Modulation of Noradrenergic and Serotonergic Systems by Cannabinoids: Electrophysiological, Neurochemical and Behavioral Evidence.

The main noradrenergic and serotonergic nuclei in the central nervous system (CNS) are the locus coeruleus (LC) and the dorsal raphe nucleus (DRN). These brain areas, located in the brainstem, play a pivotal role in the control of various functions and behaviors that are altered by cannabinoids (i.e., pain, arousal, mood, anxiety, or sleep-wake cycle). Anatomical, neurochemical, and functional data suggest that cannabinoids regulate both central noradrenergic and serotonergic neurotransmission. Thus, strong evidence has shown that the firing activity of LC and DRN monoamine neurons or the synthesis/release of noradrenaline (NA) and serotonin (5-HT) in the projection areas are all affected by cannabinoid administration. Herein, we propose that interaction between the endocannabinoid system and the noradrenergic-serotonergic systems could account for some of the anxiolytic, antidepressant, and antinociceptive effects of cannabinoids or the disruption of attention/sleep induced by these drugs.

Inhibition of rat locus coeruleus neurons by prostaglandin E2 EP3 receptors: pharmacological characterization ex vivo.

Prostaglandin E2 (PGE2) is an inflammatory mediator synthesized by the brain constitutive cyclooxygenase enzyme. PGE2 binds to G protein-coupled EP1-4 receptors (EP1 to Gq, EP2,4 to Gs, and EP3 to Gi/o). EP2, EP3 and EP4 receptors are expressed in the locus coeruleus (LC), the main noradrenergic nucleus in the brain. EP3 receptors have been explored in the central nervous system, although its role regulating the locus coeruleus neuron activity has not been pharmacologically defined. Our aim was to characterize the function of EP3 receptors in neurons of the LC. Thus, we studied the effect of EP3 receptor agonists on the firing activity of LC cells in rat brain slices by single-unit extracellular electrophysiological techniques. The EP3 receptor agonist sulprostone (0.15 nM-1.28 µM), PGE2 (0.31 nM-10.2 µM) and the PGE1 analogue misoprostol (0.31 nM-2.56 µM) inhibited the firing rate of LC neurons in a concentration-dependent manner (EC50 = 15 nM, 110 nM, and 51 nM, respectively). The EP3 receptor antagonist L-798,106 (3-10 µM), but not the EP2 (PF-04418948, 3-10 µM) or EP4 (L-161,982, 3-10 µM) receptor antagonists, caused rightward shifts in the concentration-effect curves for the EP3 receptor agonists. Sulprostone-induced effect was attenuated by the Gi/o protein blocker pertussis toxin (pertussis toxin, 500 ng ml-1) and the inhibitors of inwardly rectifying potassium channels (GIRK) BaCl2 (300 µM) and SCH-23390 (15 µM). In conclusion, LC neuron firing activity is regulated by EP3 receptors, presumably by an inhibitory Gi/o protein- and GIRK-mediated mechanism.

Cannabigerol modulates α2-adrenoceptor and 5-HT1A receptor-mediated electrophysiological effects on dorsal raphe nucleus and locus coeruleus neurons and anxiety behavior in rat.

The pharmacological profile of cannabigerol (CBG), which acid form constitutes the main precursor of the most abundant cannabinoids, has been scarcely studied. It has been reported to target α2-adrenoceptor and 5-HT1A receptor. The locus coeruleus (LC) and the dorsal raphe nucleus (DRN) are the main serotonergic (5-HT) and noradrenergic (NA) areas in the rat brain, respectively. We aimed to study the effect of CBG on the firing rate of LC NA cells and DRN 5-HT cells and on α2-adrenergic and 5-HT1A autoreceptors by electrophysiological techniques in male Sprague-Dawley rat brain slices. The effect of CBG on the novelty-suppressed feeding test (NSFT) and the elevated plus maze test (EPMT) and the involvement of the 5-HT1A receptor was also studied. CBG (30 µM, 10 min) slightly changed the firing rate of NA cells but failed to alter the inhibitory effect of NA (1-100 µM). However, in the presence of CBG the inhibitory effect of the selective α2-adrenoceptor agonist UK14304 (10 nM) was decreased. Perfusion with CBG (30 µM, 10 min) did not change the firing rate of DRN 5-HT cells or the inhibitory effect of 5-HT (100 µM, 1 min) but it reduced the inhibitory effect of ipsapirone (100 nM). CBG failed to reverse ipsapirone-induced inhibition whereas perfusion with the 5-HT1A receptor antagonist WAY100635 (30 nM) completely restored the firing rate of DRN 5-HT cells. In the EPMT, CBG (10 mg/kg, i.p.) significantly increased the percentage of time the rats spent on the open arms and the number of head-dipping but it reduced the anxiety index. In the NSFT, CBG decreased the time latency to eat in the novel environment but it did not alter home-cage consumption. The effect of CBG on the reduction of latency to feed was prevented by pretreatment with WAY100635 (1 mg/kg, i.p.). In conclusion, CBG hinders the inhibitory effect produced by selective α2-adrenoceptor and 5-HT1A receptor agonists on the firing rate of NA-LC and 5-HT-DRN neurons by a yet unknown indirect mechanism in rat brain slices and produces anxiolytic-like effects through 5-HT1A receptor.

Functional characterization of cannabidiol effect on the serotonergic neurons of the dorsal raphe nucleus in rat brain slices.

Cannabidiol (CBD), the main non-psychoactive cannabinoid found in the cannabis plant, elicits several pharmacological effects via the 5-HT1A receptor. The dorsal raphe nucleus (DRN) is the main serotonergic cluster in the brain that expresses the 5-HT1A receptor. To date, the effect of CBD on the neuronal activity of DRN 5-HT cells and its interaction with somatodendritic 5-HT1A autoreceptors have not been characterized. Our aim was to study the effect of CBD on the firing activity of DRN 5-HT cells and the 5-HT1A autoreceptor activation by electrophysiological and calcium imaging techniques in male Sprague-Dawley rat brain slices. Perfusion with CBD (30 µM, 10 min) did not significantly change the firing rate of DRN 5-HT cells or the inhibitory effect of 5-HT (50-100 µM, 1 min). However, in the presence of CBD (30 µM, 10 min), the inhibitory effects of 8-OH-DPAT (10 nM) and ipsapirone (100 nM) were reduced by 66% and 53%, respectively. CBD failed to reverse ipsapirone-induced inhibition, whereas perfusion with the 5-HT1A receptor antagonist WAY100635 (30 nM) completely restored by 97.05 ± 14.63% the firing activity of 5-HT cells. Administration of AM251 (1 µM), MDL100907 (30 nM), or picrotoxin (20 µM) did not change the blockade produced by CBD (30 µM) on ipsapirone-induced inhibition. Our study also shows that CBD failed to modify the KCl (15 mM, 4 min)-evoked increase in [Ca2+]i or the inhibitory effect of ipsapirone (1 µM, 4 min) on KCl-evoked [Ca2+]i. In conclusion, CBD does not activate 5-HT1A autoreceptors, but it hindered the inhibitory effect produced by selective 5-HT1A receptor agonists on the firing activity of DRN 5-HT cells through a mechanism that does not involve CB1, 5-HT2A, or GABAA receptors. Our data support a negative allosteric modulation of DRN somatodendritic 5-HT1A receptor by CBD.

Regulation of noradrenergic and serotonergic systems by cannabinoids: relevance to cannabinoid-induced effects.

The cannabinoid system is composed of Gi/o protein-coupled cannabinoid type 1 receptor (CB1) and cannabinoid type 2 (CB2) receptor and endogenous compounds. The CB1 receptor is widely distributed in the central nervous system (CNS) and it is involved in the regulation of common physiological functions. At the neuronal level, the CB1 receptor is mainly placed at GABAergic and glutamatergic axon terminals, where it modulates excitatory and inhibitory synapses. To date, the involvement of CB2 receptor in the regulation of neurotransmission in the CNS has not been clearly shown. The majority of noradrenergic (NA) cells in mammalian tissues are located in the locus coeruleus (LC) while serotonergic (5-HT) cells are mainly distributed in the raphe nuclei including the dorsal raphe nucleus (DRN). In the CNS, NA and 5-HT systems play a crucial role in the control of pain, mood, arousal, sleep-wake cycle, learning/memory, anxiety, and rewarding behaviour. This review summarizes the electrophysiological, neurochemical and behavioural evidences for modulation of the NA/5-HT systems by cannabinoids and the CB1 receptor. Cannabinoids regulate the neuronal activity of NA and 5-HT cells and the release of NA and 5-HT by direct and indirect mechanisms. The interaction between cannabinoid and NA/5-HT systems may underlie several behavioural changes induced by cannabis such as anxiolytic and antidepressant effects or side effects (e.g. disruption of attention). Further research is needed to better understand different aspects of NA and 5-HT systems regulation by cannabinoids, which would be relevant for their use in therapeutics.

CB(1) cannabinoid receptors inhibit the glutamatergic component of KCl-evoked excitation of locus coeruleus neurons in rat brain slices.

CB(1) cannabinoid receptors located at presynaptic sites suppress synaptic transmission in the rat brain. The aim of this work was to examine by single-unit extracellular techniques the effect of the synthetic cannabinoid receptor agonist WIN 55212-2 on KCl-evoked excitation of locus coeruleus neurons in rat brain slices. Short applications of KCl (30 mM) increased by 9-fold the firing rate of locus coeruleus cells. Perfusion with the GABA(A) receptor antagonist picrotoxin (100 microM) increased KCl-evoked effect, whereas NMDA and non-NMDA glutamate receptor antagonists (D-AP5 100 microM and CNQX 30 microM, respectively) were able to decrease KCl-evoked effect only in the presence of picrotoxin (100 microM). Bath application of WIN 55212-2 (10 microM) inhibited KCl-evoked effect; this inhibition was blocked by the CB(1) receptor antagonist AM 251 (1 microM). However, a lower concentration of WIN 55212-2 (1 microM) did not significantly change KCl effect. In the presence of picrotoxin (100 microM), perfusion with D-AP5 (100 microM) or CNQX (30 microM) blocked WIN 55212-2-induced inhibition, although picrotoxin (100 microM) itself failed to affect cannabinoid effect. In conclusion, GABAergic and glutamatergic components are both involved in KCl-evoked excitation of LC neurons, although CB(1) receptors only seem to inhibit the glutamatergic component of KCl effect in the locus coeruleus.

Excitatory regulation of noradrenergic neurons by L-arginine/nitric oxide pathway in the rat locus coeruleus in vivo.

To elucidate conflicting findings about the role of L-arginine/nitric oxide (NO) pathway in the locus coeruleus (LC), we investigated the effects of different drugs affecting NO concentrations by single-unit extracellular recordings from LC neurons in vivo and in vitro. In anesthetized rats, central (3.8-15.3 nmol i.c.v.) and local (16.5-66 pmol into the LC) administrations of the NO donor sodium nitroprusside, but not those of the inactive analogue potassium ferricyanide (16.5-66 pmol into the LC), increased by 65-84% the firing rate of LC neurons. In brain slices, low concentrations (50-200 microM) of diethylamine/NO complex, a short-lived NO releaser, also increased the neuron firing rate, although higher drug concentrations (400-800 microM) caused slowly reversible reductions of the firing activity. On the other hand, the NO synthase inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) (148-371 nmol i.c.v.) and N(omega)-nitro-L-arginine (L-NA) (46 nmol i.c.v.) gradually decreased the firing rate of LC neurons, whereas the NO synthase substrate L-arginine (0.71-1.42 micromol i.c.v. and 0.6-4.8 nmol into the LC) increased the neuron activity. The latter effect was not mimicked by the vehicle or the less active isomer D-arginine (0.6-4.8 nmol into the LC). Unexpectedly, pretreatment with high concentrations of L-NAME (371 nmol and 18.5 micromol i.c.v.) or L-NA (45.6 nmol i.c.v. and 0.24 nmol into the LC) failed to block the effect of L-arginine. The glutamate receptor antagonist kynurenic acid (1 micromol i.c.v.) strongly reduced the effect of L-arginine but not that of sodium nitroprusside. These data confirm in vivo a direct excitatory effect of NO on LC neurons and suggest a tonic regulation of noradrenergic neurons by NO in vivo. L-arginine also excites LC neurons, but this effect may be caused by a nitric-oxide-unrelated glutamate-receptor-mediated mechanism.

Characterization of functional µ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study.

BACKGROUND AND PURPOSE: Regulation of µ receptor dynamics such as its trafficking is a possible mechanism underlying opioid tolerance that contributes to inefficient recycling of opioid responses. We aimed to characterize the functional turnover of µ receptors in the noradrenergic nucleus locus coeruleus (LC). EXPERIMENTAL APPROACH: We measured opioid effect by single-unit extracellular recordings of LC neurons from rat brain slices. Immunocytochemical techniques were used to evaluate µ receptor trafficking. KEY RESULTS: After near-complete, irreversible µ receptor inactivation with ß-funaltrexamine (ß-FNA), opioid effect spontaneously recovered in a rapid and efficacious manner. In contrast, α2 -adrenoceptor-mediated effect hardly recovered after receptor inactivation with the irreversible antagonist EEDQ. When the recovery of opioid effect was tested after various inactivating time schedules, we found that the longer the ß-FNA pre-exposure, the less efficient and slower the functional µ receptor turnover became. Interestingly, µ receptor turnover was slower when ß-FNA challenge was repeated in the same cell, indicating constitutive µ receptor recycling by trafficking from a depletable pool. Double immunocytochemistry confirmed the constitutive nature of µ receptor trafficking from a cytoplasmic compartment. The µ receptor turnover was slowed down when LC neuron calcium- or firing-dependent processes were prevented or vesicular protein trafficking was blocked by a low temperature or transport inhibitor. CONCLUSIONS AND IMPLICATIONS: Constitutive trafficking of µ receptors from a depletable intracellular pool (endosome) may account for its rapid and efficient functional turnover in the LC. A finely-tuned regulation of µ receptor trafficking and endosomes could explain neuroadaptive plasticity to opioids in the LC.

Systemic effect of cannabinoids on the spontaneous firing rate of locus coeruleus neurons in rats.

Previous reports have described modulation of noradrenergic activity by cannabinoid receptors. The aim of the present research was to examine the effect of two synthetic cannabinoid CB1/CB2 receptor agonists, R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolol-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone (WIN 55212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55940), on the spontaneous activity of locus coeruleus noradrenergic neurons by single-unit extracellular recordings in vivo and in vitro. In anaesthetized rats, intravenous administrations of WIN 55212-2 (31.3-500 microg/kg) or CP 55940 (31.3-500 microg/kg) increased the firing rate of locus coeruleus neurons in a dose-dependent manner. The stimulatory effect of WIN 55212-2 was blocked by pretreatment with the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A; 2 mg/kg). Paradoxically, local administration of WIN 55212-2 (8.3-31.3 pmol) into the locus coeruleus and intracerebroventricular injections of WIN 55212-2 (10-20 microg) or CP 55940 (20-40 microg) failed to change the spontaneous firing rate of locus coeruleus neurons. Likewise, in rat brain slice preparations perfusion with WIN 55212-2 (10 microM) or CP 55940 (10-30 microM) did not specifically affect the spontaneous firing rate of locus coeruleus cells. Therefore, we conclude that synthetic cannabinoids increase the spontaneous firing activity of noradrenergic neurons in the rat locus coeruleus through cannabinoid CB1 receptors. This stimulation appears to be indirectly induced via a receptor mechanism probably located at the peripheral level.

Acute and long-term administration of citalopram desensitizes alpha2-adrenoceptors in the rat vas deferens.

The aim of this study was to investigate the effect of citalopram, a selective serotonin reuptake inhibitor, on the sensitivity of rat vas deferens alpha2-adrenoceptors and to compare it with the effects of serotonin and the dual noradrenaline-serotonin uptake inhibitor duloxetine. To this end, we studied the inhibitory effect of the alpha2-adrenoceptor agonist bromoxidine on the electrically induced contraction of the vas deferens. Citalopram (1, 3 x 10(3) and 3 x 10(4) nM) applied in-vitro significantly attenuated the concentration-response inhibition induced by activation of alpha2-adrenoceptors on the electrically evoked contraction of the vas deferens (concentration of the agonist required to promote 50% of the maximal effect, EC50, for bromoxidine increased by 232%, 421% and 818%, respectively). Similarly, serotonin also attenuated the concentration-response inhibition mediated by presynaptic alpha2-adrenoceptors (96% increase in EC50). Acute and long-term systemic administration of citalopram and duloxetine also produced a loss in the sensitivity of alpha2-adrenoceptors to bromoxidine (EC50 for bromoxidine increased by 97% and 144%, respectively, after citalopram, and by 214% and 167% after duloxetine). In addition, we observed that an increased fraction of receptors was required to be occupied to yield 50% of the inhibitory effect of bromoxidine after long-term administration of citalopram and duloxetine (KE increased by 142% and 83%). These results are indicative of early-onset and persistent down-regulation of peripheral alpha2-adrenoceptors by citalopram, which may account for some of its side effects.

alpha(2)-Adrenoceptor involvement in the in vitro inhibitory effect of citalopram on a subpopulation of rat locus coeruleus neurons.

The aim of the present study was to investigate the modulation of locus coeruleus neurons by the selective serotonin (5-HT) reuptake inhibitor citalopram using single-unit extracellular recordings in rat brain slices. Citalopram inhibited the activity of a subpopulation of locus coeruleus neurons; thus 10 microM citalopram inhibited neurons by 53+/-17% (5 out of 15 cells), whereas the inhibition due to 100 microM was 64+/-4% (32 out of 42 cells). This effect was partially reversed (47+/-11%) by the alpha(2)-adrenoceptor antagonist idazoxan (10 microM), whereas it was unaffected by antagonists for 5-HT(1A), 5-HT(2,) and 5-HT(3) receptors, and mu opioid receptors. 5-HT (50 or 200 microM), the 5-HT(1A) receptor agonist 8-OH-DPAT (+/-)-8-hydroxy-2-(DI-n-propyl-amino) tetralin hydrobromide, 10 microM) and the 5-HT(2) receptor agonist DOI ([+/-]-2,5-dimetoxy-4-iodoamphetamine) hydrochloride, 10 or 30 microM) also inhibited a subpopulation of locus coeruleus cells. In addition, citalopram but not 5-HT, enhanced by 1.7 fold the inhibitory effect of noradrenaline. Long-term treatment with citalopram (20 mg/kg/day) did not modify the effect of noradrenaline and bromoxidine. Taken together, our results indicate that citalopram exerts an inhibitory effect on locus coeruleus noradrenergic neurons. alpha(2)-adrenoceptor activation may underlie this effect as a result of elevated levels of noradrenaline in the synaptic cleft.

Contribution of nitric oxide-dependent guanylate cyclase and reactive oxygen species signaling pathways to desensitization of µ-opioid receptors in the rat locus coeruleus.

Nitric oxide (NO) is involved in desensitization of µ-opioid receptors (MOR). We used extracellular recordings in vitro to unmask the NO-dependent pathways involved in MOR desensitization in the rat locus coeruleus (LC). Perfusion with ME (3 and 10 µM) concentration-dependently reduced subsequent ME effect, indicative of MOR desensitization. ME (3 µM)-induced desensitization was enhanced by a NO donor (DEA/NO 100 µM), two soluble guanylate cyclase (sGC) activators (A 350619 30 µM and BAY 418543 1 µM) or a cGMP-dependent protein kinase (PKG) activator (8-pCPT-cGMP 30 µM). DEA/NO-induced enhancement was blocked by the sGC inhibitor NS 2028 (10 µM). A 350619 effect was also blocked by NS 2028, but not by the antioxidant Trolox. ME (10 µM)-induced desensitization was blocked by the neuronal NO synthase inhibitor 7-NI (100 µM) and restored by the PKG activator 8-Br-cGMP (100-300 µM). Paradoxically, ME (10 µM)-induced desensitization was not modified by sGC inhibitors (NS 2028 and ODQ), PKG inhibitors (H8 and Rp-8-Br-PET-cGMP) or antioxidant agents (Trolox, U-74389G and melatonin), but it was attenuated by a combination of NS 2028 and Trolox. In conclusion, MOR desensitization in the LC may be mediated or regulated by NO through sGC and reactive oxygen species signaling pathways.

Effect of ceftriaxone and topiramate treatments on naltrexone-precipitated morphine withdrawal and glutamate receptor desensitization in the rat locus coeruleus.

RATIONALE: Morphine withdrawal is associated with a hyperactivity of locus coeruleus (LC) neurons by an elevated glutamate neurotransmission in this nucleus. We postulate that reductions in the amount of glutamate in the LC by enhancing its reuptake or inhibiting its release could attenuate the behavioral and cellular consequences of morphine withdrawal. OBJECTIVES: We investigated the effect of chronic treatment with ceftriaxone (CFT), an excitatory amino acid transporter (EAAT2) enhancer, and acute administration of topiramate (TPM), a glutamate release inhibitor, on morphine withdrawal syndrome and withdrawal-induced glutamate receptor (GluR) desensitization in LC neurons from morphine-dependent rats. METHODS: Morphine withdrawal behavior was measured after naltrexone administration in rats implanted with a morphine (200 mg kg(-1)) emulsion for 3 days. GluR desensitization in the LC was assessed by performing concentration-effect curves for glutamate by extracellular electrophysiological recordings in vitro. RESULTS: Treatments with CFT or TPM reduced, in a dose-related manner, the total behavioral score of naltrexone-precipitated morphine withdrawal. CFT and TPM, at doses that were effective in behavioral tests, also reduced the induction of GluR desensitization normally occurring in LC neurons from morphine-dependent rats. Acute treatment with the specific EAAT2 inhibitor dihydrokainic acid (DHK) prevented the effect of CFT on withdrawal syndrome and GluR desensitization. Perfusion with TPM inhibited KCl-evoked but not glutamate-induced activation of LC neurons in vitro. CONCLUSIONS: Our results suggest that a reduction of synaptic concentrations of glutamate by enhancing EAAT2-mediated uptake or inhibiting glutamate release alleviates the behavioral response and the cellular changes in the LC during opiate withdrawal.

Modulation of anxiety-like behavior and morphine dependence in CREB-deficient mice.

The transcription factor cAMP-responsive element binding protein (CREB) has been shown to regulate different physiological responses including drug addiction and emotional behavior. Molecular changes including adaptive modifications of the transcription factor CREB are produced during drug dependence in many regions of the brain, including the locus coeruleus (LC), but the molecular mechanisms involving CREB within these regions have remained controversial. To further investigate the involvement of CREB in emotional behavior, drug reward and opioid physical dependence, we used two independently generated CREB-deficient mice. We employed the Cre/loxP system to generate mice with a conditional CREB mutation restricted to the nervous system, where all CREB isoforms are lacking in the brain (Crebl(NesCre)). A genetically defined cohort of the previously described hypomorphic Crebl(alphadelta) mice, in which the two major transcriptionally active isoforms (alpha and delta) are disrupted throughout the organism, were also used. First, we investigated the responses to stress of the CREB-deficient mice in several paradigms, and we found an increased anxiogenic-like response in the both Creb1 mutant mice in different behavioral models. We investigated the rewarding properties of drugs of abuse (cocaine and morphine) and natural reward (food) using the conditioned place-preference paradigm. No modification of motivational responses of morphine, cocaine, or food was observed in mutant mice. Finally, we evaluated opioid dependence by measuring the behavioral expression of morphine withdrawal and electrophysiological recordings of LC neurons. We showed an important attenuation of the behavioral expression of abstinence and a decrease in the hyperactivity of LC neurons in both Creb1 mutant mice. Our results emphasize the selective role played by neuronal CREB in emotional-like behavior and the somatic expression morphine withdrawal, without participating in the rewarding properties induced by morphine and cocaine.

Comparative study of the effects of desipramine and reboxetine on locus coeruleus neurons in rat brain slices.

Several studies have suggested that the locus coeruleus may play an important role in the pathophysiology of depression. The aim of this study was to characterize, using single-unit extracellular recordings, the in vitro effects of the noradrenaline reuptake inhibitors desipramine and reboxetine, on locus coeruleus neurons from control rats and from those chronically treated with desipramine. Bath application of desipramine (1-100 microM) and reboxetine (0.1-10 microM) decreased the firing rate of locus coeruleus neurons in a concentration-dependent manner and the alpha(2)-adrenoceptor antagonist RX 821002 (10 microM) reversed these effects. In addition, reserpine (5 mg/kg, 3 h before the experiment) almost completely blocked the inhibitory effect of desipramine. Both drugs (1 microM desipramine and 0.1 microM reboxetine) potentiated the inhibitory effect of noradrenaline (10 microM). A 7-day treatment with desipramine (3 mg/kg/12 h, i.p.) caused a decrease in sensitivity to the alpha(2)-adrenoceptor agonist bromoxidine (EC(50) increased by 3.3-fold), but not to noradrenaline or reboxetine. In contrast, this treatment potentiated the inhibitory effect of desipramine with respect to control. Moreover, 14-day treatment with desipramine (3 mg/kg/12 h, i.p.) or reboxetine (10 mg/kg/12 h, i.p.) also potentiated the in vitro effect of desipramine without modifying the in vitro effect of reboxetine. These results show that desipramine and reboxetine modulate the activity of locus coeruleus neurons by noradrenaline acting on alpha(2)-adrenoceptors, and reveal that alpha(2)-adrenoceptor-independent mechanisms may also underlie the action of noradrenaline uptake inhibitors.

Attenuation of acute and chronic effects of morphine by the imidazoline receptor ligand 2-(2-benzofuranyl)-2-imidazoline in rat locus coeruleus neurons.

1 The aim of this study was to determine if 2-(2-benzofuranyl)-2-imidazoline (2-BFI) interacts with the opioid system in the rat locus coeruleus, using single-unit extracellular recordings. 2 In morphine-dependent rats, acute administration of the selective imidazoline receptor ligands 2-BFI (10 and 40 mg kg(-1), i.p. and 100 micro g, i.c.v.) or valldemossine (10 mg kg(-1), i.p.) did not modify the naloxone-induced hyperactivity of locus coeruleus neurons compared with that observed in the morphine-dependent control group. 3 After chronic administration of 2-BFI (10 mg kg(-1), i.p., three times daily, for 5 days) and morphine, naloxone-induced hyperactivity and tolerance to morphine were attenuated. This effect was not observed when a lower dose of 2-BFI (1 mg kg(-1), i.p.) or valldemossine (10 mg kg(-1), i.p.) were used. 4 Acute administration of 2-BFI (10 and 40 mg kg(-1), i.p. and 100 micro g, i.c.v.) but not valldemossine (40 mg kg(-1), i.p.) diminished the potency of morphine to inhibit locus coeruleus neuron activity in vivo (ED(50) values increased by 2.3, 2.9; and 3.1 fold respectively). Similarly, the potency of Met(5)-enkephalin to inhibit locus coeruleus neurons was decreased when 2-BFI (100 micro M) was applied to rat brain slices (EC(50) increased by 5.6; P<0.05). 5 The present data demonstrate that there is an interaction between 2-BFI and the opioid system in the locus coeruleus. This interaction leads to an attenuation of both the hyperactivity of locus coeruleus neurons during opiate withdrawal and the development of tolerance to morphine when 2-BFI is chronically administered. These results suggest that imidazoline drugs may prove to be useful agents for the management of opioid dependence and tolerance.

Effect of agmatine on locus coeruleus neuron activity: possible involvement of nitric oxide.

1. To investigate whether agmatine (the proposed endogenous ligand for imidazoline receptors) controls locus coeruleus neuron activity and to elucidate its mechanism of action, we used single-unit extracellular recording techniques in anaesthetized rats. 2. Agmatine (10, 20 and 40 microg, i.c.v.) increased in a dose-related manner the firing rate of locus coeruleus neurons (maximal increase: 95 +/- 13% at 40 microg). 3. I(1)-imidazoline receptor ligands stimulate locus coeruleus neuron activity through an indirect mechanism originated in the paragigantocellularis nucleus via excitatory amino acids. However, neither electrolytic lesions of the paragigantocellularis nucleus nor pretreatment with the excitatory amino acid antagonist kynurenic acid (1 micromol, i.c.v.) modified agmatine effect (10 microg, i.c.v.). 4. After agmatine administration (20 microg, i.c.v.), dose-response curves for the effect of clonidine (0.625 - 10 microg kg(-1) i.v.) or morphine (0.3 - 4.8 mg kg(-1) i.v.) on locus coeruleus neurons were not different from those obtained in the control groups. 5. Pretreatment with the nitric oxide synthase inhibitors N(omega)-nitro-L-arginine (10 microg, i.c.v.) or N(omega)-nitro-L-arginine methyl ester (100 microg, i.c.v.) but not with the less active stereoisomer N(omega)-nitro-D-arginine methyl ester (100 microg, i.c.v.) completely blocked agmatine effect (10 and 40 microg, i.c.v.). 6. Similarly, when agmatine (20 pmoles) was applied into the locus coeruleus there was an increase that was blocked by N(omega)-nitro-L-arginine methyl ester (100 microg, i.c.v.) in the firing rate of the locus coeruleus neurons (maximal increase 53 +/- 11% and 14 +/- 10% before and after nitric oxide synthase inhibition, respectively). 7. This study demonstrates that agmatine stimulates the firing rate of locus coeruleus neurons via a nitric oxide synthase-dependent mechanism located in this nucleus.

Morphine withdrawal is modified in pituitary adenylate cyclase-activating polypeptide type I-receptor-deficient mice.

The pituitary adenylate cyclase-activating polypeptide type I-receptor (PAC1) is a G-protein-coupled receptor that is widely expressed in neurons of the central and peripheral nervous system. The strong expression of PAC1 in the second sensory neuron as well as in brainstem regions such as the locus coeruleus prompted us to elucidate the potential in vivo role of PAC1-mediated signalling in pain perception and opioid addiction using a PAC1-deficient mouse line. We observed a selective involvement of PAC1 in the mediation of visceral pain. While there was no impairment in acute somatic pain perception, PAC1-mutants exhibited a dramatically decreased response in the abdominal writhing test. These data in concert with data from the literature implicate PAC1 in the mediation of visceral and chronic pain. In addition, we observed that PAC1 did not influence the motivational aspects of opioid addictive properties, since morphine-induced rewarding effects and sensitization to locomotor responses were completely maintained in PAC1-deficient mice. However, there was a dramatic increase in physical withdrawal signs after naloxone-precipitated morphine withdrawal in PAC1 mutants. At the cellular level, electrophysiological examinations in locus coeruleus neurons from morphine-dependent wild-type and PAC1-deficient mice did not reveal any differences in firing rates. These data therefore suggested that most likely disruption of PAC1-mediated signalling in afferents towards the locus coeruleus but not within the intrinsic locus coeruleus system led to the enhancement of somatic withdrawal signs.

Agmatine-morphine interaction on nociception in mice.

The aim of this study was to investigate the possible participation of nitric oxide in the agmatine-mediated potentiation of morphine-induced analgesia in mice. Agmatine and L-NAME (a nitric oxide synthesis inhibitor) enhanced morphine-induced analgesia in the tail flick test, but not in the hot plate test. L-NAME did not block the agmatine-induced potentiation of morphine effect. Our results indicate that agmatine potentiates morphine-induced spinal but not supraspinal analgesia, and that this effect is not mediated by a nitric oxide-dependent mechanism.