Discharge Rate Of Locus Coeruleus Neurons Research Articles

Synergistic effect of orexin-glutamate co-administration on spontaneous discharge rate of locus coeruleus neurons in morphine-dependent rats

Various intrinsic and extrinsic factors can increase the spontaneous discharge rate of locus coeruleus (LC) neurons. Among the extrinsic ones, orexinergic neurons of the lateral hypothalamus (LH) send widespread projections to LC region. Accumulating evidence support the involvement of glutamate in mediating the excitatory effect of orexin-A on LC neurons. In addition, both orexinergic and glutamatergic systems have been shown to play critical roles in molecular changes underlying the development of morphine dependence. The present study was designed to investigate the interaction between orexin-A and glutamate in modulating the firing rate of LC neurons. Regarding the role of both orexinergic and glutamatergic systems in morphine dependence, this effect was also investigated in morphine dependent rats. For this purpose, spontaneous discharge rate of LC neurons was recorded using the whole-cell patch clamp recording method in presence of orexin-A, glutamate or orexin-A plus glutamate in acutely prepared brain slices. Results indicated that superfusion of either orexin-A or glutamate enhances the firing rate of LC neurons in both dependent and non-dependent rats. However, co-application of orexin-A and glutamate elicited a significant synergism in enhancement of firing rate only in morphine dependent animals. In conclusion, it seems that development of morphine dependence promotes adaptations in locus coeruleus neurons that potentiate the orexin-glutamate interaction.

Exploration of supraspinal mechanisms in effects of spinal cord stimulation: Role of the locus coeruleus

The neurobiological mechanisms of spinal cord stimulation (SCS) when applied for neuropathic pain are still incompletely known. Previous research indicates that brainstem circuitry is pivotal for the SCS effect. The present study aims at exploring the possible contribution to the SCS effects of the pain controlling system emanating from the locus coeruleus (LC) in the brain stem. Experiments were performed on the rat-spared nerve injury pain model. After evaluation of the attenuation of mechanical hypersensitivity induced by SCS, the effects of SCS on neuronal activity in the LC and on the noradrenaline (NA) content in the dorsal spinal cord were analyzed. SCS produced a significant increase in the discharge rate of LC neurons only in rats behaviorally responding to SCS as compared to non-responding and control animals. The NA content in the dorsal quadrant of the spinal cord ipsilateral to the nerve injury was analyzed using enzyme-linked immunosorbent assay in responding, non-responding and intact control rats both immediately following SCS and without SCS. No differences were found between these groups. In awake animals, lidocaine silencing of the ipsilateral LC or blocking of spinal noradrenergic system by intrathecal administration of α1,2 adrenoceptor antagonists failed to influence the antihypersensitivity effect of SCS. The present results indicate that the SCS-induced control of hypersensitivity in an experimental animal model of peripheral neuropathic pain may not be explained by the activation of direct spinal projections of noradrenergic LC neurons, while supraspinal projections of LC neurons still may play a role in the SCS effect.

Antinociception by motor cortex stimulation in the neuropathic rat: does the locus coeruleus play a role?

We studied whether stimulation of the primary motor cortex (M1) attenuates pain-related spinal withdrawal responses of neuropathic and healthy control rats, and whether the descending antinociceptive effect is relayed through the noradrenergic locus coeruleus (LC). The assessments of the noxious heat-evoked limb withdrawals reflecting spinal nociception and recordings of single LC units were performed in spinal nerve-ligated neuropathic and sham-operated control rats under light pentobarbital anesthesia. Electric stimulation of M1 produced equally strong spinal antinociception in neuropathic and control rats. Following microinjection into M1, a group I metabotropic glutamate receptor agonist (DHPG; 10 nmol) and a high (25 nmol) but not low (2.5 nmol) dose of glutamate slightly increased on-going discharge rates of LC neurons in neuropathic but not in control animals. Influence of electric stimulation of M1 on LC neurons was studied only in the neuropathic group, in which discharge rates of LC neurons were increased by electric M1 stimulation. Lidocaine block of the LC or block of descending noradrenergic influence by intrathecal administration of a alpha(2)-adrenoceptor antagonist failed to produce a significant attenuation of the spinal antinociceptive effect induced by electric M1 stimulation in the neuropathic or the sham group. The results indicate that stimulation of the rat M1 induces spinal antinociception in neuropathic as well as control conditions. While M1 stimulation may activate the LC, particularly in the neuropathic group, the contribution of coeruleospinal noradrenergic pathways may not be critical for the spinal antinociceptive effect induced by M1 stimulation.

Influence of peripheral nerve injury on response properties of locus coeruleus neurons and coeruleospinal antinociception in the rat

Noradrenergic locus coeruleus (LC) is involved in pain regulation. We studied whether response properties of LC neurons or coeruleospinal antinociception are changed 10–14 days following development of experimental neuropathy. Experiments were performed in spinal nerve–ligated, sham-operated and unoperated male rats under sodium pentobarbital anesthesia. Recordings of LC neurons indicated that responses evoked by noxious somatic stimulation were enhanced in nerve-injured animals, while the effects of nerve injury on spontaneous activity or the response to noxious visceral stimulation were not significant. Microinjection of glutamate into the central nucleus of the amygdala produced a dose-related inhibition of the discharge rate of LC neurons in nerve-injured animals but no significant effect on discharge rates in control groups. Assessment of the heat-induced hind limb withdrawal latency indicated that spinal antinociception induced by electrical stimulation of the LC was significantly weaker in nerve-injured than control animals. The results indicate that peripheral neuropathy induces bidirectional changes in coeruleospinal inhibition of pain. Increased responses of LC neurons to noxious somatic stimulation are likely to promote feedback inhibition of neuropathic hypersensitivity, while the enhanced inhibition of the LC from the amygdala is likely to suppress noradrenergic pain inhibition and promote neuropathic pain. It is proposed that the decreased spinal antinociception induced by direct stimulation of the LC may be explained by pronociceptive changes in the non-noradrenergic systems previously described in peripheral neuropathy. Furthermore, we propose the hypothesis that emotions processed by the amygdala enhance pain due to increased inhibition of the LC in peripheral neuropathy.

The CRF 1 receptor antagonist, NBI-35965, abolished the activation of locus coeruleus neurons induced by colorectal distension and intracisternal CRF in rats

Corticotropin-releasing factor (CRF) receptors have been reported to play a role in tonic colorectal distension (CRD)-induced activation of locus coeruleus (LC) neurons. We examined the influence of repeated phasic CRDs and intracisternal (ic) CRF on the spontaneous discharge rate of LC neurons in chloral hydrate-anesthetized rats and the role of CRF receptors using the nonselective CRF 1/CRF 2 antagonist, astressin, and the water-soluble CRF 1 receptor antagonist, NBI-35965. Two consecutive phasic CRDs (43.7 ± 1.1 mm Hg, 30 s each) at a 10-min interval increased LC activity to 184.9 ± 15% and 171.9 ± 12.2%, respectively. There was no difference in magnitude, onset (within 1 s), and duration (5–7 min) of the LC responses between the 1st and 2nd CRDs. CRF (300 ng/rat, ic) injected 10 min after the 2nd CRD increased LC activity to 191.1 ± 11.2%. Astressin (3 μg, ic) completely blocked the 2nd CRD- and ic CRF-induced LC activation. Neither ic vehicle nor astressin influenced basal LC neuronal activity. NBI-35965 (10 mg/kg, iv) prevented the 2nd CRD- and ic CRF-induced LC neuronal activation, while at 5 mg significantly reduced the LC response to the 2nd CRD by 80%, but did not block that of ic CRF injected 30 min later. These findings indicate a primary role of brain CRF interacting with CRF 1 receptors in mediating the activation of LC neurons in response to a phasic CRD within the nociceptive range (>40 mm Hg). This activation may have relevance to irritable bowel syndrome characterized by lower pain threshold to CRD and hypervigilance to colonic input.

Contribution of the stress-induced degeneration of the locus coeruleus noradrenergic neurons to the pathophysiology of depression: a study on an animal model.

A novel theory on the pathophysiology of depression would be expected to resolve a contradiction between therapeutic time lag and monoamine hypothesis. On the basis of the fact that a subgroup of depression appears during or after stress, we exposed rats to a long-term (2 weeks) forced walking stress and produced depression-model rats in one group and spontaneous recovery rats in another. The density of axon terminals of the locus coeruleus (LC) neurons in the frontal cortex stained by dopamine β-hydroxylase antiserum was lower in the depression-model rats than in the spontaneous recovery rats and in the control rats without stress. The density was higher in the model rats daily treated with imipramine than in those treated with saline. Morphological projection (MP) index (a percentage of horseradish peroxidase-positive LC cells in total number of LC cells) and electrophysiological projection index (a percentage of LC neurons activated antidromically by electrical stimulation of the cerebral cortex) were lower in the depression-model rats than in the recovery and control rats. MP index was higher in the imipramine-treated rats than that in the saline-treated rats. Electron microscopic examination of the LC disclosed such degenerative changes as low-dense areas without structure, aggregation of intracellular organs, destroyed membranes around the rough endoplasmic reticulum (rER), a decreased number of deformed subsurface cisterns, glia invaginated into the LC neurons and prominent appearance of microglia containing increased number of lipofustin or lysosome in the model rats, but not in the spontaneous recovery rats. These findings suggest that the terminals and cell bodies of the LC noradrenergic neurons degenerate in the stress-induced depression-model rats and regenerate in the imipramine-treated model rats. This degenerative change may possibly contribute to the decrease in synthesis and metabolism of noradrenaline (NA), the slowing of axonal flow, the accumulation of NA in the neurons, the decrease in discharge rate of LC neurons without stress and the increase in release of NA in response to an additional stress. It may also explain the therapeutic time lag that is required to repair the noradrenergic neurons.

Local opiate withdrawal in locus coeruleus neurons in vitro.

Noradrenergic neurons of the brain nucleus locus coeruleus (LC) become hyperactive during opiate withdrawal. It has been uncertain to what extent such hyperactivity reflects changes in intrinsic properties of these cells. The effects of withdrawal from chronic morphine on the activity of LC neurons were studied using intracellular recordings in rat brain slices. LC neurons in slices from chronically morphine-treated rats exhibited more than twice the frequency of spontaneous action potentials after naloxone compared with LC neurons from control rats. However, after naloxone treatment, the resting membrane potential (MP) of LC neurons from dependent rats was not significantly different from that in control rats. Neither resting MP nor spontaneous discharge rate (SDR) was altered by naloxone in LC neurons from control rats. Neither kynurenic acid nor a cocktail of glutamate and GABA antagonists (6-cyano-7-nitroquinoxalene-2,3-dione + 2-amino-5-phosphonopentanoic acid + bicuculline) blocked the hyperactivity of LC neurons precipitated by naloxone in slices from morphine-dependent rats. The effects of ouabain on MP and SDR were similar in LC neurons from control and morphine-dependent rats. These results indicate that an adaptive change in glutamatergic or GABAergic synaptic mechanisms or altered Na/K pump activity does not underlie the withdrawal-induced activation of LC neurons in vitro. Specific inhibitors of protein kinase A [Rp-cAMPS or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89)] partially suppressed the withdrawal hyperactivity of LC neurons, and activators of cAMP (forskolin) or protein kinase A (Sp-cAMPS) increased the discharge rate of LC neurons from control rats. These results suggest that upregulation of cAMP-dependent protein kinase A during chronic morphine treatment is involved in the withdrawal-induced hyperactivity of LC neurons.

Locus coeruleus electrophysiological activity and responsivity to corticotropin-releasing factor in inbred hypertensive and normotensive rats

The spontaneously hypertensive rat (SHR) and its normotensive progenitor, the Wistar–Kyoto rat (WKY), have been shown to be differentially responsive to the behavioral and endocrine effects of both stress and corticotropin-releasing factor (CRF), both of which increase locus coeruleus (LC) electrophysiological activity. However, the effect of central administration of CRF in these rat strains has yet to be examined. In the present studies, LC electrophysiological responsivity to intracerebroventricular infusions of CRF was assessed in SHR, an inbred strain of WKY rats (the WKY LJ rat), and an outbred normotensive rat strain, Sprague-Dawley (SD) rats. Spontaneous LC discharge rate, mean arterial blood pressure and heart rate were also examined. LC activity was increased to the same extent in the three rat strains in response to a 3 μg dose of CRF. However, WKY LJ rats showed an exaggerated LC in response to a 1 μg dose of CRF in comparison to the other rat strains tested at this dose. Spontaneous discharge rates of individual LC neurons were lower in both SHR and WKY LJ rats than in SD rats. Further, the variability of the discharge rates of LC neurons was greater in WKY LJ rats than in the other two strains. These results indicate that the WKY LJ rat may provide a useful model for assessing the role of sensitivity to CRF in stress responsiveness.

Ultrastructural Evidence for Prominent Distribution of the μ-Opioid Receptor at Extrasynaptic Sites on Noradrenergic Dendrites in the Rat Nucleus Locus Coeruleus

Physiological studies have indicated that agonists at the mu-opioid receptor (mu OR), such as morphine or the endogenous peptide methionine5-enkephalin, can markedly decrease the spontaneous activity of noradrenergic neurons in the locus coeruleus (LC). Messenger RNA and protein for mu OR are also densely expressed by LC neurons. During opiate withdrawal, increased discharge rates of LC neurons coincide with the expression of behavioral features associated with the opiate withdrawal syndrome. To better define the cellular sites for the physiological activation of mu OR in the LC and its relation to afferent terminals, we examined the ultrastructural localization of mu OR immunoreactivity in sections dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Immunogold-silver labeling for mu OR (i-mu OR) was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites, many of which also contained immunolabeling for TH. The dendrites containing exclusively i-mu OR were more numerous in the rostral pole of the LC. The i-mu OR in dendrites with and without detectable TH immunoreactivity were usually postsynaptic to unlabeled axon terminals containing heterogeneous types of synaptic vesicles and forming asymmetric synaptic specializations characteristic of excitatory-type synapses. These results provide the first direct ultrastructural evidence that mu OR is strategically localized to modulate the postsynaptic excitatory responses of catecholamine-containing neurons in the LC.

Influence of corticotropin-releasing hormone on electrophysiological activity of locus coeruleus neurons

These experiments examined the effects of corticotropin-releasing hormone (CRH) on single-unit electrophysiological activity of locus coeruleus (LC) neurons. As has been reported previously, infusion of CRH into the ventricular system of the brain (i.c.v.) of halothane-anesthetized adult male rats increased spontaneous discharge rate of LC neurons while producing no increase, and possibly a decrease, in sensory-evoked activity. However, when i.c.v. CRH was given to female rats or immature male rats, which had not been studied previously, LC activity was not altered. To attempt to understand this sex and age difference, potential mechanisms by which i.c.v. CRH elevates LC spontaneous activity in adult male rats were examined; in that i.c.v. CRH activates the pituitary-adrenal axis and autonomic nervous system, these response systems were manipulated. Adrenalectomy (with or without corticosterone replacement by pellet) did not affect the ability of i.c.v. CRH to increase LC spontaneous activity in adult male animals, but blockade of sympathetically-mediated autonomic responses, either by chlorisondamine or the beta adrenergic receptor blocker timolol, blocked this increase, indicating that afferent feedback from peripheral autonomic responses was critical for activating LC neurons following i.c.v. CRH. To determine whether CRH neurotransmission might play a role in this feedback pathway, the CRH antagonist alpha-helical CRH (α-hCRH) was microinjected into several brain regions including LC prior to i.c.v. CRH. α-hCRH microinjected into LC reduced the increase in LC activity caused by i.c.v. CRH; however, blockade of this increase was total when α-hCRH was microinjected into the lateral parabrachial nucleus ipsilateral to the LC recording site, suggesting that increased LC activity following i.c.v. CRH is mediated by CRH acting in the parabrachial region. During these studies, it was also observed that microinjection of α-hCRH into LC increased LC spontaneous discharge rate; consequently, CRH was microinjected into LC, and produced a dose-dependent decrease in LC spontaneous activity in both male and female rats, which could be blocked by microinjection of α-hCRH — these data indicated that the direct influence of CRH on LC neurons is to decrease their spontaneous activity. To reconcile this with the original report that CRH applied to LC neurons increases their activity, one possibility suggested is that the CRH microinjection procedure used in the present study stimulated inhibitory receptors on LC dendrites whereas the original study stimulated excitatory receptors on LC cell bodies. It is concluded that an inhibitory influence of CRH on LC activity is consistent with recent data indicating that decreased LC activity increases anxiety and stress-related responses, but that direct influences of CRH appear rather minor in determining LC neuronal activity in comparison to other inputs to LC such as are seen after i.c.v. CRH infusion.

Sensoryr responsiveness of brain noradrenergic neurons in modulated by endogenous brain serotonin

Previous results have indicated that application of serotonin (5-HT) onto noradrenergic locus coeruleus (LC) neurons selectively attenuates the response of these cells to excitatory amino acids (EAAs). Other studies revealed that certain sensory responses of LC neurons are mediated by EAA inputs. We examined the role of endogenous 5-HT in modulating sensory responses of LC neurons that are EAA-mediated. LC neurons recorded in rats pretreated with the serotonin (5-HT) depletion, p-chlorophenylalanine (PCPA), exhibited increased responsiveness to electrical stimulation of a rear footpad. Conversely, injection of the 5-HT precursor, 5-hdyroxytryptophan (5-HTP), reversed this effect of PCPA and attenuated this sensory response of LC neurons in drug-naive animals. Neither treatment altered the spontaneous discharge rate of LC neurons. These results are consistent with previous findings indicating that 5-HT has potent but selective effects on EAA-mediated responses of LC neurons, and in addition point to a possible functional role for endogenous 5-HT in controlling sensory-evoked LC activity.

Tonic activation of locus coeruleus neurons by systemic or intracoerulear microinjection of an irreversible acetylcholinesterase inhibitor: Increased discharge rate and induction of c-fos

Recent studies in this laboratory have demonstrated that intramuscular injection of the irreversible acetylcholinesterase (AChE) inhibitor, soman (pinacolylmethylphosphonofluoridate), produces a rapid (1–2 h) and profound depletion (70% of control) of norepinephrine (NE) in the olfactory bulb and forebrain. NE is decreased only in convulsing animals. As NE-containing locus coeruleus (LC) neurons provide the only NE input to the olfactory bulb and the major NE innervation of the forebrain, the reduction of NE suggests that soman may cause tonic activation of LC neurons leading to rapid depletion of NE. Activation of LC may result from: (i) facilitation of cholinergic transmission in LC; (ii) soman-induced activation of excitatory inputs to LC; or (iii) generalized activation of LC neurons due to seizures. The present experiments were designed to assess these alternatives. We examined whether LC neuronal activity, c-fos expression, and AChE staining are altered after peripheral (systemic) or direct intracoerulear injection of soman in anesthetized rats. Both modes of soman administration rapidly and potently increase the spontaneous discharge rate of LC neurons. This activation was associated with a desynchronization of the electroencephalogram, but not with seizures. The discharge of LC neurons remained elevated at all postsoman intervals examined (up to 2 h) and was rapidly and completely reversed by systemic injection of the muscarinic receptor antagonist scopolamine hydrochloride, but not by the nicotinic receptor antagonist mecamylamine. Both systemic and intracoerulear soman administration completely inhibited AChE staining in LC and rapidly induced the expression of c-fos in LC neurons. These results demonstrate that soman potently and tonically activates LC neurons. This effect appears to be mediated by direct inhibition of AChE in LC leading to a rapid accumulation of ACh. Unhydrolyzed ACh tonically activates LC neurons via muscarinic receptors. Soman-induced activation of LC neurons does not require seizures. We conclude that depletion of forebrain and olfactory bulb NE after systemic administration of soman results from tonic hypercholinergic stimulation of LC.

The activating action of vincamine, piracetam, and hydergine on the activity of the noradrenergic neurons of the locus coeruleus

The effect of vincamine, hydergine, and piracetam on the discharge rate of noradrenergic neurons of the locus coeruleus (LC) was studied using conventional single-cell recording techniques. Experiments were performed on 103 male rats (RAI f (SPF)) anaesthetized with chloral hydrate. Intraperitoneally applied vincamine and hydergine affected LC-neuronal firing already at a dose of 0.1 mg/kg. Both compounds increased the mean discharge rate of LC neurons by about 70% at a dose of 1 mg/kg. Piracetam elicited a 30–40% increase in firing at doses of 300 and 1000 mg/kg ip, respectively. It is conceivable that the activating action of vincamine, piracetam, and hydergine on noradrenergic neurons of LC is linked with the therapeutic action of the drugs on cognitive function or mood.