*4.5. LGF and BrdU Administration*

Twenty-month-old mice received 2 weekly 100 μL i.p. injections of saline used as a vehicle (WT and APP groups) or LGF (1.6 μg/100 μL) (WT-LGF and APP-LGF groups) for 3 weeks. This optimal dose of LGF has been used in different model systems using an identical or similar schedule [13,14]. Mice were sacrificed 1 week after the last treatment with vehicle or LGF (Figure 7).

**Figure 7.** Representative scheme showing the methodological procedure followed for the administration of LGF or vehicle to 20 months old WT and APPswe mice.

To determine whether the i.p. administration of LGF increased cell proliferation, a group of WT and APP animals were injected daily for 3 weeks with the mitotic marker 5-bromodeoxyuridine (BrdU) (50 mg/kg i.p.), starting 24 h after the beginning of LGF/saline injections.

### *4.6. Behavioral Studies*

Exploratory behavior and body weight were measured before treatment initiation to allocating the animals to the experimental groups (WT, WT-LGF, APP, APP-LGF). After 3 weeks of treatment with vehicle or LGF, mice were subjected to behavioral analysis. Two behavioral tests were carried out in this study: Y-maze test and marble burying test.

#### 4.6.1. Y-Maze

Spatial working memory was measured using the Y-maze behavioral paradigm. The Y-maze test is based on the natural exploratory preference of mice to alternate arms when exploring a new environment. The spontaneous alternation behavior of mice in the Y-maze was analyzed to assess short term memory impairment in mice, following the methodology previously reported by Perucho et al. [81,82]. Testing takes place in a Y-shaped maze with three white, opaque plastic arms spaced at an angle of 120 degrees. Briefly, after acclimatizing the mice to the behavioral room (30 min), each mouse was placed at the same end of one arm of the Y-maze and allowed to move freely through the maze for 5 min. The number of arms entered, as well as the number of spontaneous alternations, defined as a sequence of entries in each of 3 consecutive arms without repetition, were recorded to determinate the alternation rate. The percentage of alternation score was calculated as follows:

$$\text{Spontaneous alteration} \%= \left(\frac{\text{number of spontaneous alternatives}}{\text{total number of arm entries} - 2}\right) \times 100\tag{1}$$

### 4.6.2. Marble Burying Test

Marble burying was assessed as an additional measure of exploratory digging. This test is a useful paradigm to study repetitive compulsive-like behaviors based on the observation of rodents burying objects in their bedding, a phenomenon dependent on hippocampal function [75]. Briefly, mice were individually housed in a cage filled with 5 cm of wood chip bedding for a 30 min testing period of acclimation, and continued throughout the test session. Tests were performed between 18:00 and 20:00 h, under standard laboratory conditions of temperature, 22 ± 2 ◦C, 12 h light:dark cycle, relative humidity 50–60% and with food and water restriction. The mice were placed individually for 30 min in plastic cages, with 9 marbles placed on top of the bedding material, in a 3 × 3 grid with 4 cm center-to-center spacing. A new cage, clean marbles and fresh bedding were used for each mouse. At the end of that time, each mouse was returned to its home cage and the number of marbles uncovered; completely buried (not visible); 2/3 covered (only top visible) and half covered, were counted. This classification was used to obtain a "buried index", as an indication of activity and interaction with their environment [83].
