Depression-like phenotype following chronic CB1 receptor antagonism.

Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.

WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one]: a novel dopamine D2 receptor partial agonist/serotonin reuptake inhibitor with preclinical antipsychotic-like and antidepressant-like activity.

The preclinical characterization of WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D(2) receptor (D(2L) K(i), 4.0 nM) and serotonin transporter (K(i), 7.1 nM), potent D(2) partial agonist activity (EC(50), 0.38 nM; E(max), 30%), and complete block of the serotonin transporter (IC(50), 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID(50), 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D(2) partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazole's reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D(2) receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants.

Preclinical characterization of BRL 44408: antidepressant- and analgesic-like activity through selective alpha2A-adrenoceptor antagonism.

Biogenic amines such as norepinephrine, dopamine, and serotonin play a well-described role in the treatment of mood disorders and some types of pain. As alpha2A-adrenoceptors regulate the release of these neurotransmitters, we examined the therapeutic potential of BRL 44408, a potent (Ki=8.5 nM) and selective (>50-fold) alpha2A-adrenoceptor antagonist (K(B)=7.9 nM). In rats, BRL 44408 penetrated the central nervous system resulting in peak brain and plasma concentrations of 586 ng/g and 1124 ng/ml, respectively. In a pharmacodynamic assay, pretreatment with BRL 44408 to rats responding under a fixed-ratio 30 operant response paradigm resulted in a rightward shift of the clonidine dose-response curve, an effect indicative of alpha2-adrenoceptor antagonism in vivo. Consistent with presynaptic autoreceptor antagonism and tonic regulation of neurotransmitter release, acute administration of BRL 44408 elevated extracellular concentrations of norepinephrine and dopamine, but not serotonin, in the medial prefrontal cortex. Additionally, BRL 44408, probably by inhibiting alpha2A heteroceptors, produced a significant increase in cortical levels of acetylcholine. In the forced swim test and schedule-induced polydipsia assay, BRL 44408 produced an antidepressant-like response by dose-dependently decreasing immobility time and adjunctive water intake, respectively, while in a model of visceral pain, BRL 44408 exhibited analgesic activity by decreasing para-phenylquinone (PPQ)-induced abdominal stretching. Finally, BRL 44408 did not produce deficits in overall motor coordination nor alter general locomotor activity. This preclinical characterization of the neurochemical and behavioural profile of BRL 44408 suggests that selective antagonism of alpha2A-adrenoceptors may represent an effective treatment strategy for mood disorders and visceral pain.

Acid sensing ion channel (ASIC) inhibitors exhibit anxiolytic-like activity in preclinical pharmacological models.

RATIONALE: Acid sensing ion channels (ASICs) are proton-gated ion channels located in the central and peripheral nervous systems. Of particular interest is ASIC1a, which is located in areas associated with fear and anxiety behaviors. Recent reports suggest a role for ASIC1a in preclinical models of fear conditioning and anxiety. OBJECTIVES: The present experiments evaluated various ASIC inhibitors in preclinical models of autonomic and behavioral parameters of anxiety. In addition, neurochemical studies evaluated the effects of an ASIC inhibitor (A-317567) on neurotransmitter levels in the amygdala. RESULTS: In electrophysiological studies using hippocampal primary neuronal cultures, three ASIC inhibitors (PcTX-1, A-317567, and amiloride) produced concentration-dependent inhibition of acid-evoked currents. In the stress-induced hyperthermia model, acute administration of psalmotoxin 1 (PcTX-1; 10-56 ng, i.c.v.), A-317567 (0.1-1.0 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) prevented stress-induced elevations in core body temperature. In the four-plate test, acute treatment with PcTX-1 (10-56 ng, i.c.v.) and A-317567 (0.01-0.1 mg/kg, i.p.), but not amiloride (3-100 mg/kg, i.p.), produced dose-dependent and significant increases in the number of punished crossings relative to vehicle-treated animals. Additionally, PcTX-1 (56-178 ng, i.c.v.), A-317567 (0.1-10 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) lacked significant anxiolytic-like activity in the elevated zero maze. In neurochemical studies, an infusion of A-317567 (100 microM) into the amygdala significantly elevated the extracellular levels of GABA, but not glutamate, in this brain region. CONCLUSIONS: These findings demonstrate that ASIC inhibition produces anxiolytic-like effects in some behavioral models and indicate a potential role for GABAergic mechanisms to underlie these anxiolytic-like effects.

Metabolite identification in rat brain microdialysates by direct infusion nanoelectrospray ionization after desalting on a ZipTip and LTQ/Orbitrap mass spectrometry.

Analyzing brain microdialysate samples by mass spectrometry is challenging due to the high salt content of the artificial cerebral spinal fluid (aCSF), low analyte concentrations and small sample volumes collected. A drug and its major metabolites can be examined in brain microdialysates by targeted approaches such as selected reaction monitoring (SRM) which provides selectivity and high sensitivity. However, this approach is not well suited for metabolite profiling in the brain which aims to determine biotransformation pathways. Identifying minor metabolites, or metabolites that arise from brain metabolism, remains a challenge and, for a drug in early discovery, identification of metabolites present in the brain can provide useful information for understanding the pharmacological activity and potential toxicological liabilities of the drug. A method is described here for rapid metabolite profiling in brain microdialysates that involves sample clean-up using C18 ZipTips to remove salts followed by direct infusion nanoelectrospray with an LTQ/Orbitrap mass spectrometer using real-time internal recalibration. Full scan mass spectra acquired at high resolving power (100 K at m/z 400) were examined manually and with mass defect filtering. Metabolite identification was aided by sub-parts-per-million mass accuracy and structural characterization was accomplished by tandem mass spectrometry (MS/MS) experiments in the Orbitrap or LTQ depending on the abundance of the metabolite. Using this approach, brain microdialysate samples from rats dosed with one of four CNS drugs (imipramine, reboxetine, citalopram or trazodone) were examined for metabolites. For each drug investigated, metabolites, some of which not previously reported in rat brain, were identified and characterized.

Novel 1-aminoethyl-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines are potent 5-HT(6) agonists.

A series of 1-aminoethyl-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines 10a-z was prepared as novel 5-HT(6) ligands. The best compounds were high affinity, full agonists at 5-HT(6) receptors. Several agonists demonstrated good selectivity over other serotonergic and dopaminergic receptors. Acute administration of selective agonist 10e significantly increased extracellular GABA concentrations in rat frontal cortex. This compound also reduced adjunctive drinking behavior in the rat schedule-induced polydipsia assay, possibly predictive of efficacy in obsessive compulsive disorder and other anxiety related disorders.

Neuropharmacological profile of novel and selective 5-HT6 receptor agonists: WAY-181187 and WAY-208466.

One of the most recently identified serotonin (5-hydroxytryptamine (5-HT)) receptor subtypes is the 5-HT6 receptor. Although in-depth localization studies reveal an exclusive distribution of 5-HT6 mRNA in the central nervous system, the precise biological role of this receptor still remains unknown. In the present series of experiments, we report the pharmacological and neurochemical characterization of two novel and selective 5-HT6 receptor agonists. WAY-181187 and WAY-208466 possess high affinity binding (2.2 and 4.8 nM, respectively) at the human 5-HT6 receptor and profile as full receptor agonists (WAY-181187: EC50=6.6 nM, Emax=93%; WAY-208466: EC50=7.3 nM; Emax=100%). In the rat frontal cortex, acute administration of WAY-181187 (3-30 mg/kg, subcutaneous (s.c.)) significantly increased extracellular GABA concentrations without altering the levels of glutamate or norepinephrine. Additionally, WAY-181187 (30 mg/kg, s.c.) produced modest yet significant decreases in cortical dopamine and 5-HT levels. Subsequent studies showed that the neurochemical effects of WAY-181187 in the frontal cortex could be blocked by pretreatment with the 5-HT6 antagonist, SB-271046 (10 mg/kg, s.c.), implicating 5-HT6 receptor mechanisms in mediating these responses. Moreover, the effects of WAY-181187 on catecholamines were attenuated by an intracortical infusion of the GABA A receptor antagonist, bicuculline (10 microM), confirming a local relationship between 5-HT6 receptors and GABAergic systems in the frontal cortex. In the dorsal hippocampus, striatum, and amygdala, WAY-181187 (10-30 mg/kg, s.c.) elicited robust elevations in extracellular levels of GABA without producing similar effects on concentrations of norepinephrine, serotonin, dopamine, or glutamate. In contrast to these brain regions, WAY-181187 had no effect on the extracellular levels of GABA in the nucleus accumbens or thalamus. Additional studies showed that WAY-208466 (10 mg/kg, s.c.) preferentially elevated cortical GABA levels following both acute and chronic (14 day) administration, indicating that neurochemical tolerance does not develop following repeated 5-HT6 receptor stimulation. In hippocampal slice preparations (in vitro), 5-HT(6) receptor agonism attenuated stimulated glutamate levels elicited by sodium azide and high KCl treatment. Furthermore, in the rat schedule-induced polydipsia model of obsessive compulsive disorder (OCD), acute administration of WAY-181187 (56-178 mg/kg, po) decreased adjunctive drinking behavior in a dose-dependent manner. In summary, WAY-181187 and WAY-208466 are novel, selective, and potent 5-HT6 receptor agonists displaying a unique neurochemical signature in vivo. Moreover, these data highlight a previously undescribed role for 5-HT6 receptors to modulate basal GABA and stimulated glutamate transmission, as well as reveal a potential therapeutic role for this receptor in the treatment of some types of anxiety-related disorders (eg OCD).

Differentiating antidepressants of the future: efficacy and safety.

There have been significant advances in the treatment of depression since the serendipitous discovery that modulating monoaminergic neurotransmission may be a pathological underpinning of the disease. Despite these advances, particularly over the last 15years with the introduction of selective serotonin and/or norepinephrine reuptake inhibitors (SNRI), there still remain multiple unmet clinical needs that would represent substantial improvements to current treatment regimens. In terms of efficacy there have been improvements in the percentage of patients achieving remission but this can still be dramatically improved and, in fact, issues still remain with relapse. Furthermore, advances are still required in terms of improving the onset of efficacy as well as addressing the large proportion of patients who remain treatment resistant. While this is not well understood, collective research in the area suggests the disease is heterogeneous in terms of the multiple parameters related to etiology, pathology and response to pharmacological agents. In addition to efficacy further therapeutic advances will also need to address such issues as cognitive impairment, pain, sexual dysfunction, nausea and emesis, weight gain and potential cardiovascular effects. With these unmet needs in mind, the next generation of antidepressants will need to differentiate themselves from the current array of therapeutics for depression. There are multiple strategies for addressing unmet needs that are currently being investigated. These range from combination monoaminergic approaches to subtype selective agents to novel targets that include mechanisms to modulate neuropeptides and excitatory amino acids (EAA). This review will discuss the many facets of differentiation and potential strategies for the development of novel antidepressants.

ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: a novel metabotropic glutamate receptor 5-selective positive allosteric modulator with preclinical antipsychotic-like and procognitive activities.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca(2+) assays (EC(50) = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 microM) and competed for binding of [(3)H]2-methyl-6-(phenylethynyl)pyridine (K(i) = 4.3 microM), but not [(3)H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

Pharmacology of neuropeptide S in mice: therapeutic relevance to anxiety disorders.

RATIONALE: Neuropeptide S (NPS) and its receptor (NPSR) comprise a recently deorphaned G protein-coupled receptor system. Recent reports implicate NPS in the mediation of anxiolytic-like activity in rodents. OBJECTIVES: To extend the characterization of NPS, the present studies examined the in vitro pharmacology of mouse NPSR and the in vivo pharmacology of NPS in three preclinical mouse models predictive of anxiolytic action: the four-plate test (FPT), elevated zero maze (EZM), and stress-induced hyperthermia (SIH). The ability of NPS to produce antidepressant-like effects in the tail suspension test (TST) was also investigated. RESULTS: In vitro, mouse NPS 1-20 (mNPS 1-20) and the C-terminal glutamine-truncated mouse NPS 1-19 bound mNPSR with high affinity (Ki = 0.203 +/- 0.060, 0.635 +/- 0.141 nM, respectively) and potently activated intracellular calcium release (EC50 = 3.73 +/- 1.08, 4.10 +/- 1.25 nM). NPS produced effects in vivo consistent with anxiolytic-like activity. In FPT, NPS increased punished crossings (minimal effective dose [MED]: mNPS 1-20 = 0.2 microg, mNPS(1-19) = 0.02 microg), similar to the reference anxiolytic, alprazolam (MED 0.5 microg). NPS increased the percentage of time spent in the open quadrants of EZM (MED: mNPS 1-20 = 0.1 microg, mNPS 1-19 = 1.0 microg), like the reference anxiolytic, chlordiazepoxide (MED 56 microg). In SIH, NPS attenuated stress-induced increases in body temperature similar to alprazolam but with a large potency difference between the NPS peptides (MED: mNPS 1-20 = 2.0 microg, mNPS 1-19 = 0.0002 microg) and mNPS 1-20 increased baseline temperature. Unlike fluoxetine, NPS did not effect immobility time in TST, indicating a lack of antidepressant-like activity. CONCLUSIONS: These data provide an important confirmation and expansion of the anxiolytic-like effects of NPS and implicate the NPS system as a novel target for anxiolytic drug discovery.

Inhibition of uptake 2 (or extraneuronal monoamine transporter) by normetanephrine potentiates the neurochemical effects of venlafaxine.

Two distinct norepinephrine (NE) transporter mechanisms (uptake 1 and uptake 2) regulate extracellular NE concentrations. An association has been observed between the gradual improvement in patients treated with antidepressants that inhibit the NE transporter (NET/uptake 1) and increases in urinary normetanephrine, the O-methylated NE metabolite and potent inhibitor of uptake 2. These observations led to the hypothesis that increased levels of normetanephrine, and consequently inhibition of uptake 2, may partly be responsible for the clinical efficacy of some antidepressants. To investigate this hypothesis, we employed microdialysis techniques in the rat frontal cortex to monitor extracellular changes in normetanephrine following chronic administration of the clinically effective antidepressant, venlafaxine (a serotonin (5-HT) and NE reuptake inhibitor). We evaluated the neurochemical effects of inhibiting uptake 2 alone, or in conjunction with venlafaxine, on extracellular levels of NE and 5-HT. Chronic venlafaxine administration (14 days, 10 mg/kg, s.c.) elicited significant increases in cortical NE and 5-HT while producing a non-significant trend to increase cortical levels of normetanephrine. Additional studies revealed that combining normetanephrine with venlafaxine (10 mg/kg, i.p.), at a dose of normetanephrine (10 mg/kg, i.p.) that did not produce changes in extracellular levels of NE on its own, potentiated antidepressant-induced increases in extracellular NE. We also report mouse behavioral data involving the tail suspension test that complement the neurochemical observations. These preclinical findings, taken together, suggest that inhibiting both uptake 1 and uptake 2 via venlafaxine and normetanephrine, respectively, elicits a greater increase in cortical levels of NE than inhibiting either transporter alone.

Do selective serotonin reuptake inhibitors acutely increase frontal cortex levels of serotonin?

Selective serotonin uptake inhibitors (SSRIs) exert their effects by inhibiting serotonin (5-HT) re-uptake. Although blockade occurs almost immediately, the neurochemical effects on 5-HT, as measured by in vivo microdialysis, have been a matter of considerable debate. In particular, literature reports yield conflicting neurochemical results in the rat frontal cortex. Thus, while some groups consistently find increases in extracellular 5-HT levels following acute SSRI administration, others reproducibly report an absence of these acute serotonergic effects. In an attempt to unravel this apparent discrepancy, we combined published literature with in-house microdialysis experiments. When we plotted the lateral stereotaxic coordinate of the dialysis probe against published reports on the acute effects of fluoxetine a clear correlation was revealed. Whereas pronounced increases in SSRI-induced 5-HT were observed when the dialysis probe was placed 0 to 1 mm from the midline, effects diminished when the lateral probe placement was greater than 3 mm from the midline. In-house microdialysis studies corroborated these reports. Overall, these results illustrate - for the first time - that the midline stereotaxic coordinate is critical for interpreting the acute serotonergic effects of SSRIs within the frontal cortex. Moreover, the common observation that the clinical efficacy of SSRIs is not evident following acute administration complements preclinical microdialysis results in the lateral frontal cortex. The significance of this observation, along with potential explanations for the disparate neurochemical findings in the medial versus lateral cortices, will be discussed.

Discovery of N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine as a potent, selective, and orally active 5-HT(6) receptor agonist.

N1-Arylsulfonyltryptamines have been identified as 5-HT6 receptor ligands. In particular, N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine (11q) is a high affinity, potent full agonist (5-HT6 Ki = 2 nM, EC50 = 6.5 nM, Emax = 95.5%). Compound 11q is selective in a panel of over 40 receptors and ion channels, has good pharmacokinetic profile, has been shown to increase GABA levels in the rat frontal cortex, and is active in the schedule-induced polydipsia model for obsessive compulsive disorders.

Anxiolytic-like activity of the non-selective galanin receptor agonist, galnon.

Galanin's influence on monoaminergic neurotransmission, together with its discrete CNS distribution in corticolimbic brain areas, points to a potential role for this neuropeptide in mediating anxiety- and depression-like responses. To evaluate this hypothesis, the non-selective galanin receptor agonist, galnon, was tested in multiple preclinical models of anxiolytic- and antidepressive-like activity. Acute administration of galnon (0.03-1mg/kg, i.p.) dose-dependently increased punished crossings in the four plate test, with magnitude similar to the effects of the endogenous ligand, galanin (0.1-1.0 microg, i.c.v.). Moreover, the effects of galnon and galanin were blocked by central administration of the non-selective galanin receptor antagonist, M35 (10 microg, i.c.v.). Interestingly, the benzodiazepine receptor antagonist, flumazenil (1mg/kg, i.p.), reversed galnon's effect in the four plate test, implicating GABAergic neurotransmission as a potential mechanism underlying this anxiolytic-like response. In the elevated zero maze, galnon (0.3-3.0mg/kg, i.p.) and galanin (0.03-0.3 microg, i.c.v.) increased the time spent in the open arms, while in the stress-induced hyperthermia model, galnon (0.3-30 mg/kg, i.p.) attenuated stress-induced changes in body temperature. Consistent with these anxiolytic-like effects, in vivo microdialysis showed that acute galnon (3mg/kg, i.p.) treatment preferentially elevated levels of GABA in the rat amygdala, a brain area linked to fear and anxiety behaviors. In contrast to the effects in anxiety models, neither galnon (1-5.6 mg/kg, i.p.) nor galanin (0.3-3.0 microg, i.c.v.) demonstrated antidepressant-like effects in the mouse tail suspension test. Galnon (1-10mg/kg, i.p.) also failed to reduce immobility time in the rat forced swim test. In vitro, galnon and galanin showed affinity for human galanin receptors expressed in Bowes melanoma cells (K(i)=5.5 microM and 0.2 nM, respectively). Galanin displayed high affinity and functional potency for membranes expressing rat GALR1 receptors (K(i)=0.85 nM; EC(50)=0.6 nM), while galnon (10 microM) failed to displace radiolabeled galanin or inhibit cAMP production in the same GALR1 cell line. Galnon (10 microM) showed affinity for NPY1, NK2, M5, and somatostatin receptors but no affinity for galanin receptors expressed in rat hippocampal membranes. Taken together, the present series of studies demonstrate novel effects of galnon in various preclinical models of anxiety and highlight the galaninergic system as a novel therapeutic target for the treatment of anxiety-related disorders. Moreover, these data indicate rodent GALR1 receptors do not mediate galnon's in vivo activity.

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples.

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N=3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N=10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05-10nM for ACh, 0.25-50 nM for iso-ACh and 15-3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.

Synthesis and biological evaluation of novel compounds within a class of 3-aminochroman derivatives with dual 5-HT1A receptor and serotonin transporter affinity.

Compounds containing a 5-carbamoyl-8-fluoro-3-amino-3,4-dihydro-2H-1-benzopyran and a 3-alkylindole moiety linked through a common basic nitrogen were prepared and evaluated for 5-HT1A affinity, serotonin rat transporter affinity, and functional antagonist activity in vitro. 26a was found to be the most potent and selective compound in this series and was shown to possess neurochemical activity in vivo by producing acute and rapid increases in 5-HT in the rat frontal cortex.

Neurochemical changes in the RVM associated with peripheral inflammatory pain stimuli.

A greater knowledge of the neurochemical changes occurring during pain states will undoubtedly aid in the discovery of effective pain pharmacotherapies. This study highlights the acute effects of inflammatory agents on neurochemical changes in the rostral ventromedial medulla (RVM), a supraspinal site involved in the processing of painful stimuli. Consistent with previous reports, a peripheral injection of 0.1 mg prostaglandin E(2) (PGE(2)) into the intraplantar area of the rat paw produced thermal hypersensitivity that peaked 10 min after administration. In vivo microdialysis studies in the same animals revealed that this behavioral response correlated with a greater than 2-fold increase (230%) in extracellular serotonin (5-HT) levels in the RVM. In contrast, levels of other neurotransmitters measured, including norepinephrine and dopamine, were not altered in animals receiving this inflammatory agent. Similar to PGE(2), an intraplantar injection of capsaicin (0.1 mg) produced a robust thermal hypersensitivity that was paralleled by a 3-fold increase in levels of 5-HT in the RVM. The next series of experiments showed that acute administration of the opioid analgesic, morphine (5.6 mg/kg; IP), attenuated PGE(2)-induced thermal hypersensitivity and reversed the increase in extracellular 5-HT observed in the RVM. Taken together, these findings extend previous reports of central neurochemical changes during inflammatory pain conditions and show that the combination of behavioral endpoints with microdialysis can yield important insights into the neurochemical environment of the pain circuitry.

Alpha 2A-adrenoceptors enhance the serotonergic effects of fluoxetine.

The ability of subtype-preferring alpha(2)-adrenoceptor antagonists to enhance the neurochemical effects of the antidepressant, fluoxetine, was evaluated by in vivo microdialysis. Combining the selective alpha(2A)-adrenoceptor antagonist, BRL-44408 (10 mg/kg, s.c.), with fluoxetine (30 mg/kg, s.c.) elevated the extracellular levels of serotonin (5-HT) and noradrenaline in the rat frontal cortex, an effect not observed following antidepressant treatment alone. In contrast, combining fluoxetine with the alpha(2B)- or alpha(2C)-adrenoceptor antagonists, imiloxan (10 mg/kg, s.c.) or rauwolscine (10 mg/kg, s.c.), respectively, did not similarly alter biogenic amine levels. Collectively, these results reveal a specific role for the alpha(2A)-adrenoceptor subtype in augmenting the neurochemical effects of antidepressants.

Measurement of neurotransmitters from extracellular fluid in brain by in vivo microdialysis and chromatography-mass spectrometry.

During the last three decades, a great deal of information has been discovered about chemical neurotransmission. However, the most important processes, namely the complex nature of neuronal circuitry, the "cross talk" between multiple neurotransmitter systems, and the varying effects neurochemicals have at different receptors, are still being explored. Techniques such as microdialysis are routinely employed to measure neurotransmitter levels in living tissue systems. Moreover, microdialysis studies have proven to be valuable in the investigation of neurodegenerative and psychiatric disease pathology, as well as in identifying novel drugs to treat such disorders. One particular challenge in performing these experiments is the requirement to couple microdialysis to sophisticated analytical equipment. Recently, considerable attention has been focused on the development of chromatographic-mass spectrometric techniques to provide more sensitive and accurate measurements of neurochemicals collected from in vivo microdialysis experiments. This review will provide a brief overview of the microdialysis technique, as well as how microdialysis and chromatography-mass spectrometry are being used to measure extracellular levels of neurotransmitters. The primary emphasis of this review will be on how these applications are used to measure levels of acetylcholine (ACh), dopamine, norepinephrine and gamma-aminobutyric acid (GABA).

Injections of baclofen into the ventral medial prefrontal cortex block the initiation, but not the expression, of cocaine sensitization in rats.

RATIONALE: Increased excitatory output from the medial prefrontal cortex (mPFC) is thought to play a key role in the development of sensitization to cocaine. Gamma-aminobutyric acid (GABA) inhibits this excitatory output. OBJECTIVES: The present studies were designed to determine the effects of intra-mPFC injections of the GABA(B) agonist baclofen on cocaine-induced motor activity and on the development of sensitization to cocaine. METHODS: Rats received bilateral cannula implants above the ventral mPFC. Initial studies examined the dose-response effects of injection of baclofen (0.05-0.5 nmol/side) into the mPFC on the acute motor-stimulant response to cocaine (15 mg/kg, i.p.). Additional studies determined whether coadministration of intra-mPFC baclofen (0.5 nmol/side) and systemic cocaine (15 mg/kg, i.p.) could alter the initiation and/or expression of cocaine-induced behavioral sensitization. RESULTS: Intra-mPFC baclofen dose-dependently blocked cocaine-induced motor activity. In sensitization studies, intra-mPFC baclofen was able to prevent the initiation, but not the expression of cocaine-induced sensitization. CONCLUSIONS: The data suggest that the ability of GABA to modulate excitatory output from the mPFC may be attenuated in animals sensitized to cocaine.