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Gerry Herrera, Ph.D. (background; Vice President of Research and Development at Med Associates and President of Catamount Research and Development), and Alex Packanowski, B.S. (foreground; Mechanical Engineer at Catamount R&D), calibrate white noise levels before starting experiments on our recent Rat Fear Potentiated Startle project. |
Fear Potentiated Startle in Rats- a Pharmacological Assessment
Gerald M. Herrera, Ph.D.
Med Associates, Inc. and Catamount Research & Development, Inc.
St. Albans, Vermont USA
Background/Introduction
Events that cause conditioned fear, such as aversive visual, auditory, olfactory, or tactile stimuli, have a profound effect to augment the relatively simple acoustic startle reflex response (e.g. Davis, 2001; Davis, 2006, Falls, 2001). The fear-potentiated startle paradigm is a popular research application used to investigate the neural substrates contributing to acquisition of conditioned fear.
In the basic procedure, an aversive stimulus (unconditioned stimulus, US), such as a mild foot-shock, is paired with a neutral stimulus (conditioned stimulus, CS), such as a tone or light. Following this training procedure, the acoustic startle reflex response is quantified in the presence and absence of the CS. An increase in the amplitude of the startle response in the presence of the CS is interpreted as conditioned fear. Many studies have been performed to assess pharmacological sensitivity of this conditioned fear, as well as the neurological substrates attributed to expression of conditioned fear (e.g. Davis et al., 1993; Davis, 1998; Risbrough et al 2003; Davis, 2006).
In this study, we have examined the effects of three standard anxiolytic compounds for their ability to reduce fear potentiated startle in rats. Our goal was to establish benchmark standards for positive control compounds to which test compounds could be compared in preclinical drug development screening studies.
Methods
General:
Rats (male Sprague Dawley; 200-250 grams) were used for all experiments. Animals were group housed (4 per cage) in ventilated racks with a 12:12 hour light:dark cycle. Temperature was 70±2 °F and relative humidity was 30-70 %. Med Associates Startle Pro package (MED-ASR-PRO) was used for all studies. Sound levels were calibrated using a USB Sound Pressure Measurement Package (Med Associates, ANL-929A-PC). Foot-shock intensity was calibrated using an Aversive Stimulus Current Test Package (Med Associates, ENV-420). All experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Med Associates, Inc.
Training:
On Day 1, animals were placed into the startle test chambers (ENV-264A) 5 min prior to the training session. Training consisted of 10 pairings of the conditioned stimulus (CS) with the unconditioned stimulus (US) with a variable inter-trial interval (ITI) of 2-4 minutes. The CS, either a white light or 12 kHz tone, lasted 4 sec, with the US (0.4 mA foot-shock) occurring during the last 250 msec of the CS. The background white noise and fan were on throughout, resulting in ~67dB of background noise.
Testing:
Twenty-four hours after training, animals were injected with the test compound or vehicle 30 minutes prior to testing (i.p.; 1.5 to 2 ml/kg injection volume). Animals were placed into the startle test chamber 5 minutes before testing. Testing began with 10 trials consisting of a white noise startle stimulus (100 dB, 50 msec) with an ITI of 30 sec. This portion of the experiment habituates the animal to the startle stimulus. The next phase of testing consisted of 20 trials (10 CS+startle, 10 startle alone, pseudorandom). The ITI was variable at 2-4 min. The background white noise and fan were on throughout, resulting in ~67dB of background noise.
Calculations/Statistics:
Fear conditioning was measured as the difference in the startle amplitude in the presence of the startle stimulus alone and the startle stimulus in the presence of the CS divided by the startle amplitude in the presence of the startle stimulus alone multiplied by 100 % (% Fear Potentiated Startle; %FPS). Statistical significance in %FPS scores among the various groups was determined using one-way ANOVA with a post hoc Student Newman-Keuls Multiple Comparison Test. Data are expressed as means ± SEM.
Results/Discussion
The GABA-A receptor agonist chlordiazepoxide abolished expression of FPS at doses ranging from 1 to 10 mg/kg (Figure 1). Lower doses of chlordiazepoxide (1 mg/kg and 3 mg/kg) increased the amplitude of the baseline startle response (Figure 1A). This complicates analyses, as the higher startle responding may represent ceiling responding. However, at a dose of 10 mg/kg chlordiazepoxide, baseline startle was no different from vehicle (Figure 1A) whereas FPS was completely suppressed (Figure 1B).
Another GABA-A receptor agonist, diazepam, was also examined in this study. Similar to chlordiazepoxide, diazepam, at doses from 0.3 to 3mg/kg, abolished fear potentiation of the startle response (Figure 2). However, unlike chlordiazepoxide, diazepam did not affect the amplitude of the startle response in the absence of the CS (Figure 2A).
We also examined the anxiolytic buspirone, which acts on the 5HT-2A receptor (Figure 3). Buspirone at concentrations of 1 mg/kg to 10 mg/kg increased the amplitude of the startle response in the absence of the CS (Figure 3A). As with chlordiazepoxide, this complicates analyses, as the decrease in fear potentiated startle could reflect a ceiling effect in the startle response. Although a dose-dependent decrease in fear potentiated startle was observed following buspirone treatment (Figure 3B), this could reflect the buspirone-induced increase in startle responding independent of the CS.
In conclusion, the rat fear potentiated startle model represents a relatively easy model to establish in the laboratory. The necessary equipment is readily available and is economically priced, which facilitates testing multiple animals at the same time. There are several practical considerations that must be kept in mind when conducting studies on the startle reflex response. The importance of properly calibrating the various components of the system cannot be over emphasized. Most startle equipment uses a force transducer of some type that transmits the animal’s startle response to the computer for analysis. This device must be properly calibrated to ensure efficient use of the recording system’s dynamic range. When calibrating the force transducer, both the size of the animal and the range of responses expected to be observed are important factors that should be considered. Another important component of any startle apparatus is the audio system. Important considerations here include the frequency response of speakers, the amplitude of noises required, and the frequency of noises required. In addition, the function of accessories such as aversive stimulus generators (foot-shock, air puff) and lights used as visual stimuli should all be checked to ensure optimum performance. These issues are particularly important when a startle apparatus consists of more than a single test station. In multi-station systems, it is critical that all stations are functioning the same so that data can be compared across stations. Other considerations pertain to specific models and protocols. For example, in pharmacological studies, it is important to examine “background” effects of drugs. In the present study, we demonstrated that a drug can increase the startle response in the absence of any CS. This effect complicates interpretation of the result that fear potentiation of the startle response is reduced by the drug. Additional complicating effects could include sedative actions or stimulatory actions of drugs. The primary literature contains many references that should be consulted dealing with these application-specific considerations.
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Falls, W.A. Fear-potentiated startle in mice. Cur Prot Neurosci 8.11B.1-8.11B.16,
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