Stimulation of α1-adrenoceptors reduces glutamatergic synaptic input from primary afferents through GABAA receptors and T-type Ca channels

Activation of the descending noradrenergic system inhibits nociceptive transmission in the spinal cord. Although both α₁- and α₂-adrenoceptors in the spinal cord are involved in the modulation of nociceptive transmission, it is not clear how α₁-adrenoceptors regulate excitatory and inhibitory synaptic transmission at the spinal level. In this study, inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded from lamina II neurons in rat spinal cord slices. The specific α₁-adrenoceptor agonist phenylephrine significantly increased the frequency of GABAergic spontaneous IPSCs in a concentration dependent manner, and this effect was abolished by the α₁-adrenoceptor antagonist 2-(2,6-dimethoxyphenoxy)ethylaminomethyl-1,4-benzodioxane (WB4101). Phenylephrine also significantly reduced the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents. The inhibitory effect of phenylephrine on evoked monosynaptic glutamatergic EPSCs was largely blocked by the GABA_A receptor antagonist picrotoxin and, to a lesser extent, by the GABA_B receptor antagonist CGP55845. Furthermore, blocking T-type Ca²⁺ channels with amiloride or mibefradil diminished the inhibitory effect produced by phenylephrine or the GABA_A receptor agonist muscimol on monosynaptic EPSCs evoked from primary afferents. Collectively, these findings suggest that activation of α₁-adrenoceptors in the spinal cord increases synaptic GABA release, which attenuates glutamatergic input from primary afferents mainly through GABA_A receptors and T-type Ca²⁺ channels. This mechanism of presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the descending noradrenergic system.     

Testosterone is essential for α2-adrenoceptor-induced antinociception in the trigeminal region of the male rat

Activation of the α₂-adrenoceptor has been shown to produce antinociception. We have previously shown that the antinociceptive effect of clonidine, an α₂-adrenoceptor agonist, is sex-specific and is abolished by exogenous estrogen in ovariectomized rats or high level of endogenous estrogen in proestrous females. Here, we investigated whether testosterone mediates the antinociceptive effect of clonidine in the trigeminal region of the male rat. Clonidine (7 μg/5 μl) was injected intracisternally through a PE-10 cannula implanted dorsal to the trigeminal region in orchidectomized (GDX) male Sprague–Dawley rats. In separate groups, testosterone propionate (250 μg/100 μl; GDX+T) or β-estradiol benzoate (100 μg/100 μl; GDX+E) were injected subcutaneously 24 and 48 h respectively prior to the N-methyl-d-aspartic acid (NMDA)—or heat-evoked nociceptive test. NMDA-induced number of scratches or duration of scratching behavior did not change significantly in control groups with or without hormonal replacement. Clonidine significantly reduced both measures only in the GDX+T group but not in GDX or GDX+E group. Clonidine also significantly increased head withdrawal latency (HWL) in the GDX+T group, but not in GDX or GDX+E group. The antinociceptive effect of clonidine was reversed by yohimbine, an α₂-adrenoceptor antagonist, in GDX+T group. We conclude that testosterone is required for the expression of antinociception produced by selective activation of the α₂-adrenoceptor in the trigeminal region of the male rat. These findings further our understanding of sex-related differences in the modulation of nociception and may provide insight into development and administration of analgesic agents in young vs. aging men.     

Role of α2/δ subunit in the development of morphine-induced rewarding effect and behavioral sensitization

Previous data demonstrate that L-type voltage-gated calcium channel (VGCC) blockers, which bind to α₁ subunits of VGCC to suppress Ca²⁺ entry into cells, inhibit the development of psychological dependence on drugs of abuse, suggesting the upregulation of L-type VGCC in the development of psychological dependence. However, there are few available data on changes of the auxiliary subunit α₂/δ modifying L-type VGCC under such conditions. We therefore investigated here the role of α₂/δ subunits of VGCCs in the brain of mouse after repeated treatment with morphine. The treatment with morphine increased α₂/δ subunit expression in the frontal cortex and the limbic forebrain of mice showing rewarding effect and sensitization to hyperlocomotion by morphine. The morphine-induced behavioral sensitization and place preference were also suppressed by gabapentin, which binds to an exofacial epitope of the α₂/δ auxiliary subunits of VGCCs. These findings indicate that the upregulation of α₂/δ subunit as well as α₁ subunits of VGCC in the frontal cortex and the limbic forebrain plays a critical role in development of morphine-induced rewarding effect and behavioral sensitization following neuronal plasticity.     

Antagonism of α1-adrenergic and serotonergic receptors in the hypoglossal motor nucleus does not prevent motoneuronal activation elicited from the posterior hypothalamus

The perifornical (PF) region of the posterior hypothalamus plays an important role in the regulation of sleep–wake states and motor activity. Disinhibition of PF neurons by the GABA_A receptor antagonist, bicuculline, has been used to study the mechanisms of wake- and motor activity-promoting effects that emanate from the PF region. Bicuculline activates PF neurons, including the orexin-containing cells that have major excitatory projections to brainstem noradrenergic and serotonergic neurons. Since premotor aminergic neurons are an important source of motoneuronal activation, we hypothesized that they mediate the excitation of motoneurons that results from disinhibition of PF neurons with bicuculline. In urethane-anesthetized, paralyzed and artificially ventilated rats, we found that PF bicuculline injections (1 mM, 20 nl) made after combined microinjections into the hypoglossal (XII) nucleus of α₁-adrenergic and serotonergic receptor antagonists (prazosin and methysergide) increased XII nerve activity by 80 ± 16% (SE) of the control activity level. Thus, activation of XII motoneurons originating in the hypothalamic PF region was not abolished despite effective elimination by the aminergic antagonists of the endogenous noradrenergic and serotonergic excitatory drives to XII motoneurons and abolition of XII motoneuronal activation by exogenous serotonin or phenylephrine. These results show that a major component of XII motoneuronal activation originating in the posterior hypothalamus is mediated by pathways other than the noradrenergic and serotonergic projections to motoneurons.     

Adrenal α2-adrenergic receptors in the aging normotensive and spontaneously hypertensive rat

This study investigates α₂-adrenergic receptor (α₂AR) mediated feedback inhibition of catecholamine release from the adrenal medulla of adult (52 weeks) and old (98 weeks) spontaneously hypertensive rats (SHR) and normotensive controls Wistar Kyoto (WKY) rats. Adrenal epinephrine content as well as the spontaneous and the nicotinic-evoked release of epinephrine were similar between adult SHR and WKY rats. Aging produced a significant reduction in epinephrine synthesis in WKY rats. In contrast, in SHR aging produced a significant increase in epinephrine release without significant changes in epinephrine synthesis. The α₂AR agonist medetomidine abolished (80–90% inhibition) the nicotinic-evoked release of epinephrine in adult SHR and WKY rats. With aging, this effect was unaltered in WKY rats but was significantly decreased in SHR (30% inhibition). Adrenal α_2AAR mRNA levels were significantly reduced in old SHR compared with age matched WKY rats. In conclusion, in aging the α₂AR mediated feedback inhibition of epinephrine release from the adrenal medulla is preserved in WKY rats but compromised in SHR, resulting in increased epinephrine release.     

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation.     

Reflection of induced and amplified food motivation in impulse activity of the masticatory muscles during electrostimulation of the “hunger center” in the lateral hypothalamus in rabbits

We studied reflection of artificially induced and amplifi ed food motivation in impulse activity of the masticatory muscles during electrostimulation of “hunger center” of the lateral hypothalamus in the absence and presence of food. The threshold stimulation of the lateral hypothalamus in hungry and satiated animals in the absence of food induced incessant food-procuring behavior paralleled by regular generation of spike bursts in masticatory muscles with biomodal distributions of intervals between pulses. This reaction of masticatory muscles during stimulation of the lateral hypothalamus in the absence of food was an example of the anticipatory reaction reflecting characteristics of the action result acceptor. Higher level of hunger motivation during threshold stimulation of the lateral hypothalamus in hungry and satiated rabbits in the course of effective food-procuring behavior increased the incidence of spike burst generation during the food capture phase, but did not modify this parameter during the chewing phase. Impulse activity of the masticatory muscles reflected convergent interactions of food motivation and support excitation on neurons of the central generator of chewing pattern.     

Activation of α2-adrenoreceptors suppresses the excitability of C1 spinal neurons having convergent inputs from tooth pulp and superior sagittal sinus in rats

The aim of the present study was to test the hypothesis that activation of α₂-adrenoreceptors modulates the excitability of C1 neurons having convergent inputs from both the tooth pulp (TP) and the superior sagittal sinus (SSS), by using the microiontophoretic techniques of drug application and immunohistochemical approaches. Extracellular single-unit recordings were made from 38 C1 neurons responding to electrical stimulation of TP under pentobarbital-anesthetized rats. Seventy-one percent of C1 neurons (27/38) that responded to TP stimulation also responded to electrical stimulation of the SSS. In these neurons, l-glutamate-evoked C1 neuronal discharge firings were increased in a dose-dependent manner. The mean glutamate-evoked firing rates were dose-dependently inhibited after microiontophoretic application of clonidine (α₂-adrenoreceptor/imidazoline I₁ receptor agonist). The inhibition of glutamate-evoked C1 mean firings by clonidine was antagonized by the co-application of idazoxan (α₂-adrenoreceptor/imidazoline I₂ receptor antagonist), yohimbine (α₂-adrenoreceptor) but not the α₁-adrenoreceptor antagonist, prazosin with affinity for α_2B- and α_2C-adrenoreceptors. The mean spontaneous discharge frequencies were significantly inhibited by the microiontophoretic application of clonidine and this inhibition was reversed by the co-application of idazoxan, yohimbine. Microiontophoresis of clonidine also resulted in a reduction of TP-/SSS-evoked activity and this effect was reversed by the co-application of yohimbine. Immunoreactivity for α_2A-adrenoreceptor was found in the superficial layers of I–III in the C1 region. These results suggest that α₂-adrenoreceptor agonist clonidine inhibits the excitability of C1 neurons having convergent inputs from TP and SSS afferents, and that the activation of α_2A-adrenoreceptors onto C1 dorsal horn neurons may contribute as a useful therapeutic target for the alleviation of trigeminal referred pain associated with migraine and tooth pain.     

Heat loss responses and blockade of prostaglandin E2-induced thermogenesis elicited by α1-adrenergic activation in the rostromedial preoptic area

The unilateral microinjection of noradrenaline (NA), but not vehicle solution, into the rostromedial preoptic area (POA) elicited simultaneous increases in cutaneous temperatures of the tail and sole of the foot and decreases in the whole-body O₂ consumption rate, heart rate, and colonic temperature in urethane–chloralose-anesthetized rats, suggesting a coordinate increase in heat loss and decrease in heat production. The magnitude of these responses increased dose-dependently over the range of 1–100 pmol, except for the metabolic and bradycardic responses. Similar hypothermic responses were elicited by the microinjection of 40 pmol methoxamine (an α₁-adrenergic agonist), but not by that of clonidine (an α₂-agonist) or isoproterenol (a β-agonist). Sites at which microinjection of NA elicited hypothermic responses were in the vicinity of the organum vasculosum of the lamina terminalis including the median preoptic nucleus, whereas no thermal or metabolic response was elicited when NA was microinjected into the lateral POA or caudal part of the medial POA. The microinjection of 130 fmol prostaglandin (PG) E₂ into the NA-sensitive site always elicited thermogenic, tachycardic, and hyperthermic responses. Furthermore, the PGE₂-induced febrile responses were greatly attenuated by prior administration of NA at the same site. These results demonstrate that NA in the rostromedial POA exerts α₁-adrenoceptor-mediated hypothermic effects and opposes PGE₂-induced fever.     

Modulation of potassium channels via the α1B-adrenergic receptor in human osteoblasts

Recent studies have demonstrated the involvement of the sympathetic nerve system in bone metabolism. We have previously demonstrated the expression of adrenergic receptors in the human osteoblast SaM-1 cell line. The aim of this study was to reveal the function of these receptors in osteoblasts using electrophysiological methods. During whole-cell patch clamp recording, the application of noradrenaline reduced the currents induced by a voltage ramp, and the effect was larger in the positive potential range. Pretreatment with the selective α_1B-adrenergic receptor antagonist chloroethylclonidine eliminated the inhibitory effect of noradrenaline. Using Cs-based pipette solution to block potassium channels, the inhibitory effect of noradrenaline disappeared. In contrast, the effect was unaffected in the presence of tetraethylammonium (TEA), a potassium channel blocker. These results suggest that noradrenaline suppresses Cs-sensitive and TEA-insensitive potassium channels via the α_1B-adrenergic receptor in human osteoblasts.     

Activation of α2 adrenoceptor attenuates lipopolysaccharide-induced hepatic injury

Sepsis induces hepatic injury but whether alpha-2 adrenoceptor (α2-AR) modulates the severity of sepsis-induced liver damage remains unclear. The present study used lipopolysaccharide (LPS) to induce hepatic injury and applied α2-AR agonist dexmedetomidine (DEX) and/or antagonist yohimbine to investigate the contribution of α2-AR in LPS-induced liver injury. Our results showed that LPS resulted in histological and functional abnormality of liver tissue (ALT and AST transaminases, lactate), higher mortality, an increase in proinflammatory cytokines (IL-1β, IL-6 & TNF-α), as well as a change in oxidative stress (MDA, SOD). Activation of α2-AR by dexmedetomidine (DEX) attenuated LPS-induced deleterious effects on the liver and block of α2-AR by yohimbine aggravated LPS-induced liver damage. Our data suggest that α2-AR plays an important role in sepsis-induced liver damage and activation of α2-AR with DEX could be a novel therapeutic avenue to protect the liver against sepsis-induced injury.     

Therapeutic potential of α2 adrenoceptor antagonism for antipsychotic-induced extrapyramidal motor disorders

We examined the effects of JP-1302 (a selective α_2C antagonist), BRL-44408 (a selective α_2A antagonist) and yohimbine (a non-selective α₂ antagonist) on haloperidol-induced bradykinesia and catalepsy in mice to elucidate the role of α₂ adrenoceptor subtypes in modifying extrapyramidal motor disorders. JP-1302 (0.1–1 mg/kg, s.c.) dose-dependently ameliorated haloperidol-induced bradykinesia in the pole-test and reversed the catalepsy time increased by haloperidol. Antibradykinetic and anticataleptic actions of JP-1302 were statistically significant at 0.3 and 1 mg/kg, and these doses did not alter the ambulatory distance, rearing or center–perimeter residence time in the open-field test. BRL-44408 (1–10 mg/kg, s.c.) and yohimbine (0.3–3 mg/kg, i.p.) also ameliorated haloperidol-induced bradykinesia and catalepsy. However, both agents significantly decreased ambulatory distance and rearing in the open-field test, possibly reflecting their anxiogenic actions associated with α_2A antagonism. The present study shows for the first time that blockade of α_2C receptors can alleviate antipsychotic-induced extrapyramidal motor disorders without affecting gross behaviors.     

GABAergic regulation of the perifornical–lateral hypothalamic neurons during non-rapid eye movement sleep in rats

The perifornical–lateral hypothalamic area (PF–LHA) has been implicated in the regulation of behavioral arousal. The PF–LHA predominantly contains neurons that are active during behavioral and cortical activation and quiescent during non-rapid eye movement (nonREM) sleep, that is, are nonREM-off neurons. Some in vitro and in vivo studies indicate that PF–LHA neurons, including hypocretin-expressing neurons, are under GABAergic control. However, a role of GABA in suppressing the discharge of PF–LHA neurons during spontaneous nonREM sleep has not been confirmed. We recorded the sleep–wake discharge profiles of PF–LHA neurons and simultaneously assessed the contributions of local GABA_A receptor activation and blockade on their wake- and nonREM sleep-related discharge activities by delivering GABA_A receptor agonist, muscimol (500 nm, 5 μM, and 10 μM) and its antagonist, bicuculline (5 μM, 10 μM, and 20 μM), adjacent to the recorded neurons via reverse microdialysis. Muscimol dose-dependently decreased the discharge of PF–LHA neurons including nonREM-off neurons. Muscimol-induced suppression of discharge during nonREM sleep was significantly weaker than the suppression produced during waking. In the presence of bicuculline, PF–LHA neurons, including nonREM-off neurons, exhibited elevated discharge, which was dose-dependent and was significantly higher during nonREM sleep, compared to waking. These results suggest that GABA_A receptor mediated increased GABAergic tone contributes to the suppression of PF–LHA neurons, including nonREM-off neurons, during spontaneous nonREM sleep.     

A1 receptor mediated adenosinergic regulation of perifornical–lateral hypothalamic area neurons in freely behaving rats

The perifornical–lateral hypothalamic area (PF-LHA) plays a central role in the regulation of behavioral arousal. The PF-LHA contains several neuronal types including wake-active hypocretin (HCRT) neurons that have been implicated in the promotion and/or maintenance of behavioral arousal. Adenosine is an endogenous sleep factor and recent evidence suggests that activation and blockade of adenosine A₁ receptors within the PF-LHA promote and suppress sleep, respectively. Although, an in vitro study indicates that adenosine inhibits HCRT neurons via A₁ receptor, the in vivo effects of A₁ receptor mediated adenosinergic transmission on PF-LHA neurons including HCRT neurons are not known. First, we determined the effects of N⁶-cyclopentyladenosine (CPA), an adenosine A₁ receptor agonist, on the sleep–wake discharge activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe used for its delivery. Second, we determined the effects of CPA and that of an A₁ receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX) into the PF-LHA on cFos-protein immunoreactivity (Fos-IR) in HCRT and non-HCRT neurons around the microdialysis probe used for their delivery. The effect of CPA on Fos-IR was studied in rats that were kept awake during lights-off phase, whereas the effect of CPDX was examined in undisturbed rats during lights-on phase. CPA significantly suppressed the sleep–wake discharge activity of PF-LHA neurons. Doses of CPA (50 μM) and CPDX (50 μM) that suppressed and induced arousal, respectively, in our earlier study [Alam MN, Kumar S, Rai S, Methippara M, Szymusiak R, McGinty D (2009) Brain Res 1304:96–104], significantly suppressed and increased Fos-IR in HCRT and non-HCRT neurons. These findings suggest that wake-promoting PF-LHA system is subject to increased endogenous adenosinergic inhibition and that adenosine acting via A₁ receptors, in part, inhibits HCRT neurons to promote sleep.     

Activation of the skeletal α-actin promoter during muscle regeneration

Little is known conerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5–10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal -actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal -actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal -actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal - actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by –424 skeletal -actin and –99 skeletal -actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal -actin promoter activities were similar to control values. Thus, initial activation of the skeletal - actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal -actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal -actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box. © Kluwer Academic Publishers.     

Activation of nicotinic α7 acetylcholine receptor enhances long term potentation in wild type mice but not in APPswe/PS1ΔE9 mice

Amyloid β (Aβ) plays a central role in Alzheimer's disease (AD) and binds to the nicotinic α₇ receptor (α₇ nAChR). Little is known about the degree to which the binding of Aβ to the α₇ nAChR influences the role of this receptor in long-term potentiation (LTP), however. We have studied the effect of the partial α₇ nAChR agonist SSR180711 on hippocampal slice preparations from normal wild type (Wt) and APP_swe/PS1ΔE9 transgenic (Tg) mice. In the hippocampal slices from the 6 months old Wt mice, the application of both nicotine (5 μM) and SSR180711 (300 nM) resulted in a significant enhancement of LTP expressed in area CA1. However, in the Tg mice the application of SSR180711 did not result in an increase in LTP beyond control levels. The amount of binding of the α₇ nAChR ligand 125-I-α-bungarotoxin was not different between in Tg and Wt mice. These findings indicate that the α₇ nAChR is functionally blocked in the hippocampal neurons, downstream of the α₇ nAChR, and that this is likely due to an interaction between the receptor and Aβ, which leads to changes in LTP.     

Effects of central FGF21 infusion on the hypothalamus–pituitary–thyroid axis and energy metabolism in rats

The aim of this study was to evaluate the impact of intracerebroventricular chronic fibroblast growth factor 21 (FGF21) infusion on hypothalamic–pituitary–thyroid (HPT) axis, energy metabolism, food intake and body weight. Thirty male Wistar albino rats were used and divided into three groups including control, sham (vehicle) and FGF21 infused groups (n = 10). Intracerebroventricularly, FGF21 and vehicle groups were infused for 7 days with FGF21 (0.72 µg/day) and artificial cerebrospinal fluid, respectively. During the experimental period, changes in food intake and body weight were recorded daily. Serum thyroid stimulating hormone (TSH), Triiodothyronine (T3) and thyroxine (T4) levels were measured using ELISA. TRH and uncoupling protein 1 (UCP1) gene expressions were analyzed by using RT-PCR in hypothalamus and adipose tissues, respectively. Chronic infusion of FGF21 significantly increased serum TSH (p < 0.05), T3 (p < 0.05) and T4 (p < 0.001) levels. Additionally, hypothalamic TRH (p < 0.05) and UCP1 gene expressions (p < 0.05) in white adipose tissue were found to be higher than in the vehicle and control groups. While FGF21 infusion did not cause a significant change in food consumption, it caused a reduction in the body weight of rats (p < 0.05). Our findings indicate that FGF21 may have an effect on energy metabolism via the HPT axis.