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1 K a r e n L . S m i t h ( P h D ) , 1 G e m m a K . F o r d ( P h D ) , 2 D a v i d S . J e s s o p ( P h D ) , 1 * D a v i d P . F i n n ( P h D )
1 P h a r m a c o l o g y a n d T h e r a p e u t i c s , S c h o o l o f M e d i cine and NCBES Neuroscience Cluster, National University of Ireland, Galway, University Road, Galway, Ireland; 2Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY, UK.
Correspondence:
Dr David P. Finn,
Pharmacology and Therapeutics,
School of Medicine,
National University of Ireland, Galway,
University Road,
Galway,
Ireland.
Tel: +353 (0)91 495280
Fax: +353 (0)91 525700
Email: HYPERLINK "mailto:David.Finn@nuigalway.ie" David.Finn@nuigalway.ie
Sources of Support: This publication has emanated from research conducted with the financial support of Science Foundation Ireland under Grant Number 06/RFP/BIM022.
Abstract: The putative endogenous imidazoline binding site ligand harmane enhances neuronal activation in response to psychological stress and alters behaviour in animal models of anxiety and antidepressant efficacy. However, the neurobiological mechanisms underlying harmanes psychotropic effects are poorly understood. We investigated the effects of intraperitoneal injection of harmane (2.5 and 10 mg/kg) on fear-conditioned (FC) behaviour, hypothalamo-pituitary-adrenal axis activity, and monoaminergic activity within specific fear-associated areas of the rat brain. Harmane had no significant effect on the duration of contextually induced freezing or 22 kHz ultrasonic vocalizations and did not alter the contextually-induced suppression of motor activity, including rearing. Harmane did reduce the duration of rearing and tended to increase freezing in non-fear-conditioned controls, suggesting potential sedative effects. Harmane increased plasma ACTH and corticosterone concentrations, and serotonin (in hypothalamus, amygdaloid cortex, prefrontal cortex and hippocampus) and noradrenaline (prefrontal cortex) content, irrespective of fear-conditioning. Furthermore, harmane reduced dopamine and serotonin turnover in the PFC and hypothalamus, and serotonin turnover in the amygdaloid cortex in both fear-conditioned and non-fear-conditioned rats. In contrast, harmane increased dopamine and noradrenaline content and reduced dopamine turnover in the amygdala of fear-conditioned rats only, suggesting differential effects on catecholaminergic transmission in the presence and absence of fear. The precise mechanism(s) mediating these effects of harmane remain to be determined but may involve its inhibitory action on monoamine oxidases. These findings support a role for harmane as a neuromodulator, altering behaviour, brain neurochemistry and neuroendoc r i n e f u n c t i o n .
K e y w o r d s : I m i d a z o l i n e b i n d i n g s i t e s ; b e t a - c a r b o l i n e ; h a r m a n e ; f e a r c o n d i t i o n i n g ; m o n o a m i n e ; H P A a x i s ; b r a i n ; r a t .
I n t r o d u c t i o n
T h e - c a r b o l i n e h a r m a n e i s a p u t a t i v e e n d o g e n o u s a g o n i s t a t i m i d a z o l i n e b i n d i n g s i t e s A D D I N E N . C I T E A D D I N EN.CITE.DATA ( HYPERLINK \l "_ENREF_36" \o "Musgrave, 2000 #316" Musgrave et al., 2000, HYPERLINK \l "_ENREF_43" \o "Robinson, 2003 #350" Robinson et al., 2003), possesses binding affinity for monoamine oxidase-A (MAO-A) ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_27" \o "Kim, 1997 #292" Kim et al., 1997, HYPERLINK \l "_ENREF_23" \o "Herraiz, 2005 #291" Herraiz et al., 2005) at which it acts as an allosteric inhibitor, and for the GABAA receptor ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_35" \o "Muller, 1981 #406" Muller et al., 1981, HYPERLINK \l "_ENREF_34" \o "Mousah, 1986 #1242" Mousah et al., 1986, HYPERLINK \l "_ENREF_56" \o "Splettstoesser, 2005 #1239" Splettstoesser et al., 2005) where it has an inverse agonistic effect ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_38" \o "Petersen, 1983 #326" Petersen, 1983, HYPERLINK \l "_ENREF_31" \o "McMahon, 2006 #325" Mcmahon et al., 2006, HYPERLINK \l "_ENREF_14" \o "Evans, 2007 #324" Evans et al., 2007). There is a well established role for MAO-A and GABAA receptors in the regulation of emotional processing and a significant body of evidence suggesting that imidazoline binding sites (I1 and I2 subtypes) may represent a novel therapeutic target for the treatment of psychiatric disorders ADDIN EN.CITE Smith2009524(Smith, 2009)52452417Smith, K. L.Jessop, D. S.Finn, D. P.Department of Pharmacology and Therapeutics, NCBES Neuroscience Cluster, National University of Ireland, Galway, Ireland.Modulation of stress by imidazoline binding sites: implications for psychiatric disordersStressStress97-1141222008/11/1420091607-8888 (Electronic)19006007http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19006007905454977 [pii]
10.1080/10253890802302908eng( HYPERLINK \l "_ENREF_53" \o "Smith, 2009 #524" Smith et al., 2009). Thus, increased understanding of the behavioural and neurobiological effects of harmane is warranted.
Previous studies have demonstrated that systemic administration of exogenous harmane dose-dependently reduces immobility in the rat and mouse forced swim test ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_4" \o "Aricioglu, 2003 #375" Aricioglu et al., 2003, HYPERLINK \l "_ENREF_15" \o "Farzin, 2006 #243" Farzin et al., 2006) and increases time spent in the open arms of the elevated plus maze ADDIN EN.CITE Farzin2006243(Farzin, 2006)24324317Farzin, D.Mansouri, N.Department of Pharmacology, School of Medicine, Mazandaran University of Medical Sciences, Sari 48168, Iran. davoodfarzin@yahoo.comAntidepressant-like effect of harmane and other beta-carbolines in the mouse forced swim testEur NeuropsychopharmacolEur Neuropsychopharmacol324-8165Animals*Antidepressive AgentsBehavior, Animal/drug effectsCarbolines/*pharmacologyDose-Response Relationship, DrugDrug InteractionsFlumazenil/pharmacologyGABA Modulators/pharmacologyHarmine/*analogs & derivatives/pharmacologyMaleMiceMonoamine Oxidase Inhibitors/pharmacologyMotor Activity/drug effectsReserpine/pharmacologySwimming/*psychologySympatholytics/pharmacology2006Jul16183262http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16183262 ( HYPERLINK \l "_ENREF_15" \o "Farzin, 2006 #243" Farzin et al., 2006). Direct administration of harmane into the globus pallidus has been shown to increase time spent in the illuminated sector of the light-dark box without inducing motor impairment ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_58" \o "Talalaenko, 2006 #323" Talalaenko et al., 2006). Furthermore, the anxiolytic-like effect of ethanol is potentiated by pretreatment with imidazoline ligands including agmatine (I1/I2 receptor agonist), moxonidine and clonidine (I1 receptor agonists) and 2-BFI (I2 receptor agonist) in the elevated plus maze ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_57" \o "Taksande, 2010 #955" Taksande et al., 2010), suggesting that imidazoline binding site ligands may augment or act synergistically with other receptors to modulate anxiety-related behaviour. Taken together, these data suggest that imidazoline ligands, including harmane, may have anxiolytic and antidepressant-like activity. However, there is a paucity of studies investigating the effects of harmane on conditioned fear responding and associated neurochemical and neuroendocrine alterations which we sought to address here.
Fear conditioning (FC) paradigms are used to study the expression of fear and formation of fear HYPERLINK "http://en.wikipedia.org/wiki/Memory" \o "Memory" memory; in particular, the deficits or dysregulation that may lead to anxiety disorders including HYPERLINK "http://en.wikipedia.org/wiki/Phobia" \o "Phobia" phobias and post-traumatic stress disorder ( HYPERLINK "http://en.wikipedia.org/wiki/Post-traumatic_stress_disorder" \o "Post-traumatic stress disorder" PTSD). The neural circuitry underpinning the acquisition and expression of contextual fear is well understood and involves the amygdala, hippocampus and medial prefrontal cortex (mPFC) ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_46" \o "Sanders, 2003 #1223" Sanders et al., 2003, HYPERLINK \l "_ENREF_40" \o "Quirk, 2006 #572" Quirk et al., 2006, HYPERLINK \l "_ENREF_28" \o "LeDoux, 2007 #1219" Ledoux, 2007). Connectivity between the brain regional components of this fear circuitry and the paraventricular nucleus of the hypothalamus facilitates the neuroendocrine response to fear in the form of hypothalamo-pituitary-adrenal (HPA) axis activation ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_22" \o "Herman, 1997 #24" Herman et al., 1997, HYPERLINK \l "_ENREF_39" \o "Prewitt, 1998 #1226" Prewitt et al., 1998, HYPERLINK \l "_ENREF_45" \o "Rodrigues, 2009 #1227" Rodrigues et al., 2009).
Multiple neurotransmitter systems have been implicated in coordination of the behavioural response to conditioned fear, with a particularly important role for the monoamines. Fear conditioning specifically activates the mesoamygdaloid dopaminergic system; increasing the rate of dopaminergic neuron firing, dopamine (DA) content and metabolism in the hypothalamus, amygdala and medial prefrontal cortex ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_10" \o "Coco, 1992 #1210" Coco et al., 1992, Goldstein et al., 1994, HYPERLINK \l "_ENREF_24" \o "Inoue, 1994 #593" Inoue et al., 1994, HYPERLINK \l "_ENREF_47" \o "Sasaki, 1998 #1211" Sasaki et al., 1998). The serotoninergic and noradrenergic systems also play an important role. Conditioned-fear stress induces intense Fos expression in the noradrenergic locus coeruleus and serotonergic dorsal raphe neurons ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_25" \o "Ishida, 2002 #1214" Ishida et al., 2002). Activation of monoaminergic receptor subtypes or inhibition of neurotransmitter re-uptake alters the rat freezing response in a conditioned fear paradigm ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_62" \o "Wislowska-Stanek, 2008 #1215" Wislowska-Stanek et al., 2008) and in a conditioned fear stress test ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_33" \o "Mochizuki, 2002 #1246" Mochizuki et al., 2002). Both selective serotonin- and serotonin-noradrenaline reuptake inhibitors are useful first-line agents for most anxiety disorders ADDIN EN.CITE Baldwin20101254(Baldwin, 2010)1254125417Baldwin, D. S.Ajel, K. I.Garner, M.Clinical Neuroscience Division, School of Medicine, University of Southampton, Southampton, UK. dsb1@soton.ac.ukPharmacological treatment of generalized anxiety disorderCurr Top Behav NeurosciCurr Top Behav Neurosci453-6722011/02/11Adrenergic Uptake Inhibitors/*therapeutic useAnti-Anxiety Agents/*therapeutic useAntidepressive Agents/*therapeutic useAnxiety Disorders/*drug therapy/epidemiology/*prevention & controlDrug ToleranceHumansRecurrence/prevention & controlSerotonin Uptake Inhibitors/*therapeutic useTime FactorsTreatment Outcome20101866-3370 (Print)
1866-3370 (Linking)21309121http://www.ncbi.nlm.nih.gov/pubmed/21309121eng( HYPERLINK \l "_ENREF_6" \o "Baldwin, 2010 #1254" Baldwin et al., 2010).
Both I1 and I2 binding sites and their putative endogenous ligand harmane are found throughout the limbic system in stress-responsive brain regions ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_29" \o "Lione, 1998 #258" Lione et al., 1998, HYPERLINK \l "_ENREF_1" \o "Anderson, 2005 #465" Anderson et al., 2005, HYPERLINK \l "_ENREF_2" \o "Anderson, 2006 #257" Anderson et al., 2006, HYPERLINK \l "_ENREF_3" \o "Anderson, 2006 #1255" Anderson et al., 2006). Our previous work has shown that harmane is capable of modulating neuronal responses to restraint stress in rats ADDIN EN.CITE Smith2009464(Smith, 2009)46446417Smith, K. L.Roche, M.Jessop, D. S.Finn, D. P.Department of Pharmacology and Therapeutics, National University of Ireland-Galway, University Road, Galway, Ireland.The effects of synthetic and endogenous imidazoline binding site ligands on neuronal activity in discrete brain regions of naive and restraint-stressed ratsEur NeuropsychopharmacolEur Neuropsychopharmacol371-801952009/02/272009May1873-7862 (Electronic)19243923http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19243923S0924-977X(09)00010-8 [pii]
10.1016/j.euroneuro.2009.01.005eng( HYPERLINK \l "_ENREF_54" \o "Smith, 2009 #464" Smith et al., 2009) and that I2 binding site ligands potentiate the HPA axis ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_17" \o "Finn, 2004 #352" Finn et al., 2004) and central noradrenergic ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_19" \o "Finn, 2002 #242" Finn et al., 2002) response to stress. Moreover, agonism of I1/I2 binding sites with the putative endogenous imidazoline binding site ligand agmatine has been shown to induce a dose-dependent impairment in the magnitude of learned contextual fear in rats ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_30" \o "McKay, 2002 #600" Mckay et al., 2002). In addition, intra-amygdala injections of c l o n i d i n e , a n 2 - a d r e n o c e p t o r a n d I 1 b i n d i n g s i t e a g o n i s t , b l o c k e d t h e e x p r e s s i o n o f f e a r i n r a t s A D D I N E N . C I T E <