Fred J. Helmstetter

Effects of muscimol applied to the basolateral amygdala on acquisition and expression of contextual fear conditioning in rats

The amygdala is known to be important for normal aversive Pavlovian learning in the rat. The relative contribution of the amygdala to the learning vs. performance of conditional fear with the GABAa agonist muscimol was assessed. Rats were prepared with cannulas aimed at the basolateral amygdala and trained in a contextual fear conditioning paradigm in which each subject received a series of footshocks in a distinctive observation chamber. Conditional responses evoked after exposure to the observation chamber were assessed 24 hr later. Rats that were pretreated with muscimol before performance showed a significantly attenuated fear response, and injections made before acquisition resulted in a much smaller decrement in conditional fear measured 24 hr after training. These results indicate that acquisition-related processes that may be occurring within the amygdala are more difficult to disrupt than those associated with performance.

Neural Substrates Mediating Human Delay and Trace Fear Conditioning

Previous functional magnetic resonance imaging (fMRI) studies with human subjects have explored the neural substrates involved in forming associations in Pavlovian fear conditioning. Most of these studies used delay procedures, in which the conditioned stimulus (CS) and unconditioned stimulus (UCS) coterminate. Less is known about brain regions that support trace conditioning, a procedure in which an interval of time (trace interval) elapses between CS termination and UCS onset. Previous work suggests significant overlap in the neural circuitry supporting delay and trace fear conditioning, although trace conditioning requires recruitment of additional brain regions. In the present event-related fMRI study, skin conductance and continuous measures of UCS expectancy were recorded concurrently with whole-brain blood oxygenation level-dependent (BOLD) imaging during direct comparison of delay and trace discrimination learning. Significant activation was observed within the visual cortex for all CSs. Anterior cingulate and medial thalamic activity reflected associative learning common to both delay and trace procedures. Activations within the supplementary motor area (SMA), frontal operculum, middle frontal gyri, and inferior parietal lobule were specifically associated with trace interval processing. The hippocampus displayed BOLD signal increases early in training during all conditions; however, differences were observed in hippocampal response magnitude related to the accuracy of predicting UCS presentations. These results demonstrate overlapping patterns of activation within the anterior cingulate, medial thalamus, and visual cortex during delay and trace procedures, with additional recruitment of the hippocampus, SMA, frontal operculum, middle frontal gyrus, and inferior parietal lobule during trace conditioning. These data suggest that the hippocampus codes temporal information during trace conditioning, whereas brain regions supporting working memory processes maintain the CS-UCS representation during the trace interval.

Translational control via the mammalian target of rapamycin pathway is critical for the formation and stability of long-term fear memory in amygdala neurons.

The mammalian target of rapamycin kinase (mTOR) regulates protein synthesis in neurons at the translational level through phosphorylation of several intracellular targets. Recent work in invertebrates indicates that mTOR-dependent translational control may be critical for the induction and maintenance of activity-dependent synaptic plasticity underlying memory formation. Here, we report that training rats in a simple fear conditioning procedure evokes a time-dependent increase in the phosphorylation of p70s6 kinase, a major direct downstream target of mTOR. When the activation of mTOR was prevented by posttraining injection of rapamycin into the amygdala, formation of the memory and the increase in p70s6 kinase phosphorylation was attenuated. Furthermore, when rapamycin was applied to the amygdala after the recall of a previously stored fear memory, subsequent retention was disrupted, indicating that local translational control at active synapses is required for the stability as well as the formation of long-term memory in this system.

Conditional analgesia, defensive freezing, and benzodiazepines

When rats are placed in a situation that has come to be associated with footshock through the process of Pavlovian conditioning, they react with the species-specific defensive response of freezing and a reduction in sensitivity to painful stimulation. In the present experiments, the effects of three benzodiazepines on both of these responses were examined. Pain sensitivity was measured with the formalin test. Concurrent observations of formalin-induced recuperative behavior and freezing were recorded while the animals were in the presence of shock-associated contextual stimuli. It was found that midazolam (Experiments 1 and 2), chlordiazepoxide (Experiment 3), and diazepam (Experiment 4) were capable of significantly attenuating the conditional analgesia. Midazolam and diazepam also reduced the freezing response. The finding that these anxiolytic agents attenuate both conditional responses suggests that the freezing and analgesia are mediated by a common fearlike process.

The amygdala is essential for the expression of conditional hypoalgesia.

Two experiments were conducted to determine whether the amygdala is involved in the performance of hypoalgesia as a Pavlovian conditional response. Rats were trained by pairing a distinctive observation chamber with a series of 3 footshocks. Rats were returned to the chamber 24 hr later, and the time spent engaged in freezing behavior and stereotyped behavioral reactions to a subcutaneous injection of dilute formalin was recorded. Sham-operated subjects spent large amounts of time freezing and were hypoalgesic on the formalin test in relation to nonshocked controls. Small electrolytic lesions of the amygdala eliminated both defensive freezing behavior and hypoalgesia without altering baseline reactions to formalin. Larger lesions made with ibotenic acid produced a similar pattern of results implicating neurons intrinsic to the amygdala. These results indicate that the amygdala may represent a forebrain site critical for the activation of descending antinociceptive systems in response to certain classes of environmental stressors.

Amygdala and hippocampal activity during acquisition and extinction of human fear conditioning.

Previous functional magnetic resonance imaging (fMRI) studies have characterized brain systems involved in conditional response acquisition during Pavlovian fear conditioning. However, the functional neuroanatomy underlying the extinction of human conditional fear remains largely undetermined. The present study used fMRI to examine brain activity during acquisition and extinction of fear conditioning. During the acquisition phase, participants were either exposed to light (CS) presentations that signaled a brief electrical stimulation (paired group) or received light presentations that did not serve as a warning signal (control group). During the extinction phase, half of the paired group subjects continued to receive the same treatment, whereas the remainder received light alone. Control subjects also received light alone during the extinction phase. Changes in metabolic activity within the amygdala and hippocampus support the involvement of these regions in each of the procedural phases of fear conditioning. Hippocampal activity developed during acquisition of the fear response. Amygdala activity increased whenever experimental contingencies were altered, suggesting that this region is involved in processing changes in environmental relationships. The present data show learning-related amygdala and hippocampal activity during human Pavlovian fear conditioning and suggest that the amygdala is particularly important for forming new associations as relationships between stimuli change.

Acquisition of fear conditioning in rats requires the synthesis of mRNA in the amygdala.

In this study, the role of mRNA synthesis in the amygdala was studied during the acquisition of conditional fear. Rats with cannulas placed in the basolateral region of the amygdala were trained with a series of noise-shock pairings in a distinctive observation chamber. One half of the rats were pretreated with the mRNA synthesis inhibitor actinomycin-D (act-D). Responding to the training context and the auditory stimulus in a novel context measured by defensive freezing was assessed. Pretreatment with act-D significantly attenuated fear responses to both stimuli. Animals receiving act-D injections exhibited normal reactions to the conditioned stimulus-unconditioned stimulus pairings in the initial training session and displayed normal learning when retrained 7 days after injections. These results indicate that the transcription of new mRNA and subsequent protein synthesis in the amygdala may be essential for neural plasticity during this form of associative learning.

Antinociception following opioid stimulation of the basolateral amygdala is expressed through the periaqueductal gray and rostral ventromedial medulla.

The amygdala, periaqueductal gray (PAG), and rostral ventromedial medulla (RVM) are critical for the expression of some forms of stress-related changes in pain sensitivity. In barbiturate anesthetized rats, microinjection of agonists for the mu opioid receptor into the amygdala results in inhibition of the tail flick (TF) reflex evoked by radiant heat. We tested the idea that TF inhibition following opioid stimulation of the amygdala is expressed through a serial circuit which includes the PAG and RVM. Rats were anesthetized and prepared for microinjection of DAMGO (0.5 microg/0.25 microl) into the basolateral amygdala (BLA) and lidocaine HCl (2.5%/0.4-0.5 microl) into either the ventrolateral PAG or RVM. Lidocaine did not significantly alter baseline values for TF latency or TF amplitude. When injected into the PAG prior to DAMGO application in the BLA, lidocaine significantly attenuated DAMGO-induced antinociception for the entire 40 min testing session. Similar treatment in the RVM also resulted in an attenuation of antinociception although rats showed significant recovery of TF inhibition by 40 min after lidocaine injection. Since acute injection of lidocaine into the RVM also affected baseline heart rate, separate animals were prepared with small electrolytic lesions placed in the RVM. Chronic RVM lesions also blocked TF inhibition produced by amygdala stimulation but did not affect heart rate. These results, when taken together with similar findings in awake behaving animals, suggest that a neural circuit which includes the amygdala, PAG, and RVM is responsible for the expression of several forms of hypoalgesia in the rat.

Functional MRI of Human Amygdala Activity During Pavlovian Fear Conditioning: Stimulus Processing Versus Response Expression

Although laboratory animal studies have shown that the amygdala plays multiple roles in conditional fear, less is known about the human amygdala. Human subjects were trained in a Pavlovian fear conditioning paradigm during functional magnetic resonance imaging (fMRI). Brain activity maps correlated with reference waveforms representing the temporal pattern of visual conditional stimuli (CSs) and subject-derived autonomic responses were compared. Subjects receiving paired CS-shock presentations showed greater amygdala activity than subjects receiving unpaired CS-shock presentations when their brain activity was correlated with a waveform generated from their behavioral responses. Stimulus-based waveforms revealed learning differences in the visual cortex, but not in the amygdala. These data support the view that the amygdala is important for the expression of learned behavioral responses during Pavlovian fear conditioning.

Lesions of the amygdala block conditional hypoalgesia on the tail flick test

Exposure to an innocuous stimulus that has been paired with footshock during Pavlovian conditioning results in the activation of descending antinociceptive systems in the rat. Several recent studies indicate that the hypoalgesia observed when contextual stimuli are paired with shock and the formalin test is used to measure antinociception depends on the integrity of a neural circuit which includes the amygdala and the periaqueductal gray. The present experiment was designed to determine if the amygdala is also critical for hypoalgesia in response to a discrete auditory signal for footshock when hypoalgesia is measured with the radiant heat tail flick test. Groups of rats were exposed to a series of paired presentations of a tone and footshock or associative control treatments. After training, one half of the animals received large electrolytic lesions of the amygdala. Lesions of the amygdala blocked the time dependent elevation in tail flick latency following tone presentation in animals given paired training, but did not alter baseline tail flick responding. These data indicate that the amygdala is also essential for fear-related modulation of spinally mediated nociceptive reflexes, and provide further support for our current model in which amygdalo-mesencephalic projections are critical for the expression of certain forms of stress-induced hypoalgesia.