The effects of intra-amygdaloid infusions of a D2 dopamine receptor antagonist on Pavlovian fear conditioning.

The present study examined the effects of bilateral intra-amygdaloid infusions of the D2 receptor antagonist, eticlopride, on the acquisition and expression of Pavlovian fear conditioning as measured by freezing to acoustic and background contextual stimuli in the rat. Infusions of eticlopride before acquisition or before both acquisition and retention testing significantly attenuated conditioned freezing to tone presentations during the retention test 24 hr later. No effects, however, were observed on freezing that emerged during acquisition. Furthermore, these effects were not attributable to state-dependent learning effects or alterations in baseline activity or shock reactivity. In conclusion, these results suggest that amygdaloid dopamine transmission at D2 receptors contributes to the formation and/or consolidation of fear memories.

Arousal-related associative response characteristics of dorsal lateral geniculate nucleus neurons during acoustic Pavlovian fear conditioning.

This research determined whether fear-conditioned, acoustic stimuli induce thalamic arousal reflected in associative responses in dorsal lateral geniculate nucleus (dLGN) neurons. Rabbits received a Pavlovian discriminative fear conditioning procedure in which one tone conditioned stimulus (CS +) was always paired with an aversive unconditioned stimulus (US) and another tone (CS-) was never paired with the US. Responses of single dLGN neurons to random CS+ and CS- presentations were then recorded. Nine of 15 recorded neurons demonstrated significantly greater firing during the CS+ versus the CS-. Their spontaneous activity demonstrated tonic firing during increased neocortical arousal and burst firing during decreased neocortical arousal. The results demonstrate that dLGN neurons show associative responses to fear-conditioned, acoustic stimuli and present a model for investigating the neural circuits by which such stimuli affect sensory processing at the thalamic level.

An electrophysiological characterization of ventral tegmental area dopaminergic neurons during differential pavlovian fear conditioning in the awake rabbit.

Recent research has suggested that the mesencephalic dopaminergic (DA) system is activated by stress. For example, alterations in DA metabolites have been found in the ventral tegmental area (VTA) following footshock and immobilization in the rat [15,37]. Furthermore, this activation appears selective to DA neurons within the VTA since no changes were observed within the substantia nigra [15,16]. While this research suggests that DA neurons in the VTA are activated by aversive events, there has been a paucity of electrophysiological research designed to examine the sensory response characteristics of these DA neurons, and in particular their response to stimuli which predict aversive events. The present study was conducted to investigate the response characteristics of DA neurons within the VTA of the awake rabbit to acoustic stimuli which, via Pavlovian aversive conditioning procedures, came to predict the occurrence of a mild shock to the pinna. 45%, of the neurons meeting pre-established criteria for DA neurons demonstrated either significant excitation or inhibition to conditioned aversive stimuli. These neurons responded differentially to CS+ and CS- presentations. Some of these neurons (65%) demonstrated a greater increase in activity during the CS+ compared to the CS-, some (22%,) demonstrated a greater decrease in activity during the CS+ compared to the CS- and some (13%) demonstrated a greater increase in activity during the CS- compared to the CS+. Further, conditioned heart rate responses in the rabbits occurred during the recording of a majority of these neurons. These overall results suggest that conditioned aversive stimuli can affect the firing of VTA DA neurons and that these neurons comprise a heterogenous population with respect to their response profiles.

Amygdaloid D1 dopamine receptor involvement in Pavlovian fear conditioning.

The amygdala has long been implicated in conditioned fear. The mesencephalic dopaminergic system provides a rich innervation to the amygdala [J.H. Fallon, P. Ciofi, Distribution of monoamines within the amygdala, in: J.P. Aggleton (Ed.), The Amygdala: Neurobiological Aspects of Emotion, Memory and Mental Dysfunction, Wiley, New York, 1992, pp. 97-114; L.J. Freedman, M.D. Cassell, Distribution of dopaminergic fibers in the central division of the extended amygdala of the rat. Brain Research 633 (1994) 243-252; E. Asan, The catecholaminergic innervation of the rat amygdala. Advances in Anatomy Embryology and Cell Biology 142 (1996) 1-107]. Specific activation of the mesoamygdaloid dopaminergic system has been reported to occur in response to conditioned fear-arousing stimuli [M.L. Coco, C.M. Kuhn, T.D. Ely, C.D. Kilts, Selective activation of mesoamygdaloid dopamine neurons by conditioned stress: attenuation by diazepam. Brain Research 590 (1992) 39-47] suggesting that dopamine release in the amygdala may contribute to the acquisition and/or expression of conditioned fear. Using a 2x2 factorial design, Experiment 1A investigated the effects of bilateral intra-amygdaloid infusions of the selective D1 receptor antagonist, SCH 23390 (2.0 microgram 0.5 microliter-1 side-1), on the acquisition and expression of Pavlovian conditioned fear measured by freezing to acoustic and background contextual stimuli. Infusions of SCH 23390 prior to acquisition training, prior to retention testing or prior to both significantly attenuated conditioned freezing during retention testing. Experiment 1B investigated the dose-dependent effects of pre-training infusions of SCH 23390 (0.5, 1.0 and 2.0 microgram) on conditioned fear. Pre-training infusions of SCH 23390 dose-dependently attenuated conditioned freezing during retention testing. Experiment 2A investigated the effects of bilateral infusions of the selective D1 receptor agonist, SKF 82958 (2.0 microgram 0.5 microliter-1 side-1) on the acquisition and expression of conditioned fear. Infusions of SKF 82958 prior to training facilitated conditioned freezing during retention testing. Experiment 2B investigated the dose-dependent effects of pre-training infusions of SKF 82958 (1.0, 2.0 and 4.0 microgram) on conditioned fear. Pre-training infusions of SKF 82958 dose-dependently facilitated conditioned freezing during retention testing. In conclusion, these results suggest that dopamine transmission within the amygdala contributes to the acquisition and expression of Pavlovian fear conditioning.

Amygdaloid modulation of mesopontine peribrachial neuronal activity: implications for arousal.

This research sought to determine if activation of the amygdaloid central nucleus (ACe) modulates the activity of arousal-related neurons within the peribrachial (PB) region. Two categories of neurons were identified. Neurons of the 1st category demonstrated low spontaneous rates and responded with a burst of spikes to acoustic stimuli, characteristics similar to those reported for cholinergic ponto-geniculo-occipital (PGO) wave generator neurons. Neurons of the 2nd category demonstrated significant correlations between their spontaneous activity and the power of delta wave activity in the electroencephalogram (EEG) and responded to acoustic stimuli. Electrical stimulation of the ACe activated 43% of the PGO-related neurons but only 6% of the EEG-related neurons. The results suggest that the ACe modulates the activity of neurons that form the substrate for PGO waves, which are recognized correlates of arousal.

Effect of lesions of the ventrolateral periaqueductal gray on the Pavlovian conditioned heart rate response in the rabbit.

The present experiment sought to evaluate the contribution of the ventrolateral periaqueductal gray to the expression of the conditioned bradycardic heart rate response in the rabbit. This response occurs in the presence of a fear-arousing conditioned stimulus. Lesions of the ventrolateral periaqueductal gray made after training failed to affect retention of the conditioned response. The results suggest that this region of the midbrain plays a nonessential role in the expression of the conditioned bradycardic response.

Neuronal activity within the nucleus basalis and conditioned neocortical electroencephalographic activation.

The relationship between neuronal activity within the nucleus basalis (NB) and conditioned neocortical EEG activation was investigated in New Zealand rabbits during Pavlovian differential conditioning. Twenty-seven of 56 neurons recorded in conditioned animals demonstrated a significantly greater change in activity to a tone (CS+) that predicted the occurrence of a mildly aversive unconditioned stimulus when compared to a tone (CS-) that did not. Twenty-four of these 27 neurons demonstrated a significant increase in activity to the CS+ compared to the CS-, while the remaining three neurons demonstrated a significant decrease in activity to the CS+ compared to the CS-. In 24 of these 27 neurons (89%) these changes in neuronal activity during CS presentations correlated significantly with a decrease in the power of delta activity in the EEG. In addition, 13 of these 24 neurons (54%) demonstrated significant correlations between neuronal activity and the power of delta activity during CS-free periods. In experimentally naive animals, the activity of 10 of 22 neurons (45%) recorded within the region of the NB correlated with the power of delta activity in the EEG during stimulus-free periods. These results complement a growing body of evidence and provide strong support for the hypothesis that the NB contributes to neocortical activation in the conscious animal.

Effects of electrical stimulation of the amygdaloid central nucleus on neocortical arousal in the rabbit.

This study sought to determine whether electrical stimulation of the amygdaloid central nucleus (ACe) produces cholinergically mediated neocortical arousal manifested in the suppression of frontal cortex delta wave (1-4 Hz) activity. Stimulation in both anesthetized and conscious rabbits produced a suppression of delta activity that was accompanied by bradycardia and blocked by cholinergic antagonists. Stimulation of the adjacent putamen did not produce delta suppression, whereas stimulation of the adjacent ventral globus pallidus produced a suppression of shorter duration than that produced by ACe stimulation. The results suggest that the ACe influences neocortical arousal, which may be mediated by its influence on the activity of cholinergic neurons of the nucleus basalis.

Anatomical and physiological relationships between the anterior cerebellar vermis and the pontine parabrachial nucleus in the rabbit.

The anatomical connections between the midline cerebellum and the pontine parabrachial nucleus (PBN) were investigated in the rabbit using anterograde and retrograde axonal transport techniques. Small injections (20-50 nl) of cholera toxin conjugated to horseradish peroxidase (CT-HRP) or wheat germ agglutinin conjugated HRP (WGA-HRP) into the cortex of the anterior cerebellar vermis resulted in retrograde and anterograde-like label in the PBN. Focal injections of tracer into the PBN resulted in anterogradely labeled processes in the ACV and retrogradely labeled a small, but distinct group of Purkinje cells within the anterior vermis. Focal injections into the rostral fastigial nuclei (FN) resulted in anterograde-like label in the PBN, and PBN injections labeled FN neurons. Furthermore, the projection from the PBN to ACV is effective in driving cerebellar neurons as electrical microstimulation of the PBN evoked short-latency, phasic responses in ACV Purkinje cells. These experiments generated anatomical and physiological evidence for the existence of a neuroanatomical circuit connecting the midline cerebellum (ACV, FN) with the PBN, that may serve as a functional interface between the midline cerebellum and other brain stem nuclei with cardiovascular function, particularly with respect to the midline cerebellar role in classically conditioned cardiovascular responses.

The anterior cerebellar vermis: essential involvement in classically conditioned bradycardia in the rabbit.

The effects of lesions of the cerebellum on the acquisition and retention of aversive Pavlovian conditioned bradycardia were examined in rabbits. Lesions of the anterior cerebellar vermis severely attenuated the acquisition of simple conditioned bradycardia without disrupting baseline heart rate (HR), or unconditioned HR responses. Also, lesions of the vermis performed after the acquisition of conditioned bradycardia eliminated evidence of prior conditioning. Bilateral lesions of the cerebellar hemispheres did not affect conditioned or unconditioned HR responses. These results were interpreted to indicate that anterior vermis lesions specifically disrupted part of an essential conditioned response pathway without interfering with the neural circuits that mediate unconditioned HR responding. These lesion data, coupled with recent electrophysiological evidence of learning-related changes in neuronal activity within the anterior vermis of the fear-conditioned rabbit, suggest that the anterior cerebellar vermis is critically involved in the acquisition and retention of this rapidly learned autonomic conditioned response.

Purkinje cell responses in the anterior cerebellar vermis during Pavlovian fear conditioning in the rabbit.

Extracellular single-unit recordings of Purkinje cells in the anterior cerebellar vermis (ACV) of the rabbit found evidence of short-latency (20-30 ms) differential responses to discriminatively-conditioned auditory stimuli during Pavlovian fear conditioning procedures. These differential unit responses appeared to be a function of learning as differential ACV Purkinje cell responses were not observed in naive (untrained) animals. Some of these evoked neurophysiological responses were also correlated with the behavioral conditioned autonomic response. (CAR); a conditioned bradycardiac response. These electrophysiological data, coupled with previous lesion results, suggest that the ACV is part of an important neural circuit for Pavlovian conditioned bradycardia.

Electrophysiology of the dorsolateral mesopontine reticular formation during Pavlovian conditioning in the rabbit.

Extracellular single-unit recording methods were used to study the activity of neurons within a restricted portion of the dorsolateral mesopontine reticular formation, an area which includes the parabrachial, pedunculopontine tegmental and cuneiform nuclei. Recordings were obtained during presentations of unfamiliar and familiar sensory stimuli, during Pavlovian differential conditioning procedures that elicited conditioned bradycardia, and while stimulating the amygdaloid central nucleus to identify neurons that projected to, or received projections from, the amygdaloid central nucleus. Activity in most dorsolateral mesopontine reticular neurons was altered during sensory stimulation, and the convergence of auditory and somatic inputs onto single neurons was common. Moreover, neural responses were often of a different magnitude and/or direction to auditory stimuli that were unfamiliar vs familiar vs reinforced (paired with pinna stimulation), and many of these differentially responsive neurons were activated orthodromically by stimulation of the amygdaloid central nucleus. In contrast, neurons activated antidromically by stimulation of the amygdaloid central nucleus were relatively quiescent during all phases of the experiment. Results are discussed in relation to current hypotheses concerning the functional significance of various neuronal subpopulations within the dorsolateral mesopontine reticular formation during Pavlovian conditioning.

Midbrain periaqueductal gray projections to the dorsomedial medulla in the rabbit.

The present study sought to determine the existence of projections from the midbrain periaqueductal gray (PAG) to the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMN) in the rabbit. Fast Blue injections into the NTS/DMN complex revealed a population of retrogradely labeled cells within the ventrolateral PAG. Deposits of wheat germ agglutinin/horseradish peroxidase (WGA/HRP) into the ventrolateral PAG revealed terminal label within the dorsomedial, lateral, ventrolateral, intermediate, and commissural subnuclei of the NTS. Label was also observed within the DMN and a heavy concentration encapsulated this nucleus. These data suggest that the projection from the PAG to the NTS/DMN complex may represent a substrate by which the PAG may influence autonomic and cardiovascular regulation, particularly during emotional arousal.

Lesions of the amygdaloid central nucleus block conditioned cardiac arrhythmias in the rabbit receiving digitalis.

We have previously reported [15] that malignant cardiac arrhythmias can be evoked in the rabbit receiving digitalis by cardiovascular changes in response to (a) a Pavlovian aversively conditioned stimulus (CS) or (b) electrical stimulation of the amygdaloid central nucleus (ACE), a structure which contributes to Pavlovian-conditioned cardiovascular responses in the rabbit. The present study was designed to examine further the role of the ACE in arrhythmogenesis by determining the effect of lesions of the ACE on the occurrence of CS-evoked arrhythmias during digitalis administration in the rabbit. Two groups of rabbits (ACE lesion and SHAM) received electrodes implanted bilaterally in the ACE, while a third group (UNOP) served as an unoperated control. All rabbits were given Pavlovian conditioning trials in which a tone conditioned stimulus (CS) was paired with an eyelid shock as an unconditioned stimulus (US). Twenty-four h later, rabbits in the lesion group received bilateral lesions of the ACE. Twenty-four h after the lesion, rabbits in all three groups were given a retention test in which an i.v. infusion of ouabain was delivered, followed by 20 CS alone trials. Presence or absence of arrhythmias was scored during the pre-CS baseline and CS periods for each trial. All three groups exhibited few instances of pre-CS baseline arrhythmias, the frequency of which did not differ between groups. The SHAM and UNOP control groups exhibited a significant increase in the occurrence of CS-arrhythmias compared to pre-CS levels. This increase was blocked in the group receiving lesions of the ACE, as was CS-induced bradycardia which typically occurs in response to the CS in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)

Contributions of the amygdaloid central nucleus to the modulation of the nictitating membrane reflex in the rabbit.

The contributions of the amygdaloid central nucleus (ACe) to the modulation of the amplitude of the nictitating membrane reflex (NMR) were determined. Experiment 1 demonstrated that low-level electrical stimulation of the ACe enhances the amplitude of the NMR when administered immediately preceding the elicitation of the reflex by an eyelid stimulus. In Experiment 2 the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase determined that the ACe projects to the entire rostrocaudal extent of the lateral tegmental field (LTF), the brainstem area in which the multisynaptic component of the unconditioned NMR pathway is believed to be located. These results are consistent with the hypothesis that the ACe, via its projections to the LTF, modulates reflex sensitivity during conditioned arousal and may contribute to the associative enhancement of the unconditioned NMR that occurs early during Pavlovian nictitating membrane conditioning.

Response characteristics of neurons in the medial component of the medial geniculate nucleus during Pavlovian differential fear conditioning in rabbits.

Recent evidence suggests that the amygdaloid central nucleus (ACE) may contribute significantly to Pavlovian fear-conditioned bradycardic responses during the presentation of conditioned emotional stimuli. Because the medial component of the medial geniculate nucleus (MGm) is a major source of input to the region of the ACE, the extracellular single-unit responses of MGm neurons were examined during Pavlovian differentially conditioned bradycardic responding in rabbits. Conditioning involved pairing one tone (CS+) with paraorbital shock and presenting another tone (CS-) in the absence of shock. Two general classes of MGm neurons were identified based on their conditioned-response characteristics. Both groups responded differentially to the CSs. One group responded with greater increases in activity and at a shorter latency to the CS+ compared with the CS-, whereas the other group responded with greater increases in activity and at a shorter latency to the CS- compared with the CS+. Recordings from MGm neurons in naive rabbits prior to conditioning provided evidence that the acoustic stimuli used subsequently as the CS+ and CS- did not evoke differential responses. These results suggest that the MGm along with the ACE may be forebrain components of a neural circuit involved in the acquisition and/or expression of Pavlovian fear-conditioned bradycardic responses.

Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala.

The present investigation was undertaken to examine the organization of peptidergic projections that exist between the parabrachial nuclear complex (PB) and the central nucleus of the amygdala (CNA). The retrograde tracer True Blue was injected into the CNA of adult rats. The brain tissue was then reacted immunocytochemically to localize neurotensin (NT), substance P (SP), methionine enkephalin (ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SS), and cholecystokinin octapeptide (CCK). Following microinjection of True Blue in the CNA, retrogradely-labeled neurons were located primarily in the external lateral subnucleus, abutting the brachium conjunctivum. In animals that received colchicine pretreatment, two populations of neurons, containing either SP or NT, were found to project to the CNA. In addition, cells containing CCK, ENK, VIP, or SS were not found to be a part of this projection system. These data suggest that neurons in the PB project to the CNA and are, in part peptide-containing.

The organization of dorsal medullary projections to the central amygdaloid nucleus and parabrachial nuclei in the rabbit.

The amygdaloid central nucleus and the pontine parabrachial nucleus receive direct, ascending projections from autonomic regulatory nuclei of the dorsal medulla and are recognized as important components of a forebrain system which contributes to autonomic regulation. The present study was designed to provide more detailed information on the anatomical organization of this ascending system in the rabbit by determining (a) the extent to which separate populations of neurons within the solitary complex project to the central nucleus and parabrachial nucleus, (b) the topographical distribution of the projections of the solitary complex within the amygdaloid central nucleus and parabrachial nucleus and (c) the extent to which projections from the solitary complex to the parabrachial nucleus terminate in the region of origin of projections from the parabrachial nucleus to the amygdaloid central nucleus. A fluorescent dye, double retrograde-labeling technique demonstrated that separate populations of neurons in the solitary complex projected to the amygdaloid central nucleus and parabrachial nucleus. Neurons of both populations were more heavily concentrated within the caudal two thirds of nucleus of the solitary tract and were most numerous within the commissural, medial and dorsomedial subnuclei. Labeled neurons were also located within the dorsal motor nucleus of the vagus nerve. Autoradiographic experiments demonstrated that injections of amino acids into the solitary complex resulted in terminal labeling in the central nucleus. This labeling extended rostrally into the adjacent sublenticular substantia innominata and lateral component of the bed nucleus of the stria terminalis. Label was also observed within the lateral, medial, and Kolliker-Fuse regions of the parabrachial nucleus. A particularly dense field was observed overlying cells located within the ventrolateral region of the lateral parabrachial nucleus. This region contained the majority of labeled neurons within the parabrachial nucleus following fluorescent dye injections into the central nucleus. Furthermore, injections of amino acids into this region resulted in terminal labeling within the central nucleus, with a particularly dense area observed within the medial aspect of the nucleus. The results demonstrate that separate populations of neurons within the solitary complex of the rabbit project to the central amygdaloid and parabrachial nuclei and that the majority of these are located within the caudal two-thirds of the complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurobehavioral contributions to cardiac arrhythmias during aversive pavlovian conditioning in the rabbit receiving digitalis.

Clinical observations suggest that a prevalent condition for the occurrence of cardiac arrhythmias is the synergistic interaction of several risk factors including digitalis glycosides, myocardial ischemia and psychological stress. However, little research has been directed toward controlled, systematic investigations of such synergistic interaction, particularly with respect to psychological stress and digitalis glycosides. The present research was undertaken to develop an animal model with which to study the behavioral and neurobiological contributions to arrhythmias during psychological stress in the presence of digitalis. The model used the rabbit as the experimental animal, a pavlovian aversive conditioning procedure and ouabain, a digitalis glycoside. Rabbits received pavlovian conditioning trials in which a tone as a conditioned stimulus (CS) was paired with an eyelid shock as an unconditioned stimulus (US). Twenty-four hours later, a retention test was given in which either of two doses of ouabain or saline was given, followed by 20 CS presentations alone. The CS presentations were given every 120 s (+/- 10 s). During ouabain infusion, the CS significantly increased the frequency of arrhythmic episodes compared to pre-CS baseline frequencies. These arrhythmic episodes were invariably preceded by CS-induced bradycardia. Both parasympathetic and sympathetic involvement were suggested in arrhythmogenesis, since the CS-induced increase in arrhythmic episodes was blocked by pretreatment with either atropine methylnitrate or atenolol. Furthermore, electrical stimulation of the amygdaloid central nucleus, an area implicated in the expression of bradycardia to a pavlovian CS in the rabbit produced arrhythmic episodes in the presence of ouabain. These arrhythmic episodes were similar in topography to CS-induced arrhythmias within any one animal and were decreased by atropine methylnitrate pretreatment. Insights into central and peripheral nervous system mechanisms by which psychological stress is capable of evoking cardiac arrhythmias in the presence of ouabain should ultimately lead to more effective treatment strategies for the prevention of these arrhythmias.