Neurons in Medial Prefrontal Cortex Signal Memory for Fear Extinction, Article Review Example

Introduction

Neurons in the mammalian brain, although similar in structure, function in different ways depending upon their location. It is now well established that specific areas of the brain have special functions such as memory, emotions, balance of the body, comprehension of sensory stimuli, etc. Pavlov, in his classical experiment has already proven the ability of the brain to elicit a conditioned response when stimulated by a specific auditory input (A dog’s salivation was automatically increased when a bell, which was always rung when food was given, rang alone minus the food). The above study tries to identify the specific area of the brain which is responsible for fear extinction in rats which have been trained to respond to a noxious stimulus in a quantitatively measurable manner (Milad & Quirk, 2002).

Hypothesis

The authors have hypothesized that the medial prefrontal cortex (mPFC), an area of the brain lying in front of the frontal lobes of the cerebrum and towards its medial (inner side) may be responsible for storing long term extinction memory. By extinction memory, the authors mean the memory which assists the animal in recognizing that a painful stimulus which it was getting after a typical auditory input to which the animal was conditioned, is not necessarily associated with that sound. This is a sort of learned behavior which the animal understands after repetition of the sound is no longer accompanied by the painful stimulus. By the destruction of the ventral medial prefrontal cortex in rats, the authors have attempted to prove experimentally that it is this part of the brain that is associated with long term extinction memory. According to the authors’ the ventral medial prefrontal cortex is made up of infralimbic and prelimbic cortices (Milad & Quirk, 2002).

Study Design & Experiments

The authors designed a well planned and methodical study to prove their point by conducting experiments on male Sprague-Dawley rats, a specific strain developed for laboratory studies,  which satisfied strictly defined and specific criteria of uniformity – like weight between 300-350 gm., kept on a restricted diet to attain 85% of their original body weight, and which were the most suitable and the accepted laboratory animals for this kind of study. One of the end points to be used as an analysis criterion was the ‘freezing’ behavior of the animals in response to the noxious stimulus. In order to enable correct observation of the freezing behavior, the animals were pre-trained to press a mechanical bar for food at a variable interval schedule in order to bring about uniformity in all the selected experimental  animals. Standard housing in the operant chamber, wherein the experiments were conducted, were used for all animals so as to provide equal space for locomotion and observation of physical responses. In an aim to focus on the specific area of the brain which was to be studied, a standard surgical procedure was undertaken on the experimental rats to implant micro-electrodes in their brain, which could stimulate the specific area through electric stimuli administered artificially. Before any recording was done, the rats were allowed to recover from the trauma of the surgical procedure for one week. The rats were conditioned for fear by providing an auditory stimulus in the form of a tone at 4 kilohertz frequency which generated a sound of 80 decibels (dB) for a total duration of 30 seconds. This was accompanied by an unconditioned stimulus in the form of an electric shock of an intensity equal to 0.5 milliamperes (mA) for 0.5 seconds. The timing of both the auditory and the electric stimuli was adjusted in such a manner that they both ended simultaneously. When the trials were carried out, care was taken so as to keep the trial interval within an average span of 4 minutes. Continuous video monitoring was used to record the freezing incidents in the rats during the experiment. During the experiment, recording from the implanted electrodes was done using adequate amplification by specialized equipment and recording the waveform patterns on a personal computer. The recording was done during conditioning for both types of stimuli, auditory and electrical and individual cells in the brain were recorded for their electrical activity. Stimulating electrodes were used to provide impulses to the specific areas of the brain which were being investigated for their role in producing the extinction behavior in rats. The sequence of experiments followed the following pattern:

Auditory fear conditioning by a 30-s tone along with an unconditional stimulus of 5 s foot shock.

Day 1: 5 tones for habituation; 5 tones+foot shock for conditioning; after 1 hour- 20 tones without foot shock for extinction.

Day 2: 15 tones to test for recall of extinction learning.

During these two days, 74 neurons from mPFC from different locations viz. the infralimbic cortex (IL), prelimbic cortex (PL) and medial orbital cortex(MO) were recorded for electrical activity.

Based on the results obtained in these experiments, artificial electrical stimulation experiments were conducted on the IL neurons which further substantiated the initial significant findings.

Statistical Tools Employed

Analysis of Variance (ANOVA) and Student’s t test were employed to compare the neural tone responses, firing rates as well as spontaneous recovery of freezing. Comparisons were done using Tukey’s HSD (Honestly Significant Difference) method (Milad & Quirk, 2002). Parameters like spontaneous recovery of freezing, peak acquired freezing, tone induced activity and firing rates of neurons were calculated by appropriate mathematical formulae which included z-score transformation.

Results and Discussion

It was observed that during the conditioning phase, freezing to tone increased to a level of 75% which fell to 10% by the end of the extinction phase. A good recall of extinction learning was observed 24 hours later when the extinguished rats showed a level of 35% freezing as compared to 80% of the control (non extinguished) group. Initial spontaneous firing rates for neurons from different areas were 4.6, 5.5 & 4.2 Hz for IL, PL and MO cortex cells respectively which were not significantly different when analyzed by ANOVA. However, when the tone-elicited activity of neurons was evaluated, cells especially from the IL area showed robust activity by day 2, from the start of the extinction phase. The cells were unresponsive during the habituation and conditioning phases. The tone elicited activity of the IL cells translated into the occurrence of least freezing in the rats. After substantiation of data and further experimentation, it was proved that the IL cells, particularly reflect extinction memory due to the higher level of tone responses as compared to the cells from PL and MO cortical areas. The authors inferred that anatomical specificity does exist in the mPFC for the neurons from a specific area, the IL cortex to signal extinction memory. This analysis was substantiated by the fact that when specific lesions are present in certain areas of the mPFC when there is destruction of more than 70% of the IL neurons, the rats fail to show any extinction response and regain the freezing behavior. Further experiments carried out by the authors involved specific electrical stimulation of the cortical areas with calculated optimum levels of stimuli in the IL cortical regions, which remarkably resulted in the promotion of the extinction response. Rats receiving tones paired with electrical stimulation of IL cells showed significant reduction in freezing activity as compared to the control animals. In fact the IL stimulation resulted in the acceleration of the extinction learning process in a set of four trials conducted by the authors. Low freezing behavior in the IL stimulated rats continued even for the third day of the experiment. The authors, therefore opined that the IL cortical neurons might be responsible for long term extinction memory and might do so by the stimulation of the N-methyl-D-aspartate receptors in the brain. Moreover as the basolateral amygdala complex (BLA), an area in the brain known to play an important role in the learning and expression of auditory fear conditioning sends direct excitatory projections and enhanced input to the IL. The amygdala is connected to higher centers in the brain and the hypothalamus, and the process of extinction mediated through the IL region might be having a more broad spectrum and intricate mechanism of action.

Conclusion

The series of experiments conducted by the authors have generated enough data to show that a significant increase in the infralimbic cortex activity is associated with extinction learning and might be responsible for the inhibition of fear in rats leading to less freezing activity. This implies that in humans, this specific area of the brain might be subject to therapeutic interventions aimed at handling fear in depressed and psychiatric patients.

Works Cited

Milad, M.R. & Quirk, G.J. Neurons in medial prefrontal cortex signal memory for fear extinction, Nature,2002 Vol 420, Pgs. 70-73