Single unit activity of locus coeruleus neurons in the freely moving cat: II. Conditioning and pharmacologic studies

A series of experiments were carried out in which the single unit activity of presumed noradrenergic (NE) neurons in the area of the locus coeruleus (LC) was examined in freely moving cats during two conditioning paradigms and in response to pharmacologic manipulations relevant to anxiety. LC unit activity was examined during conditioned emotional response (CER) and conditioned food reward (CFR) training. During CER training, LC units showed a large increase in activity in response to a stimulus paired with a noxious air puff, whereas no increase in unit activity was seen in response to a stimulus not paired with the air puff (both comparisons relative to their appropriate control conditions). By contrast, during CFR training, a stimulus paired with a rewarding food delivery did not elicit a significant increase in LC unit activity relative to its appropriate control condition. Therefore, NE units in the LC greatly increase their activity in response to a stimulus that predicts the occurrence of a noxious event, but not in response to a stimulus that predicts the occurrence of a rewarding event. Administration of the anxiolytic drug diazepam (0.25 and 2.0 mg/kg, i.p.) had no significant effect on the spontaneous activity of LC neurons, but significantly reduced the excitatory response of LC neurons to simple stimuli. Administration of the anxiogenic drug yohimbine (2.0 mg/kg, i.p.) significantly increased the spontaneous activity of LC neurons. These results support the hypothesis that LC neurons play a role in aversive but not appetitive conditioning, and are consistent with the hypothesis that these neurons may play a role in anxiety. These data are discussed within the broader context of LC neurons mediating the organism's adaptive response to environmental or physiological challenges.     

Differential Frequency Conditioning Enhances Spectral Contrast Sensitivity of Units in Auditory Cortex (Field Al) of the Alert Mongolian Gerbil

Differential aversive auditory conditioning in the awake Mongolian gerbil was performed during single- and multi-unit recording in field Al of the primary auditory cortex. Presentations of pure tone stimuli of a given frequency (reinforced conditioned stimulus; CS+) paired with electrocutaneous stimulation (unconditioned stimulus) were combined with several other non-reinforced tone stimuli (non-reinforced conditioned stimulus; CS-). Stimulus presentation during training and testing was optimized for constancy of the probability of occurrence of both the CS+ and the CS- stimulus. The paradigm led to a reorganization of both the spectral and temporal response characteristics of auditory cortical neurons with the following basic results. First, tone-evoked responses of Al neurons recorded after multiple acoustic stimulation under these conditions varied statistically around a mean value (stationarity). Conditioning produced a shift in mean values of evoked responses. The altered tone responses were also stationary (stability of the plastic effects). Second, the frequency-receptive fields (FRFs) of neurons were reorganized in a frequency-specific way such that the CS+ frequency became located in a local minimum of the FRF after training. This resulted from a training-induced increase in the responses to frequencies adjacent to the CS+ frequency in the FRF relative to the CS+ response. The effect can be interpreted as an enhancement of the ‘spectral contrast’ sensitivity of the unit in the CS+ neighbourhood. Third, apart from this frequency-specific plastic effect, responses to other frequencies also underwent changes during training. The non-frequency-specific changes were not generally predictable but the post-trial responses were stationary. Fourth, the analysis of the long-term behaviour of FRF reorganization revealed the stability of plastic effects under retention training and the gradual re-establishment of the pretrial FRF during extinction training. Fifth, not only the spectral characteristics but also the temporal structure of the tone-evoked responses could be affected by the training. In most cases the training-induced changes measured within the first tens of milliseconds of the response corresponded to the response changes obtained by integration over the total response period. There were some cases, however, in which the direction of the response change varied with time, indicating that excitatory and inhibitory influences on the temporal response pattern were differently affected by training.     

Stimulation of the locus coeruleus decreases arterial pressure

Unilateral microinjection of L-glutamate (L-Glu, 0.5-5 nmol) into the locus coeruleus (LC) of rats anesthetized with chloralose elicited decreases in blood pressure and heart rate. The maximal depressor response elicited by injection of L-Glu into the LC was approximately 30 mmHg, and the maximal bradycardic response was approximately 35 bpm. Microinjections into the LC of DL-homocysteic acid (1 nmol) and carbachol (1 nmol), agents which (like L-Glu) stimulate neurons within the LC, also produced depressor and bradycardic responses. Vagal blockade with methyl atropine eliminated the bradycardic response, but had no effect on the decrease in arterial pressure elicited by stimulation of neurons within the LC. Destruction of the noradrenergic neurons of the LC by local administration of 6-hydroxydopamine eliminated the cardiovascular responses elicited by injection of L-Glu or carbachol into the LC. These results are in direct contrast to the pressor response elicited by electrical stimulation of the region containing the LC. Destruction of the LC with 6-hydroxydopamine, which totally eliminated the depressor response to L-Glu, slightly potentiated the pressor response elicited by electrical stimulation of this region. Electrical stimulation of the region containing the LC has also been reported to increase plasma vasopressin levels; this response could not be reproduced by by stimulation of the LC by microinjection of L-Glu. These results indicate that stimulation of the noradrenergic neurons of the rat LC decreases arterial pressure and heart rate. Furthermore, these results suggest that the pressor response elicited by electrical stimulation of this region is not due to stimulation of neurons of the LC.     

Locus coeruleus activation shortens synaptic drive while decreasing spike latency and jitter in sensorimotor cortex. Implications for neuronal integration

Chronic recording of locus coeruleus (LC) neurons in rat and monkey have pointed out that brief, phasic LC discharges, but not sustained activity, are specifically related to salient stimuli and attention. However, the sensory consequences of phasic activation of the noradrenergic system by a brief conditioning stimulation of the LC have not been fully investigated. This study examined the effect of LC activation on synaptic and neuronal responses to a tactile stimulus in the sensorimotor cortex of the anaesthetized rat, by analysing the fine temporal structure of sensory discharges and current source-density profiles recorded from the same electrodes. LC stimulation, with minimal EEG effects, consistently reduced the synaptic input in layers IV and V-VI, by decreasing the amplitude and duration of short-latency current sinks, but not the slope of their early rising phase. Simultaneously, most multiple and single unit excitatory responses were shortened by the suppression of their late component after 25-30 ms, whereas robust temporal facilitation of the early discharge was found for spike latency mean and variance, spike timing and synchronization to the stimulus, but leaving the number of spikes unaffected. These two apparently opposite effects on the synaptic drive and neuronal response are reminiscent of the noradrenergic depression of afferent synaptic potentials observed with an increased neuronal excitability in vitro. They are interpreted as a noradrenergic sharpening of thalamocortical processing consistent with a presumed role of synchronous discharges in perception that would depend on activated states, particularly when LC activity is correlated with vigilance or attention.     

Conditioned enhancement and suppression in the developing auditory midbrain

Neural responses in the adult central auditory system to binaural stimuli can be altered by preceding acoustic events, including auditory motion. To determine whether the juvenile auditory system also exhibits this feature, we have examined interaural level difference (ILD) processing in the developing gerbil. A long binaural stimulus was followed without interruption by modulation of the level difference (virtual acoustic motion), which in turn was followed smoothly by a new steady state ILD. Auditory responses of single neurons in the inferior colliculus (IC) were assessed for sensitivity to the final steady state ILD. The response of EI neurons (excited by contralateral stimulation and inhibited ipsilaterally) was examined at postnatal (P) days 17-18, P24-25, and in adult animals. In adult animals, a sudden reduction of the inhibitory stimulus level resulted in a long-lasting (median = 4.3 s) enhanced discharge rate (conditioned enhancement). In P17-18 animals, conditioned enhancement only lasted for 1.2 s. When the inhibitory stimulus level was suddenly increased, adult neurons often displayed a conditioned suppression of discharge rate (median = 4.5 s), whereas P17-18 neurons remained suppressed for a much briefer period (median = 1.2 s). Moreover, the difference between conditioned responses and control discharge rates was three-four times greater in adult neurons compared to those recorded in P17-25 animals. Because conditioned responses are sensitive to the relative balance of contralateral excitation and ipsilateral inhibition, we examined the relationship between excitatory and inhibitory thresholds. In adult animals, excitatory thresholds were an average of 12 dB lower than inhibitory thresholds, while at P17-25 excitatory and inhibitory thresholds were roughly the same. These results indicate that computational properties of juvenile and adult IC neurons differ quantitatively, and this may reflect an imbalance between excitation and inhibition. The developmental differences described herein may limit the ability of young animals to locate a sound source with the latency and accuracy of an adult.     

Spontaneous and auditory-evoked activity of medial agranular cortex as a function of arousal state in the freely moving rat: interaction with locus coeruleus activity

To characterize the electrophysiological properties of neurons in the medial agranular frontal cortex (Fr2) with respect to arousal level and locus coeruleus (LC) activity, we recorded spontaneous and auditory-evoked single unit activity in these areas simultaneously during different states of arousal in the rat. In the low-arousal state, as determined by EEG, 14/56 Fr2 neurons showed a tonic increase in spontaneous firing rate and 9/56 presented a clear inhibitory response to tone (onset latency 37 ms, duration 200 ms). The inhibitory response was not clear during the high-arousal state. Cross-correlation analysis of pairs of Fr2 and LC units, excluding activity during the actual tone, also showed a negative correlation from 120 ms before, to 170 ms after, Fr2 discharge in 5/63 pairs, only during low arousal. Significantly, 4/5 of the Fr2 neurons having this negative correlation with LC were included in that population which showed a tonic increase in spontaneous firing rate and inhibited to tone during low arousal. LC neurons, on the other hand, all showed excitation to tone stimulation (peak latency 30 ms), and response amplitude was not affected by changes in arousal level. The negative correlation in spontaneous activity, as well as their differential responses to tone, suggests an interaction between a select population of Fr2 neurons and the LC during the low-arousal state. Future studies with lesion or pharmacological manipulations would be necessary to confirm the presence of this interaction.     

Activation of lateral geniculate neurons by locus coeruleus or dorsal noradrenergic bundle stimulation: Selective blockade by the alpha1-adrenoceptor antagonist prazosin

Presentation of a stimulus train to the locus coeruleus (LC) or dorsal noradrenergic bundle (DB) resulted in a facilitation of thev spontaneous firing of single units in the dorsal lateral geniculate nucleus (LGNd) of the rat. These stimulation effects were blocked by the alpha₁-adrenoceptor antagonists WB-4101 and prazosin. Both drugs also blocked the activation of LGNd neurons by iontophoretic norepinephrine (NE). The cholinergic agonists acetylcholine (ACh) and carbachol (CCh) activated LGNd neurons in a similar fashion to NE, however, these responses were selectively blocked by the muscarinic antagonist scopolamine. The response to ACh was also sensitive to WB-4101 suggesting that the drug possesses some cholinergic blocking activity. In contrast to WB-4101, prazosin displayed a high degree of selectivity for noradrenergic but not cholinergic response. On the basis of the observation that prazosin selectively antagonizes both the stimulation effects and iontophoretic NE (but not CCh), we conclude that activation of LGNd neurons by LC or DB stimulation is mediated predominantly via the release of NE from coeruleo-geniculate fibers, rather than the inadvertent activation of a cholinergic pathway. Moreover, inasmuch as the systemic administration of prazosin effectively blocks central noradrenergic neurotransmission at dosescomparable to those uses clinically, the possibility that prazosin exerts its antihypertensive action in part via a central mechanism requires further investigation.     

Opposing influence of basolateral amygdala and footshock stimulation on neurons of the central amygdala.

BACKGROUND: The basolateral complex (BLA) and the central nucleus of the amygdala (CeA) are believed to mediate the expression of affective responses. After affective learning, conditioned stimulus-related information is thought to be conveyed from the BLA to the CeA; the medial CeA (Cem), in turn, projects to hypothalamic and brainstem structures involved with induction of affective responses. Although the conditioned stimulus and unconditioned stimulus both evoke affective responses, the precise response often differs. It is unknown whether this difference is represented by distinct activity patterns of single Cem neurons. Furthermore, the nature of the interaction between the BLA and Cem is unknown. METHODS: Using in vivo extracellular and intracellular recordings, we examined how the BLA affects the Cem and compared this with effects induced by footshock (unconditioned stimulus) in the same neurons. RESULTS: Our results demonstrate that, contrary to conventional views, BLA stimulation primarily inhibits Cem neurons by a polysynaptic circuit, and show that single Cem neurons respond to both BLA input and footshock in an opposite manner. CONCLUSIONS: These results demonstrate the predominantly inhibitory nature of the BLA-Cem interaction. These data further demonstrate the distinct cellular events that might lead to differential modulation of conditioned and unconditioned affective responses.     

Neuronal plasticity in the limbic system during classical conditioning of the rabbit nictitating membrane response. I. The hippocampus

Hippocampal unit responses were recorded throughout classical conditioning of the rabbit nictitating membrane response to a tone conditioned stimulus (CS) using a corneal air-puff unconditioned stimulus (UCS). Multiple unit analysis revealed that a rapidly developing increase in cell discharges (relative to spontaneous activity) occurs within the first block of paired trials and continues to increment with subsequent training, initially in the UCS period and then in the CS period. The pattern of hippocampal activity within paired trials closely parallels the amplitude-time course of the behavioral response and precedes it temporally. Identical recordsings from animals given unpaired CS-alone and UCS-alone presentations showed no such changes. These control results and additional lines of evidence point to the critical necessity of the learning paradigm for the development of the hippocampal response seen in conditioning animals. A single unit analysis indicates that not all hippocampal neurons exhibit the described conditioned discharge pattern. Hippocampal long-term potentiation is considered as a possible mechanism for mediating this early and rapid neuronal plasticity dependent on specific 'contingent' patterns of stimulation.     

Single-unit response of noradrenergic neurons in the locus coeruleus of freely moving cats. I. Acutely presented stressful and nonstressful stimuli

The present experiment was designed to explore the stress-relatedness of activity in noradrenergic neurons of the locus coeruleus (LC) of behaving cats. A stressor was defined as a stimulus that elicited a significant sympathoadrenal activation as measured by plasma norepinephrine level and heart rate. According to this definition, exposure to 15 min of 100 dB white noise or 15 min of restraint was stressful in cats. In contrast, exposure to inaccessible rats for 15 min was behaviorally activating but nonstressful. The single-unit activity of noradrenergic neurons in the LC of behaving cats was examined under these conditions. The stressful stimuli elicited a significant increase in LC neuronal activity for the entire 15 min stressor duration, whereas the behaviorally activating but nonstressful stimulus elicited no significant change in the activity of these neurons. These results provide evidence that behavioral activation per se is not sufficient to evoke a tonic activation of these neurons. Rather, these data support the hypothesis that the LC is involved in the CNS response to stress and provide additional evidence that the activity of LC noradrenergic neurons increases in association with sympathoadrenal activation.     

Classical conditioning does not occur when direct stimulation of the red nucleus or cerebellar nuclei is the unconditioned stimulus

The involvement of the cerebellum and the red nucleus in the classically conditioned nictitating membrane/eyeblink response in the rabbit is investigated using direct stimulation of the interpositus or the red nucleus as the unconditioned stimulus. Stimulation of either of these structures produced eye closure in naive animals, and this eye closure was paired with a tone in the standard Pavlovian conditioning paradigm. The results indicate that eyelid closure due to stimulation of either the red nucleus or the interpositus nucleus is not sufficient for the development of conditioned responses to the tone. Animals which had received interpositus stimulation as the unconditioned stimulus acquired the conditioned response to tone significantly faster following the substitution of air puff for stimulation than did those animals that had received red nucleus stimulation, or controls that did not receive any stimulation. However, animals that had been trained to tone-air puff could not retain the conditioned response after being switched to tone-interpositus stimulation. Lesions of the interpositus and the red nucleus through the stimulating electrodes were effective in impairing or abolishing conditioned responses. The results are interpreted to indicate that the red nucleus and interpositus are elements of the circuit that carries out the expression of the conditioned response. In addition the interpositus, but not the red nucleus, may be critical in the formation of the memory trace for the conditioned stimulus-unconditioned stimulus association, by virtue of the greatly accelerated learning that results from its stimulation.     

Target cell stimulation and inhibition of norepinephrine uptake in dissociated rat locus coeruleus cultures

Primary cultures from dissociated locus coeruleus (LC) neurons of 14-day-old (E14) fetal rats were grown in vitro in serum complemented medium. Noradrenergic cells were identified using immunocytochemical staining for tyrosine hydroxylase (TH) antibody. Maturation of noradrenergic neurons was assessed by measuring the high-affinity uptake of [3H]norepinephrine (NE). The presence of hippocampal cells stimulated the specific uptake of [3H]NE by LC cells only when plated at low density. Increasing the concentration of hippocampal cells resulted in a 50% decrease in NE uptake by LC cells. A similar inhibitory effect was observed with striatal cells. The inhibition exerted by striatal cells appears to be developmentally regulated, with E18 exerting a stronger inhibitory effect than E15 striatum. The decrease in [3H]NE uptake in hippocampal-LC cocultures was due to a decrease in uptake by individual noradrenergic neurons. For a given plating density, the decrease in uptake of [3H]NE per noradrenergic cell in LC culture was only half the decrease in the cocultures, suggesting a target-associated effect rather than density-derived toxic effect. In culture conditions which favored neuronal but not glial survival, the stimulatory target effect was evident, and the inhibitory effect was absent. Medium conditioned by target glial cells had a marked stimulatory effect on [3H]NE uptake. Glial feader-layer had a strong inhibitory effect on [3H]NE uptake in serum-containing medium. We suggest that both neurons and glia mediate the target-stimulatory effect, whereas the inhibitory effect is mediated by direct contact between target glia and LC neurons.     

Locus coeruleus stimulation potentiates local inhibitory processes in rat cerebellum

We previously reported that a low threshold action of norepinephrine (NE) on the cerebellar circuitry is expressed as an amplification of the inhibitory action of gamma aminobutyric acid (GABA) on Purkinje cell activity. Here we examined the effects of locus coeruleus (LC) stimulation on “off-beam” inhibitions of Purkinje cell firing induced by activation of local basket and stellate cell interneurons to determine whether endogenous NE, released from synaptic terminals, could induce a comparable enhancement of GABA-mediated synaptic input to these neurons. Stimulation of LC, at current intensities which by themselves were subthreshold for directly affecting background activity of Purkinje neurons, markedly increased off-beam inhibitory neuronal responses. Iontophoretic application of the beta-adrenergic blocker sotalol reversibly antagonized this enhancement of synaptic inhibition. In comparison, the potentiative effects observed with LC stimulation were increased by iontophoresis of the alpha-adrenergic blocker phentolamine. LC -induced increases in off-beam inhibition were not observed after destruction of cerebellar noradrenergic terminals by 6-hydroxydopamine. These results suggest that noradrenergic input from the LC can augment the efficacy of conventional GABA-mediated inputs synapsing on the Purkinje cell.     

The roles of different types of glutamate receptors involved in the mediation of nucleus submedius (Sm) glutamate-evoked antinociception in the rat

It has been accepted that the descending system from the nucleus locus coeruleus (LC)/nucleus subcoeruleus (SC) plays a significant role in spinal nociceptive processing. The present study was designed to examine modulation of nociceptive processing in the caudal part of the trigeminal sensory nuclear complex, the trigeminal subnucleus caudalis which is generally considered to be involved in the relay of oral-facial nociceptive information. Experiments were performed on anesthetized Sprague-Dawley rats. The site of LC/SC stimulation was confirmed by histology using potassium ferrocyanide to produce a Prussian blue reaction product marking the iron deposited from the stimulating electrode tip. Only data from rats which had electrode placements in the LC/SC were used. Electrical stimulation was delivered at a stimulus intensity below 100 microA in the present study. Stimulation at sites inside the LC/SC produced a reduction of both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Increasing stimulation frequency in the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. At three of nine sites outside the LC/SC, electrical stimulation was effective on descending inhibition. A significant difference in the inhibitory effects was observed when the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC. These findings suggest that nociceptive processing in the subnucleus caudalis is under the control of the descending modulation system from the LC/SC. To understand the effects of repetitive stimulation with high frequency on fine unmyelinated LC/SC fibers, the existence of recurrent collateral excitation in the LC/SC may be considered.     

Augmentation of the noradrenergic system in alpha-2 adrenergic receptor deficient mice: anatomical changes associated with enhanced fear memory

We have investigated sensitivity to the conditioned fear procedure of mice is influenced by the genetic deletion of alpha2A adrenoceptors (ARs). We observed a heightened freezing response in the discrete cue memory test in alpha2A AR knockout (alpha2A AR KO) mice and in D79N mice, a transgenic mouse strain with functionally impaired alpha2A ARs. No significant differences in contextual memory were observed between control and alpha2A AR KO or D79N mice suggesting a minimal role for the noradrenergic system in contextual memory. We speculated that the increased freezing response of the alpha2A AR KO and D79N mice in the discrete cue setting was due to increased release of norepinephrine evoked by the unconditioned footshock stimulus. In alpha2A AR KO mice we measured a doubling in the number of noradrenergic neurons in the locus coeruleus (LC) and a large increase in the cell volume of tyrosine hydroxylase positive neurons, likely due to selective preservation of large, multipolar neurons in the subcoeruleus. Hyperplasia of the noradrenergic neurons in the nucleus tractus solitarius, A5 and A7, was also observed. Alpha2A AR KO mice exhibit greater c-Fos expression in the LC compared to wild type mice suggesting that the LC neurons in the alpha2A AR KO mice were spontaneously more active. This study suggests that alpha2A ARs are involved in the development of the central noradrenergic system and raises the possibility that alterations in alpha2A AR expression may contribute to variations in fear and stress responses.     

Locus coeruleus activation modulates firing rate and temporal organization of odour-induced single-cell responses in rat piriform cortex

Piriform cortex (PCx) is the primary cortical projection region for olfactory information and has bidirectional monosynaptic connections with olfactory bulb and association cortices. PCx neurons display a complex receptive field, responding to odours rather than their molecular components, suggesting that these neurons are involved in higher order olfactory processing. Neuromodulators, especially noradrenaline (NA), have important influences on sensory processing in other cortical regions and might be responsible for the plasticity observed in PCx during learning. The present study is the first attempt to examine in vivo the actions of NA on sensory responses in the PCx. Stimulation of the noradrenergic nucleus locus coeruleus (LC) was used to induce release of NA in the forebrain in urethane-anaesthetized rats. Extracellular recording of single units was made simultaneously in anterior and posterior PCx. The responses to an odour stimulus were measured over 25 trials. Twenty-five subsequent odour presentations were preceded by stimulation of the ipsilateral LC through a bipolar electrode, previously placed in the LC under electrophysiological control. This priming stimulation modified the activity of 77 of the 135 recorded neurons. For most cells, LC stimulation enhanced cortical responses to odour in terms of both spike count and temporal organization, with some differential effects in anterior and posterior regions. These results are the first to show enhancement of sensory responses in the olfactory cortex by LC activation. Spontaneous activation of LC neurons such as occurs during learning could serve to enhance olfactory perception and promote learning.     

Inhibition of the release of LH and ovulation by activation of the noradrenergic system. Effect of interrupting the ascending pathways

The effect of stimulation of the central noradrenergic system on the release of LH and ovulation was studied in the female rat. Electrochemical stimulation (ECS) (anodic DC) was applied through monopolar stainless-steel electrodes chronically implanted in animals bearing a plastic cannula inserted into the jugular vein for blood sampling.In ovariectomized, estrogen-primed rats, ECS applied in the nucleus locus coeruleus (LC) blocked the release of LH triggered by electrical stimulation of the medial preoptic area. A similar result was observed when the stimulus was applied in the A5 cell grouping but not when it was applied outside the nuclei but near them or when a cathodic current was passed through the electrode implanted into the LC. The degree of inhibition of LH release was proportional to the amount of current applied.Electrochemical stimulation of the LC, the A5 or the A1 cell grouping in non-anesthetized freely-behaving rats on the day of proestrus also blocked ovulation and the spontaneous surge of LH.Unilateral transection of the dorsal noradrenergic bundle at the level of the mesencephalon completely suppressed the inhibitory effect on LH release resulting from ECS applied to the ipsilateral LC nucleus of A5 cell grouping. On the contrary unilateral transection of the ventral noradrenergic bundle did not interfere with the inhibition elicited from the LC and only partially affected that elicited from the A5 neuronal group.These results provide evidence that endogenous release of norepinephrine elicited by activation of the central noradrenergic system can inhibit LH secretion, suggesting that the noradrenergic system may be envolved inhibitory mechanisms controlling LH release.     

A.E. Bennett Research Award. Anatomic basis for differential regulation of the rostrolateral peri-locus coeruleus region by limbic afferents.

BACKGROUND: Neurochemical and electrophysiological studies indicate that the locus coeruleus (LC)-norepinephrine system is activated by physiological and external stressors. This activation is mediated in part by corticotropin-releasing factor (CRF), the hypothalamic neurohormone that initiates the endocrine response to stress. We have previously shown that the central nucleus of the amygdala (CNA) provides CRF afferents to noradrenergic processes in the peri-LC area that may serve to integrate emotional and cognitive responses to stress. The bed nucleus of the stria terminalis (BNST) shares many anatomical and neurochemical characteristics with the CNA, including a high density of CRF-immunoreactive cells and fibers; however, recent studies have suggested that the CNA and the BNST may differentially regulate responses to conditioned and unconditioned fear, respectively, suggesting divergent neuroanatomical circuits underlying these processes. METHODS: In the present study, neuroanatomical substrates subserving regulation of the LC by the BNST were examined. Anterograde tract-tracing was combined with immunoelectron microscopy to test the hypotheses that BNST efferents target noradrenergic neurons of the LC and that these efferents exhibit immunolabeling for CRF. RESULTS: Ultrastructural analysis of sections that were dually labeled for the anterograde tracer biotinylated dextran amine (BDA) injected into the BNST and tyrosine hydroxylase (TH)-immunoreactivity demonstrated that BDA-labeled axon terminals formed synaptic specializations (primarily inhibitory) with TH-labeled dendrites and dendrites that lacked TH immunoreactivity. In contrast to CNA efferents that exhibited substantial immunolabeling for CRF, far fewer BDA-labeled terminals from the BNST in the rostrolateral peri-LC contained CRF. CONCLUSIONS: The present results indicate that the BNST may provide distinct neurochemical regulation of the peri-LC as compared to other limbic afferents such as the CNA. These data are interesting in light of behavioral studies showing that the CNA and BNST may be differentially involved in fear versus anxiety, respectively.     

Electrophysiological characteristics of amygdaloid central nucleus neurons during Pavlovian fear conditioning in the rabbit

Recent evidence suggests that the amygdaloid central nucleus (ACE) may contribute importantly to cardiovascular adjustments in response to the presentation of conditioned emotional stimuli, possibly via direct ACE projections to cardiovascular regulatory nuclei in the medulla. The present experiment was conducted to obtain additional data relevant to this suggestion. Extracellular single-unit recordings were obtained from 85 histologically-verified ACE neurons during Pavlovian differentially conditioned heart-rate responding in rabbits. Conditioning involved pairing one tone (CS+), but not a second tone (CS-), with paraorbital shock. Those ACE neurons which project to the lower brainstem were identified by their antidromic responses to stimulation of a mesencephalic region through which descending ACE projections course. Under these conditions it was possible to classify ACE neurons as conforming to one of 6 general categories based on their spontaneous activity and conditioned response characteristics. In addition, it was determined that: (1) the electrophysiological characteristics of many ACE neurons were differentially altered in response to presentations of the CS+ versus the CS-; (2) the responses of many ACE neurons to presentations of the CS+ were correlated with the magnitudes of concomitant conditioned alterations in heart rate; and (3) the activity of antidromically-identified ACE neurons which project to the lower brainstem was decreased in response to presentations of each CS. These data provide additional support for the notion that the ACE contributes to cardiovascular regulation during the presentation of emotionally-arousing stimuli.     

A role for acetylcholine in conditioning-related responses of rat frontal cortex neurons: microiontophoretic evidence

In an associative conditioning paradigm, an auditory stimulus (CS+) was paired with rewarding medial forebrain bundle stimulation or a tone of different frequency (CS-) was presented without pairing. After training, slow potential (SP) and single neuron responses were recorded from rat frontal cortex. When cortical SP responses indicated the development of discrimination between CS+ and CS- tones, single neurons could be isolated that exhibited a discriminative response to CS+. Seventy-three percent of the 56 neurons which discriminated between CS+ and CS- were excited by the paired tone while the remainder were inhibited. Iontophoretically applied acetylcholine increased spontaneous firing rate in 90% of the excited cells and 87% of the inhibited cells. Iontophoretic administration of a muscarinic receptor antagonist, either atropine or tropicamide, during trial presentation attenuated the conditioning-related response to CS+ as well as the response to acetylcholine in the majority of neurons. The largest group of discriminating neurons were excited by both CS+ and acetylcholine, and both responses were suppressed by the antagonists. The results provide evidence that conditioning-related responses of a major population of frontal cortex neurons are modulated by cholinergic input, a portion of which may originate in the basal forebrain area. There also may be a significant non-cholinergic influence on these neuronal responses.