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.

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.

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.

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.

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.

Strain differences in reversal of conditional analgesia by opioid antagonists.

The ability of both naloxone and naltrexone to block conditional analgesia produced by shock associated contextual stimuli was determined using rats from three suppliers. Naltrexone (7 mg/kg) was equally effective in reversing analgesia on the formalin test in all rats tested. Naloxone (7 mg/kg) significantly affected analgesia in Long-Evans and Holtzman Sprague-Dawley rats but had no effect on Charles River (CD) Sprague-Dawley rats. Charles River rats also differed from the other groups in the amount of time spent freezing to apparatus cues. These results indicate that genetic factors and the choice of antagonists may complicate distinctions between opioid and nonopioid analgesia.

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.

Modulation of appetitively and aversively motivated behavior by the kappa opioid antagonist MR2266.

MR2266 (MR), an opioid antagonist that binds to kappa and mu receptors, was examined for its ability to influence the aversively motivated behaviors conditioned by electric shock and the drinking induced by water deprivation or the availability of a palatable saccharin/glucose solution. The intraperitoneal (ip) and intracerebroventricular (icv) administration routes were contrasted. After both ip and icv administration, MR was able to reverse conditional analgesia as measured by the formalin test. MR enhanced the Pavlovian conditional freezing response when administered icv prior to shock exposure but reduced freezing if given ip prior to shock. A related benzomorphan-derived opioid antagonist, MR1452, also reduced freezing when given ip prior to shock. MR2266 was a potent antidipsogenic agent when administered ip but had no such effect when administered icv. It is concluded that separable opioid systems are involved in the modulation of appetitively and aversively motivated behaviors.

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.

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.

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.

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.

[D-Ala2,Leu5,Cys6]enkephalin: short-term agonist effects and long-term antagonism at delta opioid receptors.

Four experiments were conducted. Two examined the in vivo short-term effects (less than 35 min) and two examined the in vivo long-term effects (greater than 2 days) of the synthetic enkephalin analogue, [D-Ala2,Leu5,Cys6]enkephalin (DALCE). In the short term, DALCE was found to produce analgesia and transient immobility after intracerebroventricular (ICV) administration. We found a dose-related increase in paw-lick latency for rats placed on a hotplate (52 degrees C). In the second experiment, we determined that immobility was attenuated by pretreatment with naltrexone methobromide (QNTX, 0.1 microgram), and the delta selective antagonist, 16-methyl cyprenorphine (M80, 5 micrograms). QNTX and M80 significantly attenuated DALCE-induced immobility by more than 50% of control. Paw-lick latency was then measured on the hotplate to assess analgesia. Pretreatment with M80 reliably attenuated the DALCE-induced analgesia. Whereas QNTX failed to reliably attenuate paw-lick latency on the first trial, it was as effective as M80 on the second trial. We suggest that the short-term agonist effects of DALCE are produced by actions at mu and delta opioid receptors as would be predicted from prior in vitro studies showing moderate to high affinity, respectively, at these receptors. In the third experiment, DALCE was shown to display long-term behavioral antagonism that was selective for the delta receptor. Rats were injected ICV with 6.7 micrograms of DALCE and tested 48 hr later. Analgesia was measured by injecting 15% formalin subcutaneously followed 20 min later by an ICV injection of one of three selective opioid agonists (DAGO, DPDPE or U50488H).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of selective opioid peptide antagonists on the acquisition of pavlovian fear conditioning.

Pretreatment with opioid antagonists enhances acquisition of Pavlovian fear conditioning. The present experiments attempted to characterize the type of opioid receptor responsible for this effect using a procedure that assessed the fear of rats to a chamber previously associated with electric shock (1 mA, 0.75 s). Freezing, a species-typical immobility, was employed as an index of fear. Two mu opioid antagonists, CTOP (40 ng) and naloxonazine (10 micrograms), enhanced conditioning. On the other hand, the kappa antagonist nor-binaltorphimine reduced conditioning. Two delta antagonist treatments (16-methyl cyprenorphine and naltrindole) had no reliable effect on acquisition. Thus the enhancement of conditioning appears to be mediated by mu receptors. Previous research has shown that the conditional fear produced by these procedures caused an analgesia that is also mediated by mu receptors. It is argued that the enhancement effect occurs because of an antagonism of this analgesia and that the analgesia normally acts to regulate the level of 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.

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.

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.

Conditional hypoalgesia is attenuated by naltrexone applied to the periaqueductal gray.

The ventrolateral periaqueductal gray (vPAG) is an important component in a brainstem system involved in the endogenous modulation of nociception and defensive behavior. The present study was conducted to determine if opioid receptors within the vPAG contribute to the hypoalgesia seen in rats during presentation of a Pavlovian CS for footshock. Independent groups of animals received microinjections of either 5.0 micrograms naltrexone HCl or vehicle into the vPAG prior to being placed in an observation chamber in which shock had been delivered 24 h earlier. Nociceptive reactivity was measured with the formalin test. Naltrexone treatment attenuated conditional hypoalgesia but did not affect formalin-induced behavior in non-shocked rats. Naltrexone had no effect on the amount of defensive freezing behavior observed during the test session. These results indicate that conditional hypoalgesia as measured by the formalin test involves the activation of documented brainstem antinociceptive systems.