.  Aloisi AM, Albonetti ME, Lodi L, Lupo C, Carli G. Decrease of hippocampal choline acetyltransferase activity induced by formalin pain. Brain Res 1993;269:167–70.  Armario A, Hernandez J, Bluethmann H, Hidalgo J. IL-6 deficiency leads to increased emotionally in mice: evidence in transgenic mice carrying a null mutation of IL-6. J Neuroimmunol 1998;92:160– 9.  Bauer J, Strauss S, Schreiter-Gasser U, Danter U, Schlegel P, Witt I, et al. Interleukin-6 and alpha-2-macroglubulin indicate an acute-phase syate in Alzheimer’s disease cortices. FEBS Lett 1991;285:111–4.  Bellinger FP, Madamba SG, Campbell IL, Siggins GR. Reduced long-term potentation in the dentate gyrus of transgenic mice with cerebral overexpression of interleukin-6. Neurosci Lett 1995;198:95– 8.  Bernstein D, Olton DS, Ingram DK, Waller SB, Reynolds MA, London ED. Radial maze performance in young and aged mice: neurochemical correlates. Pharmacol Biochem Behav 1985;22:301– 7.  Besedowsky HO, Del Rey A. Immune-neuro-endocrine interactions: facts and hypotheses. Endocr Rev 1996;17:64–102.  Bianchi M, Ferrario P, Clavenna A, Panerai AE. Interleukin-6 affects scopolamine-induced amnesia, but not brain amino acid levels in mice. NeuroReport 1997;8:1775–8.  Bianchi M, Maggi R, Pimpinelli F, Rubino T, Parolaro D, et al. Presence of a reduced opioid response in interleukin-6 knock out mice. Eur J Neurosci 1999;11:1501–7.  Bianchi M, Panerai AE. Interleukin-2 enhances scopolamine-induced amnesia and hyperactivity in the mouse. NeuroReport 1993;4:1046– 8.  Bianchi M, Sacerdote P, Panerai AE. Cytokines and cognitive functions in mice. Biol Signals Recept 1998;7:45–54.  Bierley RA, Rixen GJ, Troster AI, Beatty WW. Preserved spatial memory in old rats survives 10 months without training. Behav Neural Biol 1986;45:223–9.  Butterweck V, Prinz S, Schwaninger M. The role of interleukin-6 in stress-induced hyperthermia and emotional behaviour in mice. Behav Brain Res 2003;44:49–56.  Campbell IL, Abraham CR, Masliah E, Kemper P, Inglis JD, Oldstone MBA, et al. Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin-6. Proc Natl Acad Sci USA 1993;90:10061–5.  Clark WM, Rinker LG, Lessov NS, Hazel K, Hill JK, Stenzel-Poore M, et al. Lack of interleukin-6 expression is not protective against focal central nervous system ischemia. Stroke 2000;31:1715–20.  D’Mello GD, Steckler T. Animal models in cognitive behavioural pharmacology: an overview. Brain Res Cogn Brain Res 1996;3:345– 52.  Dunbar GL, Rylett RJ, Schmidt BM, Sinclair RC, Williams LR. Hippocampal choline acetyltransferase activity correlates with spatial learning in aged rats. Brain Res 1993;604:266–72.  Fonnum F. A radiochemical method for the estimation of choline acetyltransferase. Biochem J 1966;100:479–84.  Fredriksson A, Archer T. Alpha-phenyl-tert-butyl-nitrone (PBN) reverses age-related maze learning performance and motor activity deficits in C57 BL/6 mice. Behav Pharmacol 1996;7:245–53. D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429  Frick KM, Gorman LK, Markowska AL. Oxotremorine infusions into the medial septal area of middle-aged rats affect spatial reference memory and ChAT activity. Behav Brain Res 1996;80:99–109.  Gadient RA, Otten U. Expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6-R) mRNAs in rat brain during postnatal development. Brain Res 1987;15:313–4.  Gallo P, Frei K, Rordorf C, Lazdins J, Tavolato B, Fontana A. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system: an evaluation of cytokines in cerebrospinal fluid. J Neuroimmunol 1989;23:109–16.  Heyser CJ, Masliah E, Samimi A, Campbell IL, Gold LH. Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain. Proc Natl Acad Sci USA 1997;94:1500–5.  Hull M, Strauss S, Berger M, Volk B, Bauer J. The participation of interleukin-6, a stress-inducible cytokine, in the pathogenesis of Alzheimer’s disease. Behav Brain Res 1996;78:37–41.  Itoh J, Ukai M, Kameyama T. Dynorphin A-(1-13) potently improves the impairment of spontaneous alternation performance induced by the mu-selective opioid receptor agonist DAMGO in mice. J Pharmacol Exp Ther 1994;269:15–21.  Kishimoto T, Akira S, Taga T. Interleukin-6 and ist receptor: a paradigm for cytokines. Science 1992;258:593–7.  Lattanzio G, Libert C, Aquilina M, Cappelletti M, Ciliberto G, Musiani P, et al. Defective development of pristane-oil-induced plasmacytoma in interleukin-6-deficient balb/c mice. Am J Pathol 1997;151:689–96.  Loddick SA, Turnbull AV, Rothwell NJ. Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. J Cereb Blood Flow Metab 1998;18:176–9.  Ma TC, Zhu XZ. Effects of intrahippocampal infusion of interleukin-6 on passive avoidance and nitrite and prostaglandin levels in the hippocampus in rats. Arzneimittelforschung 2000;50:227–31.  Maeda Y, Matsumoto M, Hori H, Kuwabara K, Ogawa S, Du-Yan S, et al. Hypoxia/reoxygeneration-mediated induction of astrocyte interleukin-6: a paracrine mechanism potentially enhancing neuron survival. J Exp Med 1994;180:2297–308.  Matsuda S, Wen TC, Morita F, Otsuka H, Igase K, Yoshimura H, et al. Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils. Neurosci Lett 1996;204:109–112. 429  McCann UD, Rabin RA, Winter JC. Use of the radial maze in studies of phencyclidine and other drugs of abuse. Physiol Behav 1987;40:805–9.  Olton DS, Papas BC. Spatial memory and hippocampal function. Neuropsychologia 1979;17:669–82.  Poli V, Balena R, Fattori E, Markatos A, Yamamoto M, Tanaka H, et al. IL-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO J 1994;13:1189–96.  Qiu Z, Sweeney BD, Netzeband JG, Gruol DL. Chronic interleukin-6 alters NMDA receptor-mediated membrane responses and enhances neurotoxicity in developing CNS neurons. J Neurosci 1998;18:10445–56.  Reinstein DK, DeBoissiere T, Robinson N, Wurtman RJ. Radial maze performance in three strains of mice: role of the fimbria/fornix. Brain Res 1983;263:172–6.  Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotent cells by retroviral vector. Nature 1986;323:445–8.  Sala M, Braida D, Calcaterra P, Leone MP, Comotti FA, Gianola S, et al. Effect of centrally atropine and pirenzepine on radial maze performance in the rat. Eur J Pharmacol 1991;194:45–9.  Smith G. Animal models of Alzheimer’s disease: experimental cholinergic denervation. Brain Res 1988;472:103–18.  Tha KK, Okuma Y, Miyazaki H, Murayama T, Uehara T, Hatakeyama R. Changes in expressions of proinflammatory cytokines IL-1␤, TNF-␣ and IL-6 in the brain of senescence accelerated mouse (SAM) P8. Brain Res 2000;885:25–31.  Ukai M, Watanabe Y, Kameyama T. Effects of endomorphins-1 and -2, endogenous mu-opioid receptor agonists, on spontaneous alternation performance in mice. Eur J Pharmacol 2000;395: 211–5.  Ukai M, Lin HP. Involvement of mu(1)-opioid receptors and cholinergic neurotransmission in the endomorphins-induced impairment of passive avoidance learning in mice. Behav Brain Res 2002;129:197– 201.  Van Snick J. Interleukin-6: an overview. Annu Rev Immunol 1990;8:253–78.  Zalcman S, Green-Johnson JM, Murray L, Nance DM, Dyck D, Anisman H, et al. Cytokine-specific central monoamine alterations induced by interleukin-1, -2 and -6. Brain Res 1994;643:40–9.