1 Maze training. In current study rats were trained in the dual-solution plus-maze task developed 1
by Tolman et al. [1]. One week before training, rats were food-restricted to 85% of their ad libitum 2
body weight plus 5 g for normal growth. Rats were handled daily for 3 min beginning 1 wk before 3
training and given three piece of cereal, which later served as the food reward during training.
4
Rats were trained to find a food reward in a four-arm plus-shaped maze with floor and walls made of 5
black Plexiglas. The arms of the maze (12.5 cm wide by 46 cm long by 7 cm high) extended radially 6
from a central square platform (sides = 13 cm); the floor of the maze was positioned 0.7 m above the 7
floor. One of the four arms blocked so that the maze formed a “T” shape throughout training. At the 8
end of each arm was a container that contained inaccessible piece of cereal to eliminate the use of 9
olfactory cues to find the reward. Food reward was placed in the container in the goal arm such that it 10
was accessible to the rat. The training room (3 m × 4 m) contained a moderate density of cues 11
including high-contrast posters and dark-colored three-dimensional objects set against a light-colored 12
wall.
13
Before training, a habituation trial was given to allow all rats to encounter the food reward on the first 14
trial. If the rat did not enter the goal arm within 2 min during the habituation trial, it was placed in the 15
goal arm. For the training trials, the maze was configured into a T with the start and goal arms 16
remaining in the same relative position throughout training (Fig. 1A). At the start of a training trial, the 17
rat was placed in the start arm facing the choice point. If no choice was made within 2 min, the rat was 18
removed from the maze and placed in the holding cage for 30 sec before another trial was begun. On 19
trials in which the rat chose the goal arm, it was allowed to eat the reward and it was removed from the 20
maze after 10 sec or after it turned to exit the goal arm. On trials in which the rat did not choose the 21
goal arm, the rat was removed from the arm after 10 sec or after it turned to exit the arm. The intertrial 22
interval was 30 sec, during which the rat was placed in the holding cage. Training was completed 23
within a single session. All rats received one day session which consists of 100 training trials and 5 24
prob trials. Probe trials were administered after each 20 training trials in which the start arm was 25
rotated 180° relative to its position during training and both choice arms were baited (Fig. 1B).
26
2 The task used in the current study can be solved by using two different effective strategies, place and 27
response, that have been mapped onto the hippocampus and striatum, respectively [2]:Use of the place 28
strategy was indicated when rats went to the arm that was in the same location of the room as it was 29
during training. Use of the response strategy was indicated when rats turned in the same direction (left 30
or right) as they did during training(a multiple memory systems approach).Rats had a maximum of 2 31
min to enter an arm during the probe trial. For all trials correct or incorrect choice were recorded.
32 33 34
Goal
Start A
Place Response
B
35
TrainingTrial Prob trial
36 37
Supplementary Fig. 1.Maze configuration.For the training trials (A), the maze was configured into a 38
T with the start and goal arms remaining in the same relative position throughout training. For probe 39
trials (B) the start arm was rotated 180° relative to its position during training.
40 41
Tissue fractionation, electrophoresis and Western immunoblotting, The animals were deeply 42
anaesthetized via the respiratory route by exposure to ~ 15 ml diethyl-ether for approximately 2 min with 43
continuous monitoring of heart rate. Rat was decapitated when heart rate was reduced to approximately 44
one beat per second. After decapitation hippocampus was removed and frozen immediately on dry ice.
45
Brain tissue samples were rapidly homogenized in a buffer: 20 mMTris-HCl (pH 7.4), 0.32 M sucrose, 1 46
mM ethylendiamintetraacetic acid, 0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 47
Start
3 acid containing cocktail of protease inhibitors and centrifuged at 1000 g for 10 min. The supernatant 48
(nuclear-free homogenate) was centrifuged for 16 000g for 20 minutes, pellet was washed under the same 49
conditions and obtained P2 membrane-mitochondrial fraction was used in subsequent experiments. A 50
concentrated solution of sodium dodecyl sulphate (SDS) was added to the homogenate fraction to give a 51
final concentration of 5%. In all fractions, protein concentration was determined in quadruplicate using a 52
micro bicinchoninic acid protein assay kit (Pierce). Aliquots containing 30 µg of protein and of equal 53
volume were applied to the gels. SDS gel electrophoresis and Western blotting were carried out as 54
described previously [3]. After protein had been transferred onto nitrocellulose membranes, the 55
membranes were stained with Ponceau S solution and analysed with Image J software to confirm transfer 56
and the uniform loading of the gels. The membranes were then washed and cut according to the molecular 57
weight standards in 2parts to carry out separate immunostaining with used antibodies. The upper part of 58
the filters were stained with antibodies against NR2B (ab65783;Abcam), and the second part with 59
antibodies against α7 subunit of nACh (ab23832;Abcam).Standard immunochemical procedures were 60
carried out using peroxidase-labelled secondary antibodies and SuperSignal West Pico Chemiluminescent 61
substrate (Pierce). The blots were then exposed with intensifying screens to X-ray films pre-flashed with 62
Sensitize (Amersham). The optical densities of bands corresponding to the NR2Band α7nACh were 63
measured using LabWorks 4.0 (UVP). The autoradiographs were calibrated by including in each gel four 64
standards comprising the P2 fraction from the brain of untreated rats. Each standard contained 15-60 µg 65
total protein. Optical densities were proportional to the amount of studied receptors. To obtain the data 66
given in Fig. 2-3 the optical density of each band from an experimental sample (e.g. Hippocampus of 67
control rats) was divided by the optical density which, from the calibration of the same autoradiograph, 68
corresponded to 30 µg of total protein in the standard. The data expressed in this way will be referred to 69
as "relative amount" of NR2Band α7nACh.Data from experimental stained bands were not normalized 70
with respect to actin or any other housekeeping protein because it cannot be guaranteed that such proteins 71
are unaffected by our experimental conditions [see also: 4-6, for discussion of the unreliability of 72
4 normalization to housekeeping proteins see also 3]. Instead, we have controlled loading by Ponceau S 73
staining and calibrated with protein standards [3]
74
References:
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2. Manseau F, Goutagny R, Danik M, Williams S. The hippocamposeptal pathway generates 78
rhythmic firing of GABAergic neurons in the medial septum and diagonal bands; An 79
investigation using a complete septohippocampal preparation in vitro. J. Neuroscience.
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3. Meparishvili M, Nozadze M, Margvelani G, McCabe BJ and Solomonia RO. A Proteomic Study 82
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reference control for neuroscience research. Life Sci. 2013;92:747–751 89
6. Chen W, Xu WH. Beta-actin as a loading control: less than 2 mu g of total proteins hould be 90
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