Janne Lähdesmäki

Behavioral and neurochemical characterization of α2A-adrenergic receptor knockout mice

Abstract Genetic manipulation of mice now provides new tools to evaluate the biological functions of the α 2 -adrenergic receptor (α 2 -AR) subtypes (α 2A , α 2B , and α 2C ). To investigate the role of the α 2A -AR in the modulation of mouse primary behavioral characteristics and brain neurochemistry, mice with targeted inactivation of the gene for the α 2A -AR were compared with wild-type C57BL/6 control animals. First, a comprehensive behavioral screen was employed to provide a detailed characterization of basic neurologic functions. Thereafter, the mice were analyzed in three models of anxiety, i.e. the elevated-plus maze test, the marble burying test and the open field test. The diurnal activity pattern of the mice was assessed in a 24-h locomotor activity test. Furthermore, receptor autoradiography of the brain was performed using the subtype-non-selective α 2 -AR antagonist radioligand [ 3 H]RS-79948-197. Lack of the α 2A -AR was associated with alterations in autonomic functions, including increased heart rate and piloerection. The mutant mice also exhibited impaired motor coordination skills, increased anxiety-like behavior and an abnormal diurnal activity pattern. In addition, neurochemical analysis of monoamine neurotransmitters revealed a considerable increase in brain norepinephrine turnover in mice lacking α 2A -AR. Our results provide further support for the crucial role of the α 2A -AR in modulating brain noradrenergic neurotransmission and many aspects of mouse behavior and physiology.

α2-Adrenergic drug effects on brain monoamines, locomotion, and body temperature are largely abolished in mice lacking the α2A-adrenoceptor subtype

α2-ARs regulate brain monoaminergic function by inhibiting neuronal firing and release of monoamine neurotransmitters, noradrenaline (NA), serotonin (5-HT) and dopamine (DA). Both α2A- and α2C-AR inhibit monoamine release in vitro in brain slices, but the in vivo roles of individual α2-AR subtypes in modulating monoamine metabolism have not been characterised. Metabolism of brain monoamine neurotransmitters, locomotor activity and body temperature were investigated in mice with targeted inactivation of the gene encoding α2A-AR (α2A-knockout, α2A-KO) and wild-type (WT) mice after treatment with the α2-AR agonist dexmedetomidine and the antagonist atipamezole. Dexmedetomidine caused profound hypothermia (up to 14.7° C mean reduction in rectal temperature) and locomotor inhibition in WT mice, and inhibited the turnover of NA, 5-HT and DA, but increased NA turnover in α2A-KO mice. α2-AR agonist-induced hypothermia and locomotor inhibition were attenuated, but not totally abolished, in α2A-KO mice. These results suggest that α2A-ARs are principally responsible for the α2-AR mediated inhibition of brain monoamine metabolism, but other α2-ARs, possibly α2C-ARs, are also involved, especially in the striatum. However, secondary effects of the physiological alterations caused by drug administration, especially hypothermia, may have contributed to the observed neurochemical changes in WT mice.

Alpha2A-Adrenoceptors are Important Modulators of the Effects of D-Amphetamine on Startle Reactivity and Brain Monoamines

Amphetamines are commonly used to treat attention-deficit hyperactivity disorder, but are also widely abused. They are employed in schizophrenia-related animal models as they disrupt the prepulse inhibition (PPI) of the acoustic startle response. The behavioral effects of amphetamines have mainly been attributed to changes in dopamine transmission, but they also involve increases in the synaptic concentrations of norepinephrine (NE). α2-Adrenoceptors (α2-ARs) regulate the excitability and transmitter release of brain monoaminergic neurons mainly as inhibitory presynaptic auto- and heteroreceptors. Modulation of acoustic startle and its PPI by the α2A-AR subtype was investigated with mice lacking the α2A-AR (α2A-KO) and their wild-type (WT) controls, without drugs and after administration of the α2-AR agonist dexmedetomidine or the antagonist atipamezole. The interaction of D-amphetamine (D-amph) and the α2-AR-noradrenergic neuronal system in modulating startle reactivity and in regulating brain monoamine metabolism was assessed as the behavioral and neurochemical responses to D-amph alone, or to the combination of D-amph and dexmedetomidine or atipamezole. α2A-KO mice were supersensitive to both neurochemical and behavioral effects of D-amph. Brain NE stores of α2A-KO mice were depleted by D-amph, revealing the α2A-AR as essential in modulating the actions of D-amph. Also, increased startle responses and more pronounced disruption of PPI were noted in D-amph-treated α2A-KO mice. α2A-AR also appeared to be responsible for the startle-modulating effects of α2-AR drugs, since the startle attenuation after the α2-AR agonist dexmedetomidine was absent in α2A-KO mice, and the α2-AR antagonist atipamezole had opposite effects on the startle reflex in α2A-KO and WT mice.

α2A adrenoceptors contribute to feedback inhibition of capsaicin-induced hyperalgesia

Background:Studies on receptor knockout mice have so far shown that of the three &agr;2-adrenoceptor subtypes, the &agr;2A adrenoceptor has a major role in mediating the powerful central analgesia induced by synthetic &agr;2-adrenoceptor agonists. However, because a knockout of the gene for the &agr;2A adrenoceptor has produced only little if any change in the pain sensitivity of control, nerve-injured, or inflamed animals, it has not been clear whether activation of &agr;2A-adrenoceptors by endogenous ligands has a significant pain regulatory role. Methods:The authors assessed spontaneous pain behavior and mechanical hypersensitivity induced by administration of capsaicin in the colon or paw of &agr;2A-adrenoceptor knockout mice versus their wild-type controls. Results:Enhanced pain hypersensitivity was observed in &agr;2A-adrenoceptor knockout mice 20 min or more after administration of capsaicin, but before, hypersensitivity and spontaneous pain were of equal magnitude in &agr;2A-adrenoceptor knockout and wild-type mice. When wild-type mice were pretreated with an &agr;2-adrenoceptor antagonist, capsaicin-induced pain hypersensitivity increased to a level equal to that in &agr;2A-adrenoceptor knockout mice. Capsaicin-induced hypersensitivity was suppressed in wild-type but not &agr;2A-adrenoceptor knockout mice by a centrally acting &agr;2-adrenoceptor agonist, whereas a peripherally acting &agr;2-adrenoceptor agonist was without effect on hypersensitivity, although it attenuated capsaicin-induced spontaneous pain behavior in wild-type mice. Conclusions:This study shows that central &agr;2A-adrenoceptors contribute to feedback inhibition of pain hypersensitivity. Also, &agr;2A-adrenoceptors are critical for not only somatic but also visceral antinociceptive effects induced by synthetic &agr;2-adrenoceptor agonists.

The α2A-adrenoceptor subtype is not involved in inflammatory hyperalgesia or morphine-induced antinociception

The purpose of the present study was to investigate the role of the alpha(2A)-adrenoceptor subtype in inflammatory hyperalgesia, and in adrenergic-mu-opioid interactions in acute pain and inflammatory hyperalgesia. Behavioral responses to mechanical and thermal stimuli were studied in alpha(2A)-adrenoceptor knockout mice and their wild-type controls. Thermal nociception was evaluated as paw withdrawal latencies to radiant heat applied to the hindpaws. Mechanical nociception was measured using von Frey monofilament applications to the hindpaws. Mechanical and thermal hyperalgesia, induced with intraplantar carrageenan (1 mg/40 microl) were compared in alpha(2A)-adrenoceptor knockout and wild-type mice. The effects of the systemically administered mu-opioid receptor agonist morphine (1-10 mg/kg) were evaluated on mechanical withdrawal responses under normal and inflammatory conditions in knockout and wild-type mice. Withdrawal responses to radiant heat and von Frey monofilaments were similar in alpha(2A)-adrenoceptor knockout and wild-type mice before and after the carrageenan-induced hindpaw inflammation. Also, the antinociceptive effects of morphine in mechanical nociceptive tests were similar before and after carrageenan-induced hindpaw inflammation. Our observations indicate that alpha(2A)-adrenoceptors are not tonically involved in the modulation of inflammation-induced mechanical and thermal hyperalgesia. In addition, alpha(2A)-adrenoceptors do not appear to play an important role in mu-opioid receptor-mediated antinociception or antihyperalgesia.

Influence of prazosin and clonidine on morphine analgesia, tolerance and withdrawal in mice

Rapid development of tolerance and dependence limits the usefulness of morphine in long-term treatment. We examined the effects of clonidine (alpha(2)-adrenoceptor agonist) and prazosin (alpha(1)-adrenoceptor antagonist) on morphine analgesia, tolerance and withdrawal. Morphine tolerance was induced using a 3-day cumulative twice-daily dosing regimen with s.c. doses up to 120 mg/kg. Tolerance was assessed on day 4, as loss of the antinociceptive effect of a test dose of morphine (5 mg/kg). After 10 h, morphine withdrawal was precipitated with naloxone (1 mg/kg). Prazosin had no analgesic effect alone but dose-dependently potentiated morphine analgesia in morphine-naive mice. Another alpha(1)-adrenoceptor antagonist, corynanthine, had similar effects. Prazosin also increased the analgesic potency of the morphine test dose in morphine-tolerant mice. Naloxone-precipitated vertical jumping was not affected, but weight loss was reduced by prazosin. Acutely administered clonidine potentiated morphine analgesia and alleviated opioid withdrawal signs, as expected. We conclude that in addition to the already established involvement of alpha(2)-adrenoceptors in opioid actions, also alpha(1)-adrenoceptors have significant modulatory role in opioid analgesia and withdrawal.

Non‐adrenergic binding of [3H]atipamezole in rat kidney–regional distribution and comparison to α2‐adrenoceptors

Atipamezole (4‐(2‐ethyl‐2,3‐dihydro‐1H‐inden‐2‐yl)‐1H‐imidazole) was first introduced as a potent and specific α2‐adrenoceptor antagonist, but in some tissues [3H]atipamezole identifies an additional population of binding sites, distinct from both classical α2‐adrenoceptors and I1‐ and I2‐imidazoline receptors identified with [3H]para‐aminoclonidine or [3H]idazoxan. In the present study we have characterized [3H]atipamezole binding sites in rat kidney by receptor autoradiography and membrane binding assays and determined whether they are pharmacologically identical with the previously described binding sites for [3H]para‐aminoclonidine and [3H]idazoxan. [3H]RX821002 and [3H]rauwolscine were used to compare the regional distribution of α2‐adrenoceptors to that of non‐adrenergic binding sites of [3H]atipamezole. Comparative autoradiographic experiments demonstrated the differential localisation of [3H]atipamezole, [3H]RX821002 and [3H]rauwolscine binding sites in rat kidney. The pattern of distribution of non‐adrenergic [3H]atipamezole binding sites is clearly distinct from that of α2‐adrenoceptors. The non‐adrenergic binding of [3H]atipamezole in rat kidney does not fall into any of the previously identified three classes of imidazoline receptors studied with [3H]para‐aminoclonidine, [3H]idazoxan and [3H]RX821002. Atipamezole had no inhibitory effect on MAO‐A or MAO‐B activity in renal membranes, which speaks against the involvement of MAOs in the observed radioligand binding.