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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,∗
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
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 [21].
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 [24].
∗ 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.
A pathological role for IL-6 on developing CNS neurons
using a culture model [22] and a chronic treatment paradigm
has been reported [35]. Several studies implicate IL-6 as an
important mediator in the development of neurologic disorders including AIDS dementia and Alzheimer’s disease [4].
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 [5]. 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 [40].
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 [1].
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
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 [31]. However, in another study, Clark et al. [15]
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 [29].
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
[16] 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 [33]. Ultimately, because cholinergic system plays an important role
in hippocampal memory [39] 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 [37]) 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 [10].
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 [10].
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.
[38] and modified to evaluate maze learning performance
in mice [19]. 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. [32], 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. [38]. 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.
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
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
[18] with slight modification as previously described [2].
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).
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
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)
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
[8]. 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 [11]. 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 [12].
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
D. Braida et al. / Behavioural Brain Research 153 (2004) 423–429
level. Armario et al. [3] and Butterweck et al. [13] 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 [23]. In addition, the bilateral
infusion of IL-6 into the hippocampus was shown to impair
retention in the passive avoidance task [29].
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 [20]. On the other hand a decreased ChAT activity
has been reported to correlate with impaired spatial memory in the radial arm maze [17]. 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 [9].
Short- and long-term memory, evaluated in a Y-maze [41]
and in a passive avoidance task [42] 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 [25] 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 [24], 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.
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