Responses Of Locus Coeruleus Neurons Research Articles

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

Reduced Sensory-Evoked Locus Coeruleus-Norepinephrine Neural Activity in Female Rats With a History of Dietary-Induced Binge Eating.

Noradrenergic pathways have been implicated in eating pathologies. These experiments sought to examine how dietary-induced binge eating influences the neuronal activity of the locus coeruleus (LC)-norepinephrine (NE) system. Young adult female Sprague Dawley rats (7–8 weeks old) were exposed to a repeated intermittent (twice weekly) cycle of 30-min access to a highly palatable sweetened fat (i.e., vegetable shortening with 10% sucrose) with or without intermittent (24 h) calorie restriction (Restrict Binge or Binge groups, respectively). Age- and weight-matched female control rats were exposed to standard chow feeding (Naive group) or intermittent chow feeding (Restrict group). The Binge and Restrict Binge groups demonstrated an escalation in sweet-fat food intake after 2.5 weeks. On week 3, in vivo single-unit LC electrophysiological activity was recorded under isoflurane anesthesia. Restrict Binge (20 cells from six rats) and Binge (27 cells from six rats) had significantly reduced (approximate 20% and 26%, respectively) evoked LC discharge rates compared with naive rats (22 cells, seven rats). Spontaneous and tonic discharge rates were not different among the groups. Signal-to-noise ratio was reduced in the groups with intermittent sweetened fat exposure. In order to investigate the neuropeptide alterations as a consequence of dietary binge eating, relative gene expression of neuropeptide Y (NPY), glucagon-like peptide 1 receptor (GLP-1r), prodynorphin, and related genes were measured in LC and hypothalamic arcuate (Arc) regions. Glp-1r, Npy2r, and Pdyn in LC region were reduced with repeated intermittent restriction. Npy1r was reduced by approximately 27% in ARC of Restrict compared with Naive group. Such data indicate that dietary-induced binge eating alters the neural response of LC neurons to sensory stimuli and dampens the neural stress response.

Persistent Stress-Induced Neuroplastic Changes in the Locus Coeruleus/Norepinephrine System.

Neural plasticity plays a critical role in mediating short- and long-term brain responses to environmental stimuli. A major effector of plasticity throughout many regions of the brain is stress. Activation of the locus coeruleus (LC) is a critical step in mediating the neuroendocrine and behavioral limbs of the stress response. During stressor exposure, activation of the hypothalamic-pituitary-adrenal axis promotes release of corticotropin-releasing factor in LC, where its signaling promotes a number of physiological and cellular changes. While the acute effects of stress on LC physiology have been described, its long-term effects are less clear. This review will describe how stress changes LC neuronal physiology, function, and morphology from a genetic, cellular, and neuronal circuitry/transmission perspective. Specifically, we describe morphological changes of LC neurons in response to stressful stimuli and signal transduction pathways underlying them. Also, we will review changes in excitatory glutamatergic synaptic transmission in LC neurons and possible stress-induced modifications of AMPA receptors. This review will also address stress-related behavioral adaptations and specific noradrenergic receptors responsible for them. Finally, we summarize the results of several human studies which suggest a link between stress, altered LC function, and pathogenesis of posttraumatic stress disorder.

The Role of Ca2+ and BK Channels of Locus Coeruleus (LC) Neurons as a Brake to the CO2 Chemosensitivity Response of Rats

The cellular mechanisms by which LC neurons respond to hypercapnia are usually attributed to an “accelerator” whereby hypercapnic acidosis causes an inhibition of K+ channels or activation of Na+ and Ca+2 channels to depolarize CO2-sensitive neurons. Nevertheless, it is still unknown if this “accelerator” mechanism could be controlled by a brake phenomenon. Whole-cell patch clamping, fluorescence imaging microscopy and plethysmography were used to study the chemosensitive response of the LC neurons. Hypercapnic acidosis activates L-type Ca2+ channels and large conductance Ca-activated K+ (BK) channels, which function as a “brake” on the chemosensitive response of LC neurons. Our findings indicate that both Ca2+ and BK currents develop over the first 2 weeks of postnatal life in rat LC slices and that this brake pathway may cause the developmental decrease in the chemosensitive firing rate response of LC neurons to hypercapnic acidosis. Inhibition of this brake by paxilline (BK channel inhibitor) returns the magnitude of the chemosensitive firing rate response from LC neurons in rats older than P10 to high values similar to those in LC neurons from younger rats. Inhibition of BK channels in LC neurons by bilateral injections of paxilline into the LC results in a significant increase in the hypercapnic ventilatory response of adult rats. Our findings indicate that a BK channel-based braking system helps to determine the chemosensitive respiratory drive of LC neurons and contributes to the hypercapnic ventilatory response. Perhaps, abnormalities of this braking system could result in hypercapnia-induced respiratory disorders and panic responses.

Paradoxical lower sensitivity of Locus Coeruleus than Substantia Nigra pars compacta neurons to acute actions of rotenone

Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01–5.0μM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1μM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.

The role of orexin type-1 receptors in the development of morphine tolerance in locus coeruleus neurons: An electrophysiological perspective

Long-term exposure to opioid agonists results in tolerance to their analgesic effects, so the effectiveness of opioid agonists in the management of pain becomes limited. The locus coeruleus (LC) nucleus has been involved in the development of tolerance to opiates. Orexin type-1 receptors (OX1Rs) are highly expressed in LC nucleus. Orexin plays a noteworthy role in the occurrence of morphine tolerance. The purpose of the present study is to investigate the role of orexin type-1 receptors in the development of morphine tolerance in LC neurons.In this study, adult male Wistar rats weighing 250–300g were utilized. Induction of morphine tolerance was obtained by single injection of morphine per day for 6 successive days. An orexin type-1 receptor antagonist (SB-334867) was injected into the lateral ventricle instantly prior to morphine injection. On day 7, the effect of morphine on the electrical activity of LC neurons was studied using in vivo extracellular single unit recording.The results demonstrate that morphine injection for 6 consecutive days led to the development of morphine-induced tolerance in LC neurons. In other words, there was a significant decrease in LC neuronal responsiveness to morphine injection. Inhibitory responses of LC neurons to intraperitoneally applied morphine can be observed with the treatment of the SB-334867 prior to morphine injection.This study showed that OX1R blockade by SB-334867 prevents the development of morphine tolerance in LC neurons. We hope that further studies will lead to considerable progress in understanding the molecular adaptations that contribute to morphine tolerance.

Temperature influences neuronal activity and CO2/pH sensitivity of locus coeruleus neurons in the bullfrog,Lithobates catesbeianus

The locus coeruleus (LC) is a chemoreceptive brain stem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole cell patch-clamp electrophysiology to measure firing rate, membrane potential (V(m)), and input resistance (R(in)) in LC neurons in brain stem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in nonchemosensitive LC neurons or neurons in a brain stem slice 500 μm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.

Analgesic antidepressants promote the responsiveness of locus coeruleus neurons to noxious stimulation: Implications for neuropathic pain

Antidepressants that block the reuptake of noradrenaline and/or serotonin are among the first-line treatments for neuropathic pain, although the mechanisms underlying this analgesia remain unclear. The noradrenergic locus coeruleus is an essential element of both the ascending and descending pain modulator systems regulated by these antidepressants. Hence, we investigated the effect of analgesic antidepressants on locus coeruleus activity in Sprague-Dawley rats subjected to chronic constriction injury (CCI), a model of neuropathic pain. In vivo extracellular recordings of locus coeruleus revealed that CCI did not modify the basal tonic activity of this nucleus, although its sensory-evoked response to noxious stimuli was significantly altered. Under normal conditions, noxious stimulation evokes an early response, corresponding to the activation of myelinated A fibers, which is followed by an inhibitory period and a subsequent late capsaicin-sensitive response, consistent with the activation of unmyelinated C fibers. CCI provokes an enhanced excitatory early response in the animals and the loss of the late response. Antidepressant administration over 7days (desipramine, 10mg/kg/day or duloxetine, 5mg/kg/day, delivered by osmotic minipumps) decreased the excitatory firing rate of the early response in the CCI group. Moreover, in all animals, these antidepressants reduced the inhibitory period and augmented the late response. We propose that N-methyl-d-aspartate and alpha-2-adrenoceptors are involved in the analgesic effect of antidepressants. Antidepressant-mediated changes were correlated with behavioral effects indicative of analgesia in healthy and neuropathic rats.

Effects of central orexin 1 receptor blockade on locus coeruleus neurons with lateral hypothalamic input

The locus coeruleus (LC) receives a major orexinergic input and is densely packed with orexin 1 receptors (OX1‐R). Here we examined the effects of central administration of an OX1‐R antagonist on lateral hypothalamic (LH) stimulation‐induced activation of LC neurons. All experiments were conducted using male Sprague Dawley rats under initial isoflurane (1.7–1.9%) anaesthesia followed by urethane (1.3–1.4 g/kg, i.v.). Electrical stimulation of the LH (0.5 Hz, twin 0.5 ms pulses, 3 ms interpulse interval, 300uA) evoked excitatory responses in all LC neurons studied (n = 24) as judged by construction of peri‐stimulus time histograms. LC neurons responded to LH stimulation with an onset latency of 6.5±0.4 ms and a peak latency of 15.3±0.8 ms. Intracerebroventricular administration of the OX1‐R antagonist SB334867A (10 nmol) but not vehicle inhibited LH stimulation‐induced activation of LC neurons by 58±14% (n = 6 neurons). Abolition of LH stimulation‐induced excitatory responses in LC neurons occasionally (n=3/6) revealed constant latency antidromic responses verified by positive collision tests. These findings indicate that the LC receives a robust excitatory input which is mediated, at least in part, by orexin acting at OX1‐Rs. Furthermore, some LC neurons that receive an orexinergic input also project to the LH region. Supported by the Austin Hospital Medical Research Foundation.

The role of Ca2+ and BK channels in the firing rate response of locus coeruleus (LC) neurons to CO2: controlling the chemosensitive gain

Chemosensitive LC neurons increase their firing rate in response to CO2 via inhibition of K+ channels. This increase gets smaller during neonatal development (from P3‐P16). Changes of this K+ channel‐based “accelerating” pathway may account for the developmental changes. We used whole cell patch clamp and fluorescence imaging microscopy to study a different basis for these developmental changes in chemosensitive gain, a “braking” pathway. We found that an L‐type Ca2+ current increased in LC neurons during development. This current was activated by CO2, resulting in increases in [Ca2+]i and the activation of hyperpolarizing Ca2+‐activated K+ currents (KCa), which should reduce the firing rate response of LC neurons to CO2. We used the BK (a large conductance KCa) channel inhibitor paxilline and voltage clamp to study this KCa current. Inhibition of the BK current removed the “brake” and increased the firing rate response to CO2 in LC neurons. Notably, the “braking” pathway increased during neonatal development in a fashion that paralleled the decrease in the chemosensitive firing rate response of LC neurons to CO2. These findings suggest a unique role for Ca2+ in controlling central chemoreceptor gain. Abnormalities in this pathway could be associated with disorders involving elevated central gain, such as sleep apnea and panic disorders.[NIH R01 HL56683, AHA Great Rivers Affiliate Predoctoral Fellowship]

Examining the effect of the CaMKII inhibitor administration in the locus coeruleus on the naloxone-precipitated morphine withdrawal signs in rats

Drug addiction is an occurrence with physiological, psychological, and social outcomes. Repeated drug exposure causes neuronal adaptations and dependency. It has been shown that CaMKIIα enzyme contributes to morphine dependency. The locus coeruleus nucleus has been implied in the morphine withdrawal syndrome. This research focuses on the behavioral and molecular adaptations that occur in the locus coeruleus neurons in response to the chronic morphine exposure. Adult male Wistar rats were injected by morphine sulfate (10 mg/kg/s.c.) at an interval of 12 h for a period of nine subsequent days. On the tenth day, naloxone (1 mg/kg/i.p.) was injected 2 h after the morphine administration. Somatic withdrawal signs were investigated for 30 min. We concluded that the inhibition of CaMKIIα by administration of KN-93, the specific inhibitor of this enzyme, significantly attenuated some of the withdrawal signs. In molecular method, the expression of CaMKIIα protein has been enhanced in locus coeruleus of the morphine dependent rats. These findings indicate that CaMKIIα may be involved in the modulation of the naloxone-induced withdrawal syndrome, and treatment with KN-93 may have some effects on this system.

Optogenetic Analysis of Area‐Specific Glial‐Neuronal Signalling

We studied glia‐neuronal signalling in two brainstem areas, the retrotrapezoid nucleus (RTN) and the locus coeruleus (LC). RTN and LC neurons were visualized using adenoviral expression of DsRed2 under control of PRSx8 promoter. For optogenetic selective stimulation of astrocytes we have generated an adenoviral vector AVV‐sGFAP‐ChR2(H134R)‐Katushka1.3. We first patched AVV‐sGFAP‐ChR2(H134R)‐Katushka1.3‐transduced astrocytes and found that flashing 470 nm light induced precisely timed depolarisations (+18.8 ± 2.0 mV, n=7, p<0.001). We then patched RTN and LC neurons and stimulated adjacent astrocytes using light. This evoked long lasting depolarization of both RTN (+3.57 ± 0.5 mV, n=9, p<0.001) and LC neurons (+ 3.08 ± 0.33 mV, n=14, p<0.001). Responses of the RTN neurons were markedly reduced by the ATP receptor antagonist MRS2179 (10 μm), depolarisation +0.7mV, n=7, p<0.001). In contrast, neither MRS 2179 nor TNP‐ATP (10 μm) had any effect on of the responses in LC neurons (n=6, p=0.71 for MRS2179 and n=9, p=0.86 for TNP‐ATP). At the same time depolarisations in LC neurons were prevented by an NMDA receptor antagonist dAPV (50 μm, n=8, p<0.01) and AMPA receptor antagonist CNQX (10 μm, n=5, p<0.001). Therefore, glia‐neuronal information transfer occurs in area‐ and transmitter‐dependent manner.

Immunohistochemical identification of K+ channel subunits in neurons from chemosensitive regions of rat brainstem

CO2‐sensitive neurons control breathing and are found in various regions of the brainstem. Neurons within these areas respond to CO2 with different percentages and different magnitudes of changes in firing rate responses to CO2. We propose that neuronal responses to CO2 are different because of differences in the ion channels expressed on neurons in these regions. The current study localized pH‐sensitive K+ channels in two different areas of the brainstem, the solitary tract (SC) (nucleus tractus solitarius, NTS, and dorsal motor nucleus, DMN) and locus coeruleus (LC). Brainstem slices (300µm) were fixed and thin sectioned (20‐40µm). Nissl staining was used to stain all cells and glial fibrillary acidic protein (GFAP)stained astrocytes. Neurons were identified as Nissl positive/GFAP negative. Sections were stained with antibodies for pH‐sensitive subunits of voltage‐sensitive (Kv1.4) and inwardly‐rectifying (Kir2.3 and Kir4.1) K+ channels. We found that Kv1.4 was prevalent on SC neurons (60‐90%), which stained less for Kir2.3 and 4.1 (30‐40%). In contrast, LC neurons stained similarly (50‐60%) for Kv1.4, Kir2.3 and Kir4.1. These data are consistent with an A current (inhibitable by 4 aminopyridine) playing a major role in the CO2 response of SC neurons but both an A current and Kir channels playing a role in the CO2 response of LC neurons.Supported by NIH R01 HL56683 (RWP) and T35 HL007805 (CM).

Electrophysiologic effects of systemic and locally infused epibatidine on locus coeruleus neurons

We evaluated the electrophysiologic response of locus coeruleus neurons to the systemic and local infusion of epibatidine. Rats were anesthetized with 2% halothane and single-unit locus coeruleus discharge was recorded after administration of systemic (2.5, 5 and 10 μg/kg subcutaneously) and intracoerulear (0.03–0.01–0.001 μg) epibatidine. The subcutaneous epibatidine activated locus coeruleus neurons only at the highest dose (10 μg/kg). The 2.5–5 μg/kg doses, previously shown to induce analgesia, did not activate locus coeruleus neurons. The intracoerulear infusion of epibatidine induced excitement of locus coeruleus neurons at every tested dose. Higher doses (0.03 and 0.01 μg) excited 100% of the recorded neurons. A significantly lower number of neurons (50% and 43% respectively) were excited when lower doses (0.005–0.001 μg) were used ( P = 0.035). The intracoerulear infusion of mecamylamine (1 μg) significantly reduced neuronal discharge rate (45%) and blocked the effects of epibatidine. The intra-dorsal raphe infusion of 0.03 μg epibatidine induced significant excitation of locus coeruleus neurons. These data show that the administration of epibatidine induces excitation of locus coeruleus neurons, which is mediated by nicotinic receptors. This activation occurs after systemic and selective local administration of epibatidine. The response of locus coeruleus neurons to systemic and locally administered epibatidine is dose-related.

Alterations of BDNF and NT-3 genes expression in the nucleus paragigantocellularis during morphine dependency and withdrawal

Locus coeruleus (LC) plays a key role in opioid dependence and withdrawal. Chronic morphine administration induces neurochemical adaptations in the noradrenergic system. The nature of signal responsible for opiate-induced adaptations of noradrenergic neurons in LC is not well defined. Neurotrophins-signaling pathways such as brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3) play a key role for regulating the noradrenergic response of LC neurons to opiates. The nucleus paragigantocellularis (PGi) is one of the two major afferents to LC. The present study was designed to evaluate the expression of BDNF and NT-3 in the context of opiate dependence and withdrawal in PGi. Such data are important because they could reveal the role of PGi as an additional source of BDNF and NT-3 in the neurochemical plasticity of LC neurons. Opiate dependence was induced by a progressive intraperitoneal treatment of morphine. In morphine dependent group PGi nucleus was extracted for gene expression assay 6h after the last injection of morphine. In spontaneous withdrawal, rats received the same chronic treatment as morphine group. PGi was extracted for gene expression assay 24, 48 and 72 h after the last injection of morphine. PGi nucleus was assayed for the expression of BDNF and NT-3 using semi-quantitative RT-PCR normalized to beta-actin gene expression. Results showed that chronic administration of morphine significantly increased BDNF and NT-3 gene expression in PGi. In spontaneous withdrawal, BDNF/NT-3 genes expression were high in comparison to control group. It seems that BDNF/NT-3 -signaling pathway originating from PGi is essential for opiate-induced adaptations of the LC neurons.

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.

The subdiaphragmatic vagus nerves mediate activation of locus coeruleus neurons by peripherally administered microbial substances

Our earlier studies demonstrated that representative microbial substances—lipopolysaccharide, peptidoglycan, and poly-inosine: poly-cytosine (poly(I):(C))—increased the spontaneous discharge rates and sensory-evoked responses of isolated locus coeruleus (LC) neurons in a dose- and time-related manner after i.p. injection into rats. We then turned our attention to the mechanism by which microbial substances administered into the peritoneal cavity affect the LC neurons. The involvement of the subdiaphragmatic vagus nerves was examined in this regard since several brain responses to peripherally administered lipopolysaccharide have been found to depend upon the integrity of these nerves. The experiments reported here show that lipopolysaccharide, peptidoglycan, and poly(I):(C) all failed to excite LC neurons after i.p. injection into rats that had previously been subjected to complete transection of the subdiaphragmatic vagus nerves. Furthermore, selective transection of the subdiaphragmatic vagus nerve trunks indicated that the dorsal trunk, and not the ventral trunk, was necessary to excite LC neurons in response to i.p. lipopolysaccharide. The inability of LC neurons to respond to i.p. lipopolysaccharide in vagotomized rats is unlikely to be attributed to a desensitization of the neurons to lipopolysaccharide since i.c.v. injection of lipopolysaccharide excited LC neurons in vagotomized rats as it did in vagus-intact rats. These findings suggest that a variety of microbial substances excited LC neurons after administration into the peritoneal cavity in a manner involving the subdiaphragmatic vagus nerves.

The effects of endogenous interleukin-1 bioactivity on locus coeruleus neurons in response to bacterial and viral substances

In a previous study, we found that microinjection of the cytokine interleukin-1 (IL-1) into the locus coeruleus (LC) increased the electrophysiological activity of LC neurons. To determine if endogenous IL-1 similarly affects the LC, brain IL-1 was induced with lipopolysaccharide (LPS), a substance derived from Gram-negative bacteria. LPS microinjected directly into the LC increased the activity of LC neurons in anesthetized rats, and this effect was blocked by microinfusion of the IL-1 receptor antagonist (IL-1RA) protein into the LC indicating the involvement of IL-1 receptors. Similarly, intraperitoneal (i.p.) LPS injection increased the activity of LC neurons in a dose- and time-related manner that was sensitive to IL-1RA. The change in the activity of LC neurons caused by a single i.p. injection of LPS was surprisingly long-lasting, and evolved over a period of at least 3 weeks. Other microbial substances—namely, peptidoglycan from Gram-positive bacteria and poly-inosine/poly-cytosine (poly(I)/(C)), which resembles RNA viruses—were used to determine the generality of the findings with LPS. Both i.p. peptidoglycan and poly(I)/(C) increased LC activity but with lesser efficacy than LPS. IL-1RA reversed the increase in the activity of LC neurons caused by i.p. peptidoglycan treatment; however, that caused by i.p. Poly(I)/(C) was not diminished by IL-1RA. Thus, the increased activity of LC neurons caused by LPS and peptidoglycan requires IL-1 receptor binding, suggesting the involvement of endogenously-produced IL-1. In contrast, poly(I)/(C) increased the activity of LC neurons but this did not critically involve IL-1 receptors in the LC.

Fos-related Antigens are involved in the Transcriptional Responses of Locus Coeruleus Neurons to Altered Gravitational Fields in Rats

Locus coeruleus (LC) neurons, which have widespread projections to the whole brain, respond to natural stimulation of macular receptors. Using immunocytochemistry we investigated whether rats exposed to altered gravitational fields showed changes in Fos and Fos-related antigen (FRA) protein levels in the LC. Fos protein is induced very rapidly and returns to basal levels within hours after stimulation, while FRAs persist for days or weeks after induction. Adult male albino rats (Fisher 344) were sacrificed at different time points during a space flight (NASA Neurolab Mission, STS-90) and the numbers of Fos- and FRA-positive cells in the LC were counted and compared to those in ground-based control rats. No significant changes in Fos protein expression were detected in the LC under different space flight conditions. In contrast, the number of FRA-positive cells increased on average to 167% of that of the controls at FD2, i.e. when gravity increased from 1 to 3 g during the launch before reaching about 0 g . FRA-labeled neurons then decreased to 46% of control values at FD14, i.e. after adaptation to 0 g , but increased again to 317% of control values at R + 1, when the animals were exposed to an increase in gravitational force from 0 to 1.5-1.8 g before reaching 1 g during landing. The number of labeled cells was 193% of the control values at R + 13, i.e. after readaptation to 1 g . Thus gravitational force appears to be very effective in inducing a long-term increase in FRA protein expression in the LC. Because activity in the noradrenergic LC neurons may increase Fos expression in several target structures, we postulate that the long-lasting induction of FRAs in the LC at FD2, and more prominently at R + 1, may contribute to the long-term molecular changes which probably occur in the brain during adaptation to 0 g and readaptation to 1 g .