Consolidation and reconsolidation of contextual fear memory requires mammalian target of rapamycin-dependent translation in the dorsal hippocampus.

The mammalian target of rapamycin (mTOR) pathway is important for regulating protein translation. The present study characterized the role of mTOR-dependent translation in the dorsal hippocampus (DH) during the consolidation and reconsolidation of contextual fear memory. We first showed that fear conditioning resulted in increased phosphorylation of p70s6 kinase (p70s6K) in the DH and that infusion of the mTOR inhibitor rapamycin (RAP) into the DH immediately after training disrupted formation of long-term contextual fear memory. Additionally we showed that p70s6K was activated after retrieval of a previously stored fear memory, and inhibition of mTOR by DH infusion of RAP blocked the reconsolidation of contextual fear memory. Together these results demonstrate that within the DH translational control through the mTOR pathway is important for consolidation as well as the stability of fear memory after retrieval.

An updated unified pharmacophore model of the benzodiazepine binding site on gamma-aminobutyric acid(a) receptors: correlation with comparative models.

A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.

The formation of auditory fear memory requires the synthesis of protein and mRNA in the auditory thalamus.

The medial geniculate nucleus of the thalamus responds to auditory information and is a critical part of the neural circuitry underlying aversive conditioning with auditory signals for shock. Prior work has shown that lesions of this brain area selectively disrupt conditioning with auditory stimuli and that neurons in the medial geniculate demonstrate plastic changes during fear conditioning. However, recent evidence is less clear as to whether or not this area plays a role in the storage of auditory fear memories. In the current set of experiments rats were given infusions of protein or messenger RNA (mRNA) synthesis inhibitors into the medial geniculate nucleus of the thalamus 30 min prior to auditory fear conditioning. The next day animals were tested to the auditory cue and conditioning context. Results showed that rats infused with either inhibitor demonstrated less freezing to the auditory cue 24 h after training, while freezing to the context was normal. Autoradiography confirmed that the doses used were effective in disrupting synthesis. Taken together with prior work, these data suggest that the formation of fear memory requires the synthesis of new protein and mRNA at multiple brain sites across the neural circuit that supports fear conditioning.

Calcitonin gene-related peptide released within the amygdala is involved in Pavlovian auditory fear conditioning.

The effects of CGRP and the CGRP receptor antagonist hCGRP(8-37) injected into the amygdala on both the acquisition and expression of fear behavior to a discrete auditory conditional stimulus (CS) and the training context were assessed. In Experiment 1, pretraining injections of CGRP but not hCGRP(8-37) produced fear-like behavior before any aversive stimuli were presented. While both compounds attenuated freezing to the contextual CS on the test day, neither affected learning about the auditory CS. In Experiment 2, pretesting injections of hCGRP(8-37) (0.63 mM) selectively attenuated freezing to the auditory CS but left freezing to the contextual CS intact. These data suggest that CGRP in the amygdala may selectively contribute to the expression of learning about auditory stimuli during fear conditioning.

Activation of kappa opioid receptors in the rostral ventromedial medulla blocks stress-induced antinociception.

Prior work has shown that kappa opioids may attenuate the effects of analgesic mu receptor agonists in some neural circuits related to pain modulation. This study examined whether hypoalgesia following exposure to a signal for shock is attenuated by infusions of the kappa agonist U69593 into the rostral ventromedial medulla (RVM). Rats were trained with paired or unpaired presentations of white noise and foot shock. On test days, tail flick latencies were measured before, during, and after exposure to the auditory conditioned stimulus (CS). One of three doses of U69593 (0.0445, 0.178 and 1.00 microg) or an equivalent volume of saline was injected into the RVM. Rats that had received noise-shock pairings displayed conditional hypoalgesia (CHA) compared to those given unpaired presentations. Expression of CHA was completely blocked by the highest dose of U69593 (1.00 microg) injected 20 min before testing, indicating an antagonistic effect of U69593 on expression of CHA. These findings are discussed in terms of the evidence for antagonism of morphine- and DAMGO-induced hypoalgesia by kappa agonists.

Antinociception produced by mu opioid receptor activation in the amygdala is partly dependent on activation of mu opioid and neurotensin receptors in the ventral periaqueductal gray.

Exposure to stressful or fear-inducing environmental stimuli activates descending antinociceptive systems resulting in a decreased pain response to peripheral noxious stimuli. Stimulating mu opioid receptors in the basolateral nucleus of the amygdala (BLA) in anesthetized rats produces antinociception that is similar to environmentally induced antinociception in awake rats. Recent evidence suggests that both forms of antinociception are mediated via projections from the amygdala to the ventral periaqueductal gray (PAG). In the present study, we examined the types of neurochemicals released in the ventral PAG that may be important in the expression of antinociception produced by amygdala stimulation in anesthetized rats. Microinjection of a mu opioid receptor agonist into the BLA resulted in a time dependent increase in tail flick latency that was attenuated by preadministration of a mu opioid receptor or a neurotensin receptor antagonist into the ventral PAG. Microinjection of a delta(2) opioid receptor antagonist or an NMDA receptor antagonist into the ventral PAG was ineffective. These findings suggest that amygdala stimulation produces antinociception that is mediated in part by opioid and neurotensin release within the ventral PAG.

Expression of antinociception in response to a signal for shock is blocked after selective downregulation of mu-opioid receptors in the rostral ventromedial medulla.

Prior work has shown that release of endogenous ligands for mu-opioid receptors in the rostral ventromedial medulla (RVM) is critical for the modulation of spinal nociceptive reflexes observed during stress. In the present study, we used antisense oligodeoxynucleotides (AS ODN) to suppress synthesis of mu-opioid receptors in the RVM prior to activating descending antinociceptive systems with a signal for foot shock. Five groups of rats with RVM cannulae were trained with paired or unpaired exposures to white noise (WN) and foot shock. Over several days, they received RVM infusions of an AS ODN probe targeting exon 1 of the cloned MOR-1 receptor, an inactive missense (MS) ODN with the same base composition in which the sequence for four bases was changed, an AS ODN probe targeting exon 4, or saline. Tail-flick latencies (TFLs) were measured before, during, and after presentation of the auditory signal for shock. Rats given paired training and saline injections displayed longer TFLs than saline control rats given unpaired exposures to WN and shock, confirming the ability of the conditional stimuli (CS) to elicit antinociception. Expression of this conditional hypoalgesia (CHA) was attenuated by pretreatment with the AS ODN probe targeting exon 1, but was unaffected by pretreatment with AS ODN probe targeting exon 4 or MS ODN sequence for exon 1. However, pretreatment with the AS ODN probe targeting exon 1 did not affect expression of conditional freezing to other shock-associated cues. Testing of the same animals several days after the ODN injections showed that the attenuating effect on expression of CHA were reversible. These results support the idea that mu-opioid receptors in the RVM are critically involved in mediating expression of hypoalgesia following stress. They also provide further evidence for dissociation in the mechanisms mediating expression of aversive conditional responses.

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.

Functional MRI of human Pavlovian fear conditioning: patterns of activation as a function of learning.

fMRI was used to study human brain activity during Pavlovian fear conditioning. Subjects were exposed to lights that either signaled painful electrical stimulation (CS+), or that did not serve as a warning signal (CS-). Unique patterns of activation developed within anterior cingulate and visual cortices as learning progressed. Training with the CS+ increased active tissue volume and shifted the timing of peak fMRI signal toward CS onset within the anterior cingulate. Within the visual cortex, active tissue volume increased with repeated CS+ presentations, while cross-correlation between the functional time course and CS- presentations decreased. This study demonstrates plasticity of anterior cingulate and visual cortices as a function of learning, and implicates these regions as components of a functional circuit activated in human fear conditioning.

The role of mu and kappa opioid receptors within the periaqueductal gray in the expression of conditional hypoalgesia.

The periaqueductal gray (PAG) is a midbrain structure involved in the modulation of pain and expression of classically conditioned fear responses. Non-selective opioid antagonists applied to the PAG block the expression of hypoalgesia in rats exposed to a Pavlovian signal for shock. This study was conducted to determine the anatomical and pharmacological specificity of the PAG's role in conditional hypoalgesia. Rat subjects received injections of either the mu opioid antagonist CTAP (6.6 nMol), the kappa opioid antagonist Nor-binaltorphimine (Nor-BNI, 6.6 nMol) or saline. Injections were made into either the dorsolateral (dlPAG) or ventrolateral (vlPAG) PAG prior to the presentation of an auditory stimulus that had previously been paired with foot shock while measuring nociception with the radiant heat tail flick (TF) test. Elevation in TF latency in response to the auditory stimulus was blocked only by administration of CTAP into the vlPAG. These results suggest that conditional hypoalgesia (CHA) is subserved by mu but not kappa opioid receptors located in the vlPAG but not the dlPAG.

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.

The effects of central injections of calcitonin gene-related peptide on fear-related behavior.

Calcitonin gene-related peptide (CGRP) has been localized in several regions of the rat brain that are known to be important for the expression of fear responses. Some evidence suggests that CGRP may act as a neurotransmitter at synapses that are believed to be important for aversive learning. In the present study, male rats were prepared with intracerebroventricular cannulae and injected with CGRP during different phases of training and testing when a distinctive environment was paired with foot shock. When injected prior to training, CGRP directly evoked fear-related behavior and postshock freezing. When pretreated with CGRP and exposed to the shock-associated environment 24 h after training rats again showed an enhanced fear response. These results support the idea that this peptide functions as a neurotransmitter at central synapses which are important for the expression of fear.

Neural systems for the expression of hypoalgesia during nonassociative fear.

A single brief exposure to moderately intense while noise is sufficient to produce opioid-mediated antinociception in rats. This form of stress-induced hypoalgesia represents a response to unconditional fear or anxiety. Three experiments compared the neural circuits responsible for learned versus unlearned fear responses. Male rats received lesions of the medial geniculate nucleus, lateral or central nuclei of the amygdala, or the ventral, dorsal lateral, or dorsal medial periaqueductal gray (PAG). Controls showed a pronounced elevation in tail-flick latency following presentation of 90-dB white noise. All lesions, with the exception of dorsolateral and dorsomedial PAG, significantly blocked this response. These results support the idea that hypoalgesia produced by aversive auditory stimuli uses a common neural circuit regardless of whether the response is a product of associative learning or unconditional fear/anxiety.

Microinfusion of mu but not delta or kappa opioid agonists into the basolateral amygdala results in inhibition of the tail flick reflex in pentobarbital-anesthetized rats.

Recent evidence suggests that certain forms of opioid-mediated hypoalgesia may depend on monosynaptic projections from the amygdala to nociceptive modulatory neurons in the midbrain. We recently demonstrated that the microinjection of morphine sulfate into the basolateral nucleus of the amygdala will result in a robust elevation of radiant heat tail flick (TF) latency in the pentobarbital-anesthetized rat. The present study was conducted to begin to clarify the opioid receptor type(s) responsible for this effect. Rats were anesthetized with sodium pentobarbital and prepared for microinfusion and TF testing. Rats received simultaneous bilateral infusions of agonists for mu ([D-Ala2, N-MePhe4, Gly-ol 5] enkephaphalin (DAMGO); 0.01, 0.05, 0.1, 1.0 or 5.0 micrograms), delta ([D-Pen2, D-Pen5]enkephalin [DPDPE]; 6.458 or 64.58 micrograms) or kappa (trans-3,4-dichloro-N-methyl-N-(2-(1-pyrolidinyl)-cyclohexyl)-benz ene acetamide methanesulfonate hydrate [U50, 488H]; 5.0, 40.0 or 84.0 micrograms) opioid receptors during TF testing. The mu agonist produced a dose- and time-dependent elevation in TF latency when injected into the basolateral amygdala. Application of the delta and kappa agonists to similar sites within the amygdala was without effect. In separate experiments, U50, 488H and DPDPE were injected into the lateral ventricle at concentrations similar to those applied to the amygdala. Intracerebroventricular administration of these compounds resulted in reliable inhibition of TF. These results indicate that mu opioid receptors in the basolateral amygdala may be able to modulate transmission in a recently identified neural circuit that is at least partially responsible for the expression of stress-related hypoalgesia in behaving animals.

Lesions of the periaqueductal gray and rostral ventromedial medulla disrupt antinociceptive but not cardiovascular aversive conditional responses.

The presentation of an auditory stimulus that signals a noxious event such as foot shock results in the simultaneous expression of multiple aversive conditional responses (CRs), which include a transient elevation of arterial blood pressure (ABP) and an opioid-mediated form of hypoalgesia. Recent evidence suggests that the neural circuits responsible for the expression of these two aversive responses may overlap. In the present study, rats were trained using a Pavlovian fear conditioning paradigm in which white noise was repeatedly paired with shock. After training, groups of animals received electrolytic lesions centered in the dorsal or ventral periaqueductal gray (PAG) or in the medial or lateral rostral medulla. In sham-lesioned animals that were given paired presentations of noise and shock, subsequent presentation of the auditory stimulus caused a significant transient elevation of ABP and time-dependent inhibition of the tail flick reflex evoked by radiant heat. Lesions of either the dorsal or the ventral PAG blocked the antinociceptive CR but did not significantly affect ABP responses. Lesions of the ventromedial, but not the lateral, rostral medulla blocked hypoalgesia. Rostral medullary lesions did not reliably affect stimulus-evoked cardiovascular responses or baseline ABP. These results indicate that antinociceptive and cardiovascular conditional responses are anatomically dissociable and support our proposal that conditional hypoalgesia is mediated by a serial neural circuit that includes the amygdala, PAG, and rostral ventromedial medulla.

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.

Hypoalgesia in response to sensitization during acute noise stress.

Three experiments examined the antinociceptive response shown by rats during exposure to loud noise. Noise exposure resulted in a time-dependent elevation of radiant heat tail flick latency that varied as a function of stimulus intensity. Noise stress hypoalgesia in response to a 90-dB stimulus was blocked by pretreatment with the opioid antagonist naltrexone (0.1-7.0 mg/kg). Systemic administration of midazolam (2 mg/kg) prior to exposure to the stressor attenuated the elevation in tail flick latency. Because topographically similar antinociceptive responses may be elicited with a low intensity noise stimulus that has served as a Pavlovian conditional stimulus for shock, the use of this paradigm may permit direct comparisons of associative and nonassociative fear responses using qualitatively similar auditory stimuli.

Functional mapping of the rat brain during drinking behavior: a fluorodeoxyglucose study.

Autoradiographic techniques using the radiolabeled glucose analog [14C]2-fluoro-2-deoxy-D-glucose (FDG) were used to map the functional activity in the CNS during drinking behavior. Rats were trained to drink water during a 1-h session each day. Half of the rats were injected with FDG and allowed to drink, while the other half were satiated prior to FDG injection. Uptake of FDG for drinking and control groups of rats was quantified in 60 brain structures from frontal cortex to cervical spinal cord. The largest percent increase in activity (96%) during drinking was in the lateral hypothalamus. Limbic structures with significant metabolic increases included the lateral septum (48%), lateral habenula (44%), and nucleus accumbens (32%). Thalamic nuclei activated included intralaminar (60%), zona incerta (51%), ventroposteromedial (50%), anterior ventral (47%), and dorsal medial (40%). Other structures with increases were the caudal caudate nucleus (53%) and the spinal trigeminal nucleus (45%). The findings were interpreted in light of related metabolic mapping studies of the effects of orofacial stimulation, dehydration, ingestion, arousal, and reward. It was concluded that this FDG study revealed primarily the involvement of structures linked to rewarding and arousal components of motivated drinking behavior, as well as sensorimotor correlates of the orofacial stimulation. The findings provide the first comprehensive functional map of brain systems related to drinking behavior in adult animals.

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.

Inhibition of the tail flick reflex following microinjection of morphine into the amygdala.

Recent evidence indicates that the amygdala plays a critical role in the activation of brain stem antinociceptive systems during stress. In the present experiment, bilateral microinjection of morphine sulfate (10 micrograms) into the amygdala of pentobarbital-anesthetized rats resulted in a time-dependent elevation in latency of the tail flick reflex evoked by radiant heat. The most effective sites within the amygdala were in or immediately adjacent to the basolateral nucleus. The relative amplitude of the tail flick reflex did not differ as a function of repeated testing or morphine treatment. These results suggest that important forebrain inputs which normally activate endogenous antinociceptive systems in behaving animals may be manipulated and studied in detail using the anesthetized rat.