Amygdaloid GABA, not glutamate neurotransmission or mRNA transcription controls footshock-associated fear arousal in the acoustic startle paradigm.

In Pavlovian conditioning the fear-evoking properties of the aversive unconditioned stimulus are represented by the conditioned stimulus. A major challenge for theories of classical fear conditioning has been to understand how associations are formed between a conditioned stimulus and unconditioned stimulus. Although the cellular mechanisms in the amygdala that underlie fear learning have received considerable attention relatively little is known about the neural substrates underlying unconditioned stimulus-associated fear. In the present study we examined the role of GABA(A), N-methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptors, and protein synthesis inhibition on the immediate fear arousal produced by footshock as measured by the shock sensitization of acoustic startle. Laboratory rats showed shock-enhanced startle after infusion into the basolateral amygdala of the N-methyl-D-aspartic acid receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (5.0 microg), the non-N-methyl-D-aspartic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione disodium (5.0 microg) and the protein synthesis inhibitor anisomycin (80.0 microg). We concluded that fear arousal provoked by footshock is not mediated by glutamate neurotransmission in the amygdala and does not involve de novo protein synthesis. Bilateral infusion into the basolateral amygdala of the GABA(A) receptor agonist muscimol in doses ranging from 0.001-0.5 microg reliably blocked the shock sensitization of acoustic startle responding. None of the muscimol doses altered shock reactivity amplitudes indicating the normal perception of footshock. The muscimol results were interpreted to suggest that decreased GABA neurotransmission in the amygdala may be essential for the neural causation of fear that is acquired and expressed by conditioned stimuli.

Extinction deficit and fear reinstatement after electrical stimulation of the amygdala: implications for kindling-associated fear and anxiety.

Generalized seizures produced by electrical kindling of the amygdala in laboratory rats are a widely used animal model of temporal lobe epilepsy. In addition to seizure evolution amygdala kindling enhances emotionality. The relative roles of electrical stimulation and seizure induction in fear responding are unclear. Here we investigate this issue using extinction and reinstatement of fear-potentiated startle. After classical conditioning (light+footshock pairings) laboratory rats were fear extinguished with each light presentation followed by nonepileptogenic amygdala stimulation. In contrast to the normal extinction learning of control subjects, amygdala stimulated animals exhibited conditioned fear after 120 presentations of the nonreinforced conditioned stimulus (CS). In a second experiment electrical stimulation of the amygdala restored extinguished fear responding and the fear reinstatement was specific to extinction context. The reinstatement effect did not involve sensitized fear to the CS produced by amygdala stimulation. The possibility that electrical activation of the amygdala produces unconditioned fear was considered. Animals uniformly failed to demonstrate fear-potentiated startle using electrical stimulation of the amygdala as the unconditioned stimulus. This was the case with a subthreshold afterdischarge stimulus and a stimulation schedule that produced kindled seizures. The extinction deficit and fear reinstatement results were interpreted to suggest that amygdala stimulation activates acquired excitatory stimulus-affect neural connections formed during Pavlovian fear conditioning. Our data supports a model in which excitation of an amygdala-based memory-retrieval system reinforces the expression of learned fear behaviors.

Inhibition of amygdaloid dopamine D2 receptors impairs emotional learning measured with fear-potentiated startle.

Considerable advances have been made in understanding the neurocircuitry underlying the acquisition and expression of Pavlovian conditioned fear responses. Within the complex cellular and molecular processes mediating fearfulness, amygdaloid dopamine (DA), originating from cells in the ventral tegmental area (VTA) of the midbrain, is thought to contribute to fear-motivated responding. Considering that blockade of DA D(2) receptors is a common mechanism of action for antipsychotic agents, we hypothesized that inhibition of D(2) receptors in the amygdala may be involved in the antiparanoid effects of these drugs. To assess the role of amygdaloid DA D(2) receptors in aversive emotionality, the D(2) receptor antagonist raclopride was infused into the amygdala prior to Pavlovian fear conditioning. Potentiated startle was used as a behavioral indicator of fear and anxiety. Classical fear conditioning and acoustic startle testing were conducted in a single session allowing for the concomitant assessment of shock reactivity with startle enhancement. Depending on dose, the results found conditioned fear acquisition and retention to be impaired following administration of raclopride into the amygdala. Additionally, the learning deficit was dissociated from shock detection and from fear expression assessed with the shock sensitization of acoustic startle. These findings further refine the known neural mechanisms of amygdala-based emotional learning and memory and were interpreted to suggest that, along with D(1) receptors, D(2) receptors in the amygdala may mediate the formation and the retention of newly-acquired fear associations.

Peripheral and intraamygdalar administration of the dopamine D1 receptor antagonist SCH 23390 blocks fear-potentiated startle but not shock reactivity or the shock sensitization of acoustic startle.

Central dopamine (DA) activity is thought to play a role in fear motivation. The aim of the present study was to assess the involvement of DA D1 receptors in emotional learning. The authors report that peripheral and intraamygdalar administration of the specific D1 receptor antagonist SCH 23390 blocked the acquisition of fear-potentiated startle. Analysis of shock reactivity during footshock administration revealed that the learning impairment could not be explained by a diminution in the aversive properties of the unconditioned stimulus. Additionally, systemic and intraamygdalar injection of SCH 23390 did not alter fear expression as measured with the shock sensitization of acoustic startle. The potential contribution of mesoamygdaloid DA to the acquisition and retrieval of conditioned fear responses is discussed.

The involvement of ventral tegmental area cholinergic muscarinic receptors in classically conditioned fear expression as measured with fear-potentiated startle.

Accumulating evidence suggests that dopamine (DA) neurons in the ventral tegmental area (VTA) contribute to the complex amygdala-based neurocircuitry that mediates fear-motivated behaviors. Because of acetylcholine's (ACh) role in DA neuronal activation, the involvement of VTA cholinergic muscarinic receptors in Pavlovian conditioned fear responding was evaluated in the present study. Fear-potentiated startle was used to assess the effects of intraVTA infused methylscopolamine on conditioned fear performance in laboratory rats. Application of this nonspecific muscarinic receptor antagonist to VTA neurons was observed to inhibit the ability of a conditioned stimulus (CS) previously paired with footshock to enhance the amplitude of the acoustic startle reflex. Doses of methylscopolamine that blocked conditioned fear expression did not alter baseline sensorimotor responding. These results identify ACh neurotransmission in the VTA as a potential excitatory mechanism underlying the fear-arousing properties of threatening environmental stimuli.

Enhanced amygdala kindling after electrical stimulation of the ventral tegmental area: implications for fear and anxiety.

Electrical kindling refers to the seizure-generating properties of brain stimulation. In addition to producing epilepsy, the reorganization of forebrain neurocircuitry associated with kindling contributes to psychiatric disturbances involving fear and anxiety. The amygdala is a limbic structure that kindles readily and regulates the complex neurocircuitry underlying emotional responding. Dopamine-containing ventral tegmental area (VTA) neurons, known to be activated by threatening environmental stimuli, are an important component of the amygdala-based fear network. Using amygdala kindling as an indicator of sensitization development, we report here that repeated low-current, high-frequency stimulation of the VTA provoked afterdischarge in the central amygdala and enhanced kindling rate. By establishing a fundamental link between VTA activation and neural excitability in the central amygdala, the present results are consistent with the possibility of a common process underlying epileptogenisis and the fear motivational consequences of amygdala and VTA kindling. Considering the established role of the VTA and the amygdala in emotional responding, such a sensitization mechanism might mediate exaggerated fearfulness.

The effects of cocaine, amphetamine, and the dopamine D1 receptor agonist SKF 38393 on fear extinction as measured with potentiated startle: implications for psychomotor stimulant psychosis.

Using Pavlovian conditioned increases in the amplitude of the acoustic startle reflex as a behavioral indicator of fear motivation, the authors previously showed a resistance to extinction after repeated associations of cocaine with the fear-evoking conditioned stimulus (CS). In Experiment 1, acute administration of cocaine, amphetamine, and the dopamine (DA) D1 receptor agonist SKF 38393 produced a similar fear enhancement. In Experiment 2, a noncontingent injection of cocaine and SKF 38393 provoked a CS potentiation of acoustic startle in fear-extinguished laboratory rats. Potential behavioral, neurochemical, and neuroendocrine explanations for the effects of psychomotor stimulants on conditional fear were discussed. It was suggested that DA agonist drugs increase fear expression possibly by activating mesoamygdaloid associative neurocircuitry involved in excitatory conditioned fear reactions.

Infusion of the dopamine D1 receptor antagonist SCH 23390 into the amygdala blocks fear expression in a potentiated startle paradigm.

Dopamine (DA) D1 receptors are distributed in the nucleus accumbens and the amygdala, two regions of the mesocorticolimbic DA system known to be activated by aversive environmental stimuli. The objective of the present study was to determine the contribution of D1 receptors in these brain regions to the expression of a fear-motivated behavior, notably, potentiated startle in rats. Injection of the DA D1 receptor antagonist SCH 23390 into the amygdala blocked the ability of a conditioned light stimulus previously paired with footshock to enhance acoustic startle amplitudes. Bilateral intracerebral administration of SCH 23390 into the nucleus accumbens had no effect on fear-potentiated startle. The observed opposing effects of amygdaloid DA D1 receptor antagonism on fear expression, along with earlier research demonstrating the involvement of ventral tegmental area (VTA) DA neurons on fear-potentiated startle, suggest a role for mesoamygdaloid activity in conditioned excitatory fear reactions.

Differential effects of interleukin (IL)-1beta, IL-2 and IL-6 on responding for rewarding lateral hypothalamic stimulation.

Bacterial endotoxin and interleukin-1 (IL-1) challenge induce a constellation of symptoms associated with illness. While such treatment may result in anhedonia, it is often difficult to dissociate this effect from the anorexia induced by these agents, particularly in paradigms that involve appetitive motivation. The present investigation assessed the effects of several systemically administered cytokines (IL-1beta, IL-2 and IL-6) on reward processes by evaluating responding for rewarding intracranial self-stimulation (ICSS) from the lateral hypothalamus. Systemic administration of interleukin-2 (IL-2) disrupted responding from the medial forebrain bundle, and this disturbance persisted as long as 1 week following initial cytokine treatment. In contrast to reinforced responding, following IL-2 treatment, non-reinforced behavior was unaffected, indicating that the cytokine did not provoke reward-unrelated performance deficits. It was suggested that the effects of IL-2 on ICSS likely do not involve motoric, soporific, attentional or cognitive changes, but instead involve specific actions on motivational arousal. Although IL-6 was previously found to produce mesolimbic dopamine (DA) changes as marked as those induced by IL-2, systemic IL-6 treatment did not influence responding for rewarding brain stimulation. Likewise, although IL-1 at the dosage used reliably induces sickness behavior, responding for rewarding brain stimulation was unaffected. Thus it seems that anhedonia is not necessarily a component of the sickness response associated with IL-1 treatment.

Lipopolysaccharide, central in vivo biogenic amine variations, and anhedonia.

Systemic administration of lipopolysaccharide (LPS), a non-specific activator of proinflammatory cytokine release from macrophages, provokes sickness characterized by anorexia, soporific effects, and disturbances of locomotor activity and exploration. In addition, endotoxin treatment may provoke an anhedonic response. Assessment of anhedonia in appetitive paradigms, however, is compromised by the anorexia provoked by the treatment. The present investigation assessed the anhedonic effects of LPS on rewarding lateral hypothalamic brain stimulation. Using a simultaneous discrimination, current titration procedure in the assessment of intracranial self-stimulation (ICSS), it was found that acute systemic administration of LPS (50 microg, 100 microg or 200 microg) reduced ICSS during the ascending sequence of current presentations, but had little effect on responding to a series of descending currents. In a parallel experiment, peripheral administration of LPS (100 microg) increased in vivo dopamine (DA) efflux from the nucleus accumbens, a region thought to be involved in goal-directed responding to positively reinforcing stimuli. It is suggested that LPS alters ICSS in a manner different than that observed following stressor exposure or peripheral IL-2 treatment. Furthermore, LPS may engender an anhedonic effect (possibly secondary to sickness), and the decline of responding reflects the relation between the cost of responding given in the face of sickness and the reward received for responding.

Infusion of quinpirole and muscimol into the ventral tegmental area inhibits fear-potentiated startle: implications for the role of dopamine in fear expression.

Dopamine (DA) D2 and gamma-aminobutyric acid (GABA)A somatodendritic receptors tonically inhibit mesolimbic projection neurons in the A10 DA cell grouping of the ventral tegmentum. In the present study we determined the contribution of the ventral tegmental area (VTA) to the expression of a classically conditioned fear-induced increase in the acoustic startle reflex. Saline applied to VTA neurons did not modify the capacity of a light previously associated with footshock to potentiate acoustic startle amplitudes; conversely, bilateral administration of the DA D2/3 agonist quinpirole or the GABAA receptor agonist muscimol into the ventral tegmentum blocked fear-potentiated startle without altering baseline acoustic startle responding. It was suggested that DA VTA neurons regulate the excitatory aspects of fear expression by gating levels of aversive emotional arousal within the amygdala-based fear system.

Contribution of ventral tegmental area dopamine neurons to expression of conditional fear: effects of electrical stimulation, excitotoxin lesions, and quinpirole infusion on potentiated startle in rats.

Potentiated startle was used in this study to determine the fear-motivational functions of the ventral tegmental area (VTA). In Experiment 1, electrical stimulation of the VTA increased acoustic startle amplitudes. In subsequent experiments fear-potentiated startle was assessed following axon-sparing N-methyl-D-aspartic acid (NMDA) lesions of the VTA and after bilateral intra-VTA infusion of the dopamine (DA) D2/3 receptor agonist quinpirole. The NMDA lesions produced substantial cell loss in the medial ventral tegmentum and suppressed fear expression. Similarly, inhibition of DA neuronal activity associated with locally administered quinpirole blocked fear-potentiated startle. It was suggested that VTA neurons and their forebrain DA projections regulate levels of aversive emotional arousal within the amygdala-based fear system.

Interleukin-2 decreases accumbal dopamine efflux and responding for rewarding lateral hypothalamic stimulation.

Systemic administration of interleukin-2 (IL-2) provoked marked alterations of responding for rewarding brain stimulation from the medial forebrain bundle (MFB). In particular, when animals were tested for ICSS immediately following IL-2 treatment only a modest disturbance of responding was evident. However, if animals were subsequently exposed to repeated daily ICSS sessions (24-168 h) in the drug-free state, rightward shifts in the rate intensity functions and significant increases in reward thresholds were apparent. These results were dependent upon the presence of IL-2 during the initial ICSS session. If animals were tested for ICSS 24 h after IL-2 administration, without an intervening test, performance was unaffected. Evaluation of nonreinforced behavior after IL-2 treatment revealed that ICSS remained under stimulus control and the cytokine did not provoke reward-unrelated performance deficits. Dopamine (DA) activity in the nucleus accumbens has been implicated in goal-directed responding to positively reinforcing stimuli and in the present investigation, using in vivo microdialysis, it was observed that IL-2 markedly reduced DA release from this region. It was suggested that the protracted consequences of IL-2 on ICSS likely do not involve motoric, soporific, attentional or cognitive changes, but may be attributable to its specific actions on motivational arousal, possibly engendered by the cytokine-induced diminution of accumbal DA efflux.

Cocaine enhances the expression of fear-potentiated startle: evaluation of state-dependent extinction and the shock-sensitization of acoustic startle.

Cocaine's effects on fear extinction and on the shock-sensitization of acoustic startle were examined. Following fear acquisition, rats exposed to the nonreinforced conditioned stimulus (CS) after cocaine administration demonstrated significant levels of fear-potentiated startle when evaluated in the drug-free state. The CS also increased startle amplitudes in subjects extinguished and tested with cocaine, indicating that mechanisms other than state-dependent learning are involved in the extinction deficit. The presentation of 10 footshocks augmented acoustic startle, and the shock enhancement was unaffected by cocaine preexposure. These data indicate that the aversive consequences of footshock relevant to the acquisition of conditional fear are not sensitized by the drug. It was suggested that cocaine reinforces fear responding to a threatening stimulus.

The effects of ventral tegmental administration of GABAA, GABAB and NMDA receptor agonists on medial forebrain bundle self-stimulation.

Using a conditioned discrimination ICSS paradigm, rate-current intensity functions were determined for both reward- and nonreward-associated responding for electrical self-stimulation of the MFB following intra-VTA infusion of baclofen, muscimol and N-methyl-D,L-aspartate (NMDLA). A low dose (0.064 microgram/0.5 microliter) of the GABAB receptor agonist, baclofen, microinjected into the VTA, ipsilateral to the lateral hypothalamic stimulating electrode, resulted in a rightward shift of the ICSS curve without significantly influencing either maximal rates of operant responding for electrical brain stimulation or nonreinforced performance levels. Increases in MFB current thresholds were also evident after infusion of higher doses of baclofen (0.128, 0.26 and 0.52 microgram) into the ventral tegmentum. Intra-VTA administration of the GABAA receptor agonist, muscimol (0.006, 0.012, 0.025 and 0.05 microgram) and NMDA receptor activation by NMDLA (0.5, 1, 2 and 5 micrograms) did not affect reward thresholds; however, the low dose of muscimol and the high dose of NMDLA elicited behavioral activation resulting in reward-unrelated performance effects. These results implicate the specific involvement of the GABAB receptor in the reward neurocircuitry of the VTA.

Cocaine preexposure sensitizes conditioned fear in a potentiated acoustic startle paradigm.

The consequences of chronic cocaine administration on fear-potentiated startle were evaluated in two experiments. Cocaine treatment (40 mg/kg) for 7 days prior to fear acquisition (light + shock pairings) had an attenuating influence on the ability of the conditioned stimulus (CS) to increase acoustic startle. When cocaine was administered in the context of the CS, following fear conditioning, a marked enhancement of potentiated startle was observed. In contrast, an extinction of the fear response was seen in saline and procaine animals repeatedly exposed to the nonreinforced CS. The results from control subjects injected with cocaine either in the shock chambers (contextual cues) or in their home cage environment, suggest that the systemic effects of this stimulant served to intensify the fear-eliciting properties acquired by the CS during fear conditioning. These findings demonstrate a cocaine sensitization of conditioned fear, and were related to the emotional and psychological disturbances associated with long-term cocaine use.

Effects of scopolamine on the rewarding and seizure-inducing properties of amygdaloid stimulation.

The relationship between amygdaloid brain-stimulation reward and the evolution of seizure activity was evaluated in this study. Current levels that maintained optimal intracranial self-stimulation (ICSS) rates were found to be lower than the minimal current intensity required to elicit an afterdischarge (AD) from the central nucleus of the amygdala. After the ICSS session, AD thresholds (ADTs) were reduced to the same levels of current used to support ICSS. Assessment of seizure stage development during ICSS testing revealed that the emergence of early-stage epileptiform events following repeated amygdaloid stimulation suppressed ICSS performance. While administration of the antimuscarinic scopolamine did not prevent the stimulation-elicited reduction in AD thresholds, it was observed to inhibit seizure progression and increase ICSS rates. These results are consistent with the excitatory function of acetylcholine in epileptogenesis and were related to the possibility that different mechanisms underlie the rewarding and seizure-inducing properties of amygdaloid stimulation.

Clozapine inhibits limbic system kindling: implications for antipsychotic action.

Clozapine and haloperidol were tested for their ability to influence the acquisition of kindled seizures following electrical stimulation of the amygdala and ventral hippocampus. While haloperidol pretreatment did not alter kindling genesis from either limbic region, preexposure to clozapine delayed the rate at which kindling evolved. Analysis of the number of seizure behaviors expressed during epileptogenesis revealed that clozapine produced a relative antagonism of seizure development arresting kindling at the stage-3 level. The kindling inhibition was dependent upon the daily administration of clozapine during the kindling process and was not evident after withdrawal from a chronic schedule of clozapine exposure. A subconvulsive dose of pilocarpine (80.0 mg/kg) produced an overall enhancement of kindling rate, a finding consistent with the excitatory role of acetylcholine (ACh) in kindling. Lower doses of pilocarpine (20.0 and 40.0 mg/kg) that did not alter seizure advancement partially antagonized the clozapine-elicited inhibition of amygdaloid kindling. Pilocarpine, however, did not affect the clozapine-induced increase in the number of stage-3 behaviors exhibited during amygdaloid kindling, suggesting that other neurochemical effects of clozapine, not related to its anticholinergic properties, modulate the kindling suppression. Clozapine's unique actions on limbic system sensitization were discussed in relation to its effectiveness as an antipsychotic agent.

The effects of amygdaloid stimulation on amphetamine-elicited locomotor sensitization.

Systemic injection of d-amphetamine (1.0 mg/kg) resulted in a progressive increase in locomotor activity as a function of repeated daily drug administration. The magnitude of the stimulant-induced sensitization effect was enhanced by low-current electrical stimulation of the central nucleus of the amygdala during open-field testing. Amygdaloid stimulation in the absence of amphetamine treatment did not influence spontaneous locomotor activity, and there was no behavioral evidence of epileptogenesis following amygdaloid stimulation over the course of the experiment. However, with continued stimulation of the amygdala, early-stage convulsive activity was apparent in animals after approximately 40 days of testing, signifying the advancement of kindling evolution. These results suggest that the processes responsible for kindling acquisition, prior to the behavioral expression of epileptiform events, interact with the underlying substrates of amphetamine sensitization.

Amphetamine and antidepressant drug effects on GABA- and NMDA-related seizures.

Research has shown a synergistic relationship between amphetamine sensitization and limbic system kindling. To explore the role of GABA and NMDA receptor activity in modulating the positive effects of amphetamine on epileptogenesis, alterations in GABA- and NMDA-related convulsions were examined after acute and chronic amphetamine administration. A single injection of d-amphetamine (7.5 mg/kg) significantly decreased latencies to generalized motor seizures induced 12 h later by the noncompetitive GABAA receptor antagonist picrotoxin (10 mg/kg). The increased sensitivity to clonus was specific to acute amphetamine treatment and was not evident following withdrawal from chronic drug exposure. Seizures induced by NMDLA (1,000 mg/kg), on the other hand, were not modified by acute amphetamine injection; however, the latency to clonus was reduced substantially after NMDLA injection to mice chronically preexposed to amphetamine. The short- and long-term amphetamine effects on GABA- and NMDA-associated convulsive activity were not paralleled by similar drug treatment schedules involving acute (20 mg/kg) and chronic administration of desipramine, zimelidine, and buproprion. These results suggest that amphetamine may be acting on inhibitory and excitatory amino acid systems independently of its monoaminergic properties. The implications of these findings were discussed in relation to amphetamine sensitization of mesolimbic functioning.