Design and Methods - Learning and Memory Mutations
High-throughput screen using fear-potentiated startle - We arel using a 24-hour FPS training and testing protocol [211] to screen 8,000 mice per year. For each deviant, data from each stage of the protocol are compared with established norms derived from the progenitor strain. Although the post-training FPS test provides the most valuable information on learning and memory, data collected during first two phases are essential to determining whether learning has taken place and provide an opportunity to begin to investigate the nature of any prospective mutation. Thus, mice whose pre-training startle amplitudes, shock reactivity (acquired during training), or post-training FPS data lie more than two s.d above or below the mean are selected for second-level screening.
Apparatus - Conditioning and FPS testing are conducted in commercially available MedAssociates startle chambers that consist of a sound-attenuating cubicle, stimulus panel, load-cell platform, and shocker. Separate and distinct cages for conditioning and startle response are mounted interchangeably on the load-cell platform. Cage movement results in displacement of the load cell generating a voltage proportional to the velocity of displacement. Startle amplitude is defined as the peak load-cell voltage that occurs during the first 100 ms after SS onset. The shock unconditioned stimulus is 0.5 s in duration and will be 0.6 mA. The stimulus panel includes a high-frequency tweeter for presentation of the SS, a mid-range speaker for presentation of the tone CS, and a florescent light for presentation of the light CS.
Procedure - The standardized set of stimulus parameters used for FPS (Startle Stimulus (SS), Conditioning Stimulus (CS) frequency/intensity, and shock duration) is based upon previous data collected in the B6 strain (Falls, W.A., et al.). The SS is a broadband noise ( 4-25 kHz, 100;105;110-dB (SPL; A scale), 20-ms in duration and a rise-fall of 0.1 msec). The CS is a pure tone delivered at a frequency of 12 kHz and intensity of 70 dB.
The FPS protocol consists of three sessions; a pre-training session (Phase 1), a conditioning session (Phase 2) and a post-training session (Phase 3).
Phase 1 - pre-training - 9 sequential SS alone trials (leaders); 9 CS-plus-SS trials and 9 SS alone trials presented randomly.
Phase 2 - training - 10-30 second CS followed by mild shock
Phase 3 - post-training - repeat of Phase 1
For Phases 1 and 3 the SS is delivered at 100,105 or 110 db (9 of each) with a 1-min inter-trial intervals (ITI). Each session has a total duration of approximately 30 minutes. For CS-plus-SS trials the SS is presented 29.5 sec after onset of the CS. Phase 2, CS-plus-shock training trials consist of the 30-sec CS tone co-terminating in a 0.5-sec 0.6 mA shock (ITI = 2.5 min).
FPS is calculated as the percentage difference in startle amplitude in the presence of the CS between the Phase 1 and Phase 3 trials. Initial criteria for selection of potential deviants are mice that show than less than a 20% increase in startle amplitude at each of the three SS intensities in CS+SS trials and less than a 20% increase in SS only trials.
Confirmation and secondary charcterizaton of potential learning and memory deviants - The main objectives of the secondary screen are to confirm the results of initial high-throughput Screen 1, to provide data for use in guiding a more comprehensive analysis of the phenotype of naïve mice in the third-level screen, and to detect mutations that specifically affect learning and memory. The properties of the CS and SS, the ability to form and retrieve an association between the CS and SS (i.e., learning and memory), and the ability to perform the fear response can all contribute to the acquisition and expression of conditioned fear. Thus, to evaluate the specificity of a mutation for learning and memory, are examining the contribution of CS, SS, and performance variables to poor or exaggerated FPS.
Apparatus - Same as in Screen 1.
Procedure - Screen 2 has three components [223]. Mice that show no FPS but normative responses in the pre-training test and on shock reactivity are given additional tone CS-plus-shock training (5 CS+shock trials every 24 hrs for a total of 30 trials) to determine whether conditioned fear can be acquired at all. Mice with exaggerated FPS are given additional tests for FPS (above test repeated at 24-hr intervals) to determine the rate of extinction, a measure of the strength of conditioning [207]. Mutants with excessively large or small startle amplitudes and those that show unusual startle-enhancing or startle-inhibiting effects of the tone CS in the pre-training test are further examined using a procedure in which the parameters of either the SS (i.e., intensity) or CS (i.e., intensity and frequency) are manipulated [217]. Unconditioned stimulus intensity contributes greatly to the strength of conditioned fear. Mice showing shock reactivity (high/low) are further examined by measuring shock reactivity across a range of intensities (0.2-1.8 mA in 0.2-mA steps).
Comprehensive phenotypic characterization of confirmed conditioned-fear mutants - Once heritability has been established we conduct several experiments with new conditioned-fear mutants to determine the association of conditioned fear with CS modality and contextual CS, to more fully examine the basis of aberrant FPS, and to determine whether mutations affect short- and/or long-term memory. Freezing, a complimentary measure of conditioned fear defined as a cessation of all bodily movements except those required for respiration [225], will be measured along with FPS. Measuring freezing addresses the possibility that a mutation is specific to FPS.
Apparatus - Same as in Screen 1. In addition to FPS, freezing will be measured using the same load cell following the procedure outlined by [226]: Freezing will be measured during training and testing.
Procedure - CS modality. Here we substitute a visual CS (light) for a tone CS; this allows conditioned-fear testing of animals with hearing problems or unconditioned effects produced by auditory stimuli, and a second CS also provides the opportunity to examine the generality of learning and memory deficits. Mice (power analysis indicates that n's of 8 are sufficient) are trained and tested as in Aim 1 with the exception of the different CS. Contextual fear. Conditioned fear can also be elicited by contextual stimuli. Contextual fear, unlike fear to an explicit CS, appears to involve both the hippocampus and the amygdala [227]. To assess the generality of learning and memory deficits in detected mutants, mice (n=8)are given shock-only training in the training chamber. Contextual fear is examined by comparing measurements of FPS/freezing in the training chamber 24 hr after training and in the startle chamber (a non-shocked context). The order of testing in the two contexts are counterbalanced and separated by 24 hrs. Contextual fear is defined as greater FPS/freezing in the training chamber than in the startle chamber. Basis of aberrant FPS. Conditioned fear is normally produced with a fixed number of training trials. One inherent problem is that mice displaying a slower acquisition or lower asymptotic levels of conditioned fear may be incorrectly identified as mice that do not learn. In addition, it is possible that poor or exaggerated FPS is the consequence of altered shock sensitivity. Thus, we will examine the number of training trials and shock intensity in a factorial experiment where groups of mice (n=8) are trained with successive 5-trial training blocks (24 hrs between blocks a total of 8 blocks), with one of three different shock intensities (0.4, 0.6 or 1.2 mA). Each block will be preceded by an FPS/freezing test. Control groups (n=8) that receive explicitly unpaired tones and shock are included to assess non-associative effects of stimulus presentation on startle and freezing. Short- and long-term memory. To examine whether a mutation affects short-term or long-term memory, or both [223, 224], two groups of mice are given five tone-plus-shock training trials followed by a test of FPS-freezing. FPS-freezing is assessed prior to training and either 1 hr or 24 hrs after training. Separate groups are used in the 1-hr and 24-hr tests to prevent extinction from confounding the test of long-term memory.
