Supplementary material for Steiner et al. (March 20, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.061445598

Mice were group-housed by gender in a temperature- and humidity-controlled specific pathogen-free vivarium. Behavioral tests were conducted during the light phase of the circadian cycle, 7 a.m.–7 p.m.. Mice used in the present experiments were identified by eartag numbers after genotyping; 11 male and 10 female galanin transgenic (GAL-tg) mice, including both homozygote and heterozygote transgenics, were age-matched from the same litters as 16 male and 9 female wild-type (WT) control mice.

Before evaluating the effects of galanin overexpression on learning and memory, we used our standardized three-tiered behavioral phenotyping strategy as previously described (1-3). All mice were first observed for incidence of aberrant home cage behaviors and on measures of general health. A battery of neurological reflexes was then conducted. Sensory abilities were tested by using standard acoustic startle, hot plate, and tail flick tests. Motor abilities were tested by using a standard Digiscan open field apparatus to measure exploratory locomotion over a 2-h period and latency to fall from an accelerating rotarod over a 5-min session. These preliminary tests were designed to avoid false positives caused by potential health problems or specific deficits in sensory or motor abilities.

Learning and memory were tested when the cohort of GAL-tg and WT littermate control mice were 8, 16, and 24 months old. Performance was assessed in the Morris water task, by using standard methods and equipment as described ( 4--11). This spatial navigation task quantitates learning of environmental cues to locate an escape platform in a large pool of water. Mice were tested on three components in the following order: (i) learning to swim to a visible platform, which measures procedural abilities required to solve the task, including vision and swimming abilities; (ii) learning to locate a hidden platform over successive training trials, by using an acquisition criterion of latencies of 15 sec or shorter to reach the hidden platform; and (iii) memory of the former location of the hidden platform when the platform is removed. This probe trial is a transfer test that provides the critical proof that environmental spatial cues were learned and remembered as the specific strategy for locating the hidden platform.

A circular pool, 1 m in diameter (Nalge) was filled with water, maintained at 22°C, and rendered opaque by the addition of nontoxic white paint. The tank was emptied, cleaned, and refilled at the end of each test day. Video tracking was conducted with a video camera focused on the full diameter of the pool. Navigation parameters were analyzed by using NIH Image WM software, originally developed by Wayne Rasband, National Institute of Mental Health (NIMH), and modified by Tsuyoshi Miyakawa, NIMH. Training on the visible and hidden platform tasks consisted of placing the mouse in a new quadrant on each successive trial facing the center of the pool, allowing a maximum of 60 sec to reach the platform, and removing the mouse from the platform after 15 sec. Four trials per day were administered for 3 days of training on the visible platform task and 5 days of training on the hidden platform task. At the end of the last day of training on the hidden platform task, the platform was removed and a 60-sec probe trial was administered. Parameters analyzed included latency to reach the platform, swim speed, swim pattern, thigmotaxis (time spent in the outer 8 cm of the pool) for the training trials, and time spent in each quadrant and number of crossings over the former location of the platform for the probe trial.

The same mice were tested at age 26 months on the social transmission of food-preference memory task. Methods have been described (12, 13). Demonstrator mice ate powdered chow flavored with either thyme or turmeric for 1 h. Observer cage mates sniffed the scent on the breath of the demonstrator mouse for 30 min. After 24 h, each observer was given a choice between chow containing the familiar flavor previously cued by the demonstrator's breath odor and chow containing the uncued novel flavor. Food consumption was then measured over a 60-min preference test.

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