Jukka Sallinen

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.

Evaluation of the alpha2C-adrenoceptor as a neuropsychiatric drug target studies in transgenic mouse models.

The functional characterization of the three distinct alpha2-adrenoceptor (Q2-AR) subtypes was for long hampered by the inavailability of subtype-selective pharmacological probes. Recent studies with gene-targeted mice have revealed that the alpha2A-AR has a major role in the mediation of many prominent effects of subtype non-selective alpha2-AR agonists, i.e. sedation, analgesia, hypothermia, sympatho-inhibition, and reduction of blood pressure. We have now employed several neuropsychopharmacological test models to investigate the effects mediated by the alpha2C-AR subtype and this receptors potential as a CNS drug target. The studies employed two genetically engineered mouse strains, having either a targeted inactivation of the alpha2C-AR gene (alpha2C-KO) or over-expressing the alpha2C-AR (alpha2C-OE). Lack of alpha2C-AR expression was associated with increased amphetamine-induced locomotor activity, startle reactivity, aggression, and activity in the forced swimming test; prepulse inhibition of the startle reflex was attenuated. Opposite changes were observed in the alpha2C-OE mice. The results suggest that the alpha2C-AR subtype has a distinct inhibitory role in the processing of sensory information and in the control of motor and emotion-related activities in the CNS. It is therefore possible that alpha2C-AR-selective drugs may have therapeutic value in the treatment of various neuropsychiatric disorders.

Pharmacological characterization and CNS effects of a novel highly selective α2C-adrenoceptor antagonist JP-1302

Pharmacological validation of novel functions for the α2A‐, α2B‐, and α2C‐adrenoceptor (AR) subtypes has been hampered by the limited specificity and subtype‐selectivity of available ligands. The current study describes a novel highly selective α2C‐adrenoceptor antagonist, JP‐1302 (acridin‐9‐yl‐[4‐(4‐methylpiperazin‐1‐yl)‐phenyl]amine).

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.

Overexpression of Alpha2C-adrenoceptors impairs water maze navigation

We investigated the role of overexpression of alpha2C-adrenoceptors in water maze navigation in mice transgenically manipulated to have a threefold overexpression of the alpha2C-adrenoreceptors. Alpha2C-adrenoreceptors overexpressing mice swam more in the peripheral annulus of the pool and did not find the hidden escape platform as well as the wild type control mice. A subtype-nonselective alpha2-adrenoreceptor antagonist, atipamezole (ATI, 1000 microg/kg, s.c.), fully reversed the deficit in platform finding and search strategy in overexpressing mice. Noradrenaline depletion (-95%) induced by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) did not impair platform finding of wild type or overexpressing mice. The DSP-4 lesion slightly increased swimming in the peripheral annulus in wild type mice, but not in overexpressing mice. The DSP-4 lesion produced a dissociable effect on the action of atipamezole to improve platform finding and search strategy in overexpressing mice: atipamezole did not alleviate the platform finding deficit in DSP-4 lesioned overexpressing mice, but normalized their abnormal search strategy. These results suggest that the abnormal search pattern and deficit in the accuracy of platform finding are mediated by constitutive activity of overexpressed alpha2C-adrenoreceptors.

Autoradiographic characterization of α2C‐adrenoceptors in the human striatum

Indirect experimental evidence suggests that drugs acting on the α2C‐adrenoceptor could be useful in the treatment of neuropsychiatric disorders such as depression and schizophrenia. In rodent brain, the highest levels of α2C‐adrenoceptors are found in the striatum, with lower levels in cerebral cortex and hippocampus. In human brain, because of the poor subtype‐selectivity of the available α2‐adrenoceptor ligands, the localization of α2C‐adrenoceptors has remained unknown. Recently, a selective α2C‐adrenoceptor antagonist, JP‐1302, was characterized, and to assess the presence of α2C‐adrenoceptors in human brain, we performed competition binding in vitro receptor autoradiography with JP‐1302 and the α2‐adrenoceptor subtype nonselective antagonist [ethyl‐3H]RS79948‐197 on rat and human postmortem brain sections. In striatum of both species, JP‐1302 vs. [ethyl‐3H]RS79948‐197 competition binding was biphasic, identifying high‐ and low‐affinity binding sites, whereas in cortex and cerebellum, only low‐affinity binding sites were detected. The results indicate that a significant portion of the α2‐adrenoceptors in striatum is of the α2C subtype, whereas non‐α2C‐adreocneptors predominate in cortex and cerebellum. Because the α2C‐adrenoceptor subtype distribution pattern appears to be conserved between rodents and humans, results obtained from studies on the role of the α2C‐adrenoceptor in rodent models of neuropsychiatric disorders may be relevant also for human diseases. Synapse 62:508–515, 2008.

Application of cross-species PET imaging to assess neurotransmitter release in brain

RationaleThis review attempts to summarize the current status in relation to the use of positron emission tomography (PET) imaging in the assessment of synaptic concentrations of endogenous mediators in the living brain.ObjectivesAlthough PET radioligands are now available for more than 40 CNS targets, at the initiation of the Innovative Medicines Initiative (IMI) “Novel Methods leading to New Medications in Depression and Schizophrenia” (NEWMEDS) in 2009, PET radioligands sensitive to an endogenous neurotransmitter were only validated for dopamine. NEWMEDS work-package 5, “Cross-species and neurochemical imaging (PET) methods for drug discovery”, commenced with a focus on developing methods enabling assessment of changes in extracellular concentrations of serotonin and noradrenaline in the brain.ResultsSharing the workload across institutions, we utilized in vitro techniques with cells and tissues, in vivo receptor binding and microdialysis techniques in rodents, and in vivo PET imaging in non-human primates and humans. Here, we discuss these efforts and review other recently published reports on the use of radioligands to assess changes in endogenous levels of dopamine, serotonin, noradrenaline, γ-aminobutyric acid, glutamate, acetylcholine, and opioid peptides. The emphasis is on assessment of the availability of appropriate translational tools (PET radioligands, pharmacological challenge agents) and on studies in non-human primates and human subjects, as well as current challenges and future directions.ConclusionsPET imaging directed at investigating changes in endogenous neurochemicals, including the work done in NEWMEDS, have highlighted an opportunity to further extend the capability and application of this technology in drug development.

Pharmacological Characterisation of a Structurally Novel α2C ‐Adrenoceptor Antagonist ORM‐10921 and its Effects in Neuropsychiatric Models

The α2‐adrenoceptors (ARs) are important modulators of a wide array of physiological responses. As only a few selective compounds for the three α2‐AR subtypes (α2A, α2B and α2C) have been available, the pharmacological profile of a new α2C‐selective AR antagonist ORM‐10921 is reported. Standard in vitro receptor assays and antagonism of α2, and α1‐AR agonist ‐evoked responses in vivo were used to demonstrate the α2C‐AR selectivity for ORM‐10921 which was tested in established behavioural models related to schizophrenia and cognitive dysfunction with an emphasis on pharmacologically induced hypoglutamatergic state by phencyclidine or MK‐801. The Kb values of in vitro α2C‐ AR antagonism for ORM‐10921 varied between 0.078–1.2 nM depending on the applied method. The selectivity ratios compared to α2A‐AR subtype and other relevant receptors were 10‐100 times in vitro. The in vivo experiments supported its potent α2C‐antagonism combined with only a weak α2A‐antagonism. In the pharmacodynamic microdialysis study, ORM‐10921 was found to increase extracellular dopamine levels in prefrontal cortex in the baseline conditions. In the behavioural tests, ORM‐10921 displayed potent antidepressant and antipsychotic‐like effects in the forced swimming test and prepulse‐inhibition models analogously with the previously reported results with structurally different α2C‐selective AR antagonist JP‐1302. Our new results also indicate that ORM‐10921 alleviated the NMDA‐antagonist‐induced impairments in social behaviour and watermaze navigation. This study extends and further validates the concept that α2C‐AR is a potential therapeutic target in CNS disorders such as schizophrenia or Alzheimers disease and suggests the potential of α2C‐antagonism to treat such disorders.

Spatial working memory improvement by an α2-adrenoceptor agonist dexmedetomidine is not mediated through α2C-adrenoceptor

Abstract 1. Aged α2C-adrenoceptor knockout and wild type mice were used to investigate whether α2C-adrenoceptors are involved in mediating the beneficial effects of α2-adrenoceptor agonist, dexmedetomidine, on spatial working memory. 2. A win-stay task in the radial arm maze was used to dissociate the effects of dexmedetomidine on working vs. reference memory. In addition, the animals were tested in simple response habit learning in the T-maze. 3. Knockout mice made more working memory errors after the change of the baited arm in radial arm maze, but after training reached again as accurate level of performance as wild type controls. Dexmedetomidine 5 and 10 μg/kg alleviated the increase in spatial working memory errors after the change of the baited arm in knockout mice. Knockout and wild type mice performed equally well in T-maze, and dexmedetomidine had no effect on this simple response learning. 4. The present results indicate that α2-adrenoceptor agonists have a selective effect on spatial working memory not only in monkeys but also in mice. Further, this study confirms our earlier finding that the presence of α2C-adrenoceptors is not necessary for the spatial working memory enhancing effect of α2-adrenoceptor agonists.