Comparative Effects of LY3020371, a Potent and Selective Metabotropic Glutamate (mGlu) 2/3 Receptor Antagonist, and Ketamine, a Noncompetitive N-Methyl-d-Aspartate Receptor Antagonist in Rodents: Evidence Supporting the Use of mGlu2/3 Antagonists, for the Treatment of Depression.

The ability of the N-methyl-d-aspartate receptor antagonist ketamine to alleviate symptoms in patients suffering from treatment-resistant depression (TRD) is well documented. In this paper, we directly compare in vivo biologic responses in rodents elicited by a recently discovered metabotropic glutamate (mGlu) 2/3 receptor antagonist 2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY3020371) with those produced by ketamine. Both LY3020371 and ketamine increased the number of spontaneously active dopamine cells in the ventral tegmental area of anesthetized rats, increased O2 in the anterior cingulate cortex, promoted wakefulness, enhanced the efflux of biogenic amines in the prefrontal cortex, and produced antidepressant-related behavioral effects in rodent models. The ability of LY3020371 to produce antidepressant-like effects in the forced-swim assay in rats was associated with cerebrospinal fluid (CSF) drug levels that matched concentrations required for functional antagonist activity in native rat brain tissue preparations. Metabolomic pathway analyses from analytes recovered from rat CSF and hippocampus demonstrated that both LY3020371 and ketamine activated common pathways involving GRIA2 and ADORA1. A diester analog of LY3020371 [bis(((isopropoxycarbonyl)oxy)-methyl) (1S,2R,3S,4S,5R,6R)-2-amino-3-(((3,4-difluorophenyl)thio)methyl)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylate (LY3027788)] was an effective oral prodrug; when given orally, it recapitulated effects of intravenous doses of LY3020371 in the forced-swim and wake-promotion assays, and augmented the antidepressant-like effects of fluoxetine or citalopram without altering plasma or brain levels of these compounds. The broad overlap of biologic responses produced by LY3020371 and ketamine supports the hypothesis that mGlu2/3 receptor blockade might be a novel therapeutic approach for the treatment of TRD patients. LY3020371 and LY3027788 represent molecules that are ready for clinical tests of this hypothesis.

The Rapidly Acting Antidepressant Ketamine and the mGlu2/3 Receptor Antagonist LY341495 Rapidly Engage Dopaminergic Mood Circuits.

Ketamine is a rapidly acting antidepressant in patients with treatment-resistant depression (TRD). Although the mechanisms underlying these effects are not fully established, inquiry to date has focused on the triggering of synaptogenesis transduction pathways via glutamatergic mechanisms. Preclinical data suggest that blockade of metabotropic glutamate (mGlu2/3) receptors shares many overlapping features and mechanisms with ketamine and may also provide rapid efficacy for TRD patients. Central dopamine circuitry is recognized as an end target for mood regulation and hedonic valuation and yet has been largely neglected in mechanistic studies of antidepressant-relevant effects of ketamine. Herein, we evaluated the changes in dopaminergic neurotransmission after acute administration of ketamine and the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid ] in preclinical models using electrophysiologic, neurochemical, and behavioral endpoints. When given acutely, both ketamine and LY341495, but not the selective serotonin reuptake inhibitor (SSRI) citalopram, increased the number of spontaneously active dopamine neurons in the ventral tegmental area (VTA), increased extracellular levels of dopamine in the nucleus accumbens and prefrontal cortex, and enhanced the locomotor stimulatory effects of the dopamine D2/3 receptor agonist quinpirole. Further, both ketamine and LY341495 reduced immobility time in the tail-suspension assay in CD1 mice, which are relatively resistant to SSRI antidepressants. Both the VTA neuronal activation and the antidepressant phenotype induced by ketamine and LY341495 were attenuated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- (9CI)-benzo[f]quinoxaline-7-sulfonamide, indicating AMPA-dependent effects. These findings provide another overlapping mechanism of action of ketamine and mGlu2/3 receptor antagonism that differentiates them from conventional antidepressants and thus support the potential rapidly acting antidepressant actions of mGlu2/3 receptor antagonism in patients.

Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia.

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

Chronic pre-treatment with nicotine enhances nicotine-evoked striatal dopamine release and alpha6 and beta3 nicotinic acetylcholine receptor subunit mRNA in the substantia nigra pars compacta of the rat.

Whilst local intrastriatal infusion of nicotine consistently elicits striatal dopamine release, systemic administration often fails to do so. Since chronic nicotine administration is known to result in desensitisation-induced upregulation of nicotinic acetylcholine receptors (nAChRs), the present study investigated whether chronic pre-treatment could enhance the response to systemic nicotine and, if so, whether increases in specific nAChR subunit mRNA levels in the substantia nigra pars compacta (SNc) may underlie this effect. In vivo microdialysis studies in male Sprague-Dawley rats revealed that following 4 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), local intrastriatal nicotine infusion (3 mM) elicited significantly higher dopamine efflux compared to vehicle pre-treated controls (peak release: 1273 +/- 199% basal versus 731 +/- 113% basal), whereas systemic nicotine challenge (0.8 mg kg(-1)s.c.) elicited no response. In contrast, following 8 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), systemic nicotine challenge (0.8 mg kg(-1)s.c.) now produced significantly higher dopamine efflux than that of vehicle pre-treated controls (147 +/- 30% basal versus 91 +/- 5% basal). Eight days pre-treatment with nicotine also significantly elevated the levels of alpha6 (approximately 55%) and beta3 (approximately 43%) nAChR subunit mRNA in the SNc, suggesting that up-regulation of these nAChR subunit genes in the nigrostriatal tract may contribute to the enhanced nicotine-evoked striatal dopamine release.

Behavioural and neurochemical responses evoked by repeated exposure to an elevated open platform.

Increased psychophysiological resistance to chronic stress has been related to increased 5-HT release in the dorsal hippocampus. This study investigated the changes in 5-HT release and turnover in the hippocampus evoked by acute and repeated exposure to an inescapable stressor, an elevated open platform, and compared them to the changes evoked in the frontal cortex. Repeated exposure to this stressor results in habituation of the plasma corticosterone response to the test, with full habituation being observed after 20 trials. Repeated exposure to the stressor for 5 or 10 occasions increased 5-HT turnover in the hippocampus. By contrast, 5-HT turnover in frontal cortex was increased by acute exposure to the stressor. Microdialysis studies showed that acute stress increased 5-HT overflow in prefrontal cortex but not dorsal hippocampus whereas repeated daily (10 days) exposure to the stressor increased basal extracellular 5-HT in the dorsal hippocampus, but not the prefrontal cortex. Prior exposure to the stressor on up to 10 occasions enhanced the plasma corticosterone response to a challenge in an elevated plus-maze performed 24h later whereas repeated, but not acute, exposure to the stressor, elicited anxiolytic-like behavioural responses in this test. It is concluded that acute exposure to this form of inescapable stress selectively stimulates the 5-HT projections to the frontal cortex; repeated stress elicits a sustained increase in 5-HT release and turnover in the hippocampus. The data are consistent with the hypothesis that increased 5-HT release in the hippocampus may be implicated in the mechanisms underlying habituation to inescapable stress.

Role of the locus coeruleus in the noradrenergic response to a systemic administration of nicotine.

Experiments were conducted using in vivo microdialysis to ascertain the role of nicotinic receptors in the terminal, or the cell body area, in the hippocampal noradrenaline response provoked by a systemic administration of nicotine. These experiments combined systemic administration of nicotine with local administration of antagonists into the hippocampus via the microdialysis probe, or close to the LC via a cannula, while continuously monitoring extracellular levels of NA in the hippocampus. Systemic administration of nicotine (0.4 mg/kg, s.c.) produced a rapid and prolonged increase in extracellular levels of noradrenaline in the hippocampus of conscious animals, reaching a maximum in the first 10 min sample. In anaesthetised animals the maximum occurred 20 min after administration, but the subsequent response profile was similar. In both anaesthetised and freely moving animals nicotine increased extracellular levels of dihydroxyphenylacetic acid and homovanillic acid in the hippocampus, but failed to alter levels of dopamine or 5-hydroxyindoleacetic acid. In anaesthetised animals intrahippocampal administration of nicotine (250 microM over 10 min via the dialysis probe) significantly increased extracellular levels of noradrenaline; the response was shortlasting, being evident only in the 10 min sample during exposure to the drug. Local administration of nicotine failed to alter extracellular levels of any other amine or metabolite measured. Mecamylamine (25 microM), a nicotinic channel blocker, administered intrahippocampally 10 min prior to an intrahippocampal administration of nicotine completely blocked the increase in noradrenaline. However, intrahippocampal administration of mecamylamine (25 microM) for 10 min, or for the duration of recording, failed to antagonise the effect of a systemic administration of nicotine (0.4 mg/kg, s.c.) on extracellular levels of noradrenaline, dihydroxyphenylacetic acid or homovanillic acid. In contrast administration of mecamylamine (50 microM) close to the locus coeruleus abolished the increase in noradrenaline levels in the ipsilateral hippocampus following a systemic administration of nicotine (0.4 mg/kg, s.c.), while trimethaphan (50 microM), a nicotine receptor antagonist, significantly reduced the response. Administration of mecamylamine also attenuated increases in dihydroxyphenylacetic acid and homovanillic acid, suggesting that the response of these metabolites may be associated with the functional metabolism of noradrenergic neurones. Locus coeruleus administration of kynurenic acid (1 mM), a non-specific excitatory amino acid antagonist, was without effect. Finally, application of nicotine (50 microM) close to the locus coeruleus significantly increased extracellular levels of noradrenaline in the ipsilateral hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of acute administration of nicotine on in vivo release of noradrenaline in the hippocampus of freely moving rats: a dose-response and antagonist study.

The effect of systemic administration of (-)-nicotine on release of noradrenaline in the hippocampus was studied by in vivo microdialysis in freely moving rats, using dialysate containing nomifensine (5 microM). (-)-Nicotine, at both 0.4 and 0.8 mg/kg but not 0.2 mg/kg, rapidly and significantly increased extracellular levels of noradrenaline. Extracellular levels of dopamine were also increased, but this was only significant after the larger dose. Both 0.4 and 0.8 mg/kg also produced a significant increase in extracellular levels of the metabolites of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Extracellular levels of the metabolite of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, increased after 0.8 mg/kg but this effect was only apparent much later. Injection of a second 0.8 mg/kg challenge of (-)-nicotine, 150 min after the first, produced similar increases in extracellular levels of noradrenaline, dopamine, 3-4-dihydroxyphenylacetic acid and homovanillic acid. Over the experimental period, there was no further increase in extracellular levels of 5-hydroxyindoleacetic acid. Increases in extracellular levels of noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in response to 0.8 mg/kg (-)-nicotine, were prevented by the systemic administration of mecamylamine, but not hexamethonium (both at 5 mg/kg). Mecamylamine also inhibited the delayed increase in extracellular levels of 5-hydroxyindoleacetic acid, produced by the first injection of (-)-nicotine. These results suggest that (-)-nicotine, dose-dependently stimulated the release and metabolism of amine transmitters by an action at central nicotinic receptors. However, the precise site of action, i.e. at nerve terminals, cell bodies or both, requires further elucidation.

Acute administration of nicotine increases the in vivo extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid and ascorbic acid preferentially in the nucleus accumbens of the rat: comparison with caudate-putamen.

Using in vivo dialysis and voltammetry, the effect of acute administration of (-)-nicotine (0.8 mg/kg free base, s.c.) on extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, 5-hydroxy-indoleacetic acid and ascorbic acid in the nucleus accumbens and caudate-putamen of chloral hydrate-anaesthetised rats has been examined. Nicotine stimulated release of dopamine only in the nucleus accumbens, measured using dialysis. After a short time delay levels of 3,4-dihydroxyphenylacetic acid in both the nucleus accumbens and caudate-putamen also increased. In both regions, 5-hydroxyindoleacetic acid was unaffected by nicotine. Using voltammetry the effect of nicotine on extracellular levels of 3,4-dihydroxyphenylacetic acid and ascorbic acid was examined. An increase in 3,4-dihydroxyphenylacetic acid was observed in both regions after nicotine. This increase was blocked by pretreatment with the central nicotinic receptor antagonist mecamylamine (5 mg/kg). Nicotine increased the level of ascorbic acid in the nucleus accumbens and caudate-putamen; while in animals pretreated with mecamylamine, nicotine decreased levels of ascorbate. These results show that acute administration of nicotine stimulated release of dopamine in the nucleus accumbens and increased the levels of DOPAC and ascorbic acid in the nucleus accumbens and caudate-putamen. This effect is probably mediated by nicotinic receptors as it was antagonised by mecamylamine.

Modulation of dopamine release in the nucleus accumbens by 5-HT1B agonists: involvement of the hippocampo-accumbens pathway.

Changes in extracellular levels of dopamine (DA), DA metabolites DOPAC and HVA, and the serotonin metabolite 5-HIAA, were measured by microdialysis in the rat nucleus accumbens (n. acc) after treatments with serotonin (5-HT)1A (8-OH-DPAT) or 5-HT1B (RU 24969 and S-CM-GTNH2) receptor agonists. Subcutaneous injections of RU 24969 (0.02-2 mg/kg) dose-dependently decreased 5-HIAA levels (0 to -38%), and also induced long-lasting increases in DA levels (0 to +37%) and DOPAC (+11% at the dose 0.5 mg/kg) in the shell of the n. acc, whereas 8-OH-DPAT (0.25 and 0.5 mg/kg) reduced 5-HIAA levels (-25%) and very slightly increased DOPAC at the lower dose (+4%), but had no effect on DA levels. Three weeks after interruption of the subicular efferent projections, the increase in DA levels previously observed after systemic injections of RU 24969 was abolished. Microinjections of RU 24969 (10 micrograms/microliter) or S-CM-GTNH2 (3 micrograms/microliter) into the ventral subicular area reproduced the effects of systemic injections of RU 24969 cn DA levels and increased DOPAC (+13%; +19%, respectively) and HVA levels (+23%; +24%), with no significant change in 5-HIAA. It is concluded that: (1) serotonin interacts with the mesolimbic dopaminergic system through 5-HT1B, but not 5-HT1A, receptors: and (2) serotonin interaction with the mesolimbic dopaminergic system involves postjunctional 5-HT1B heteroreceptors located in the ventral subicular area, which modulate the activity of glutamatergic hippocampo-accumbens pathways and only secondarily alter DA levels in the n. acc. The possible relevance of these results for schizophrenia is discussed.

Increases in tyrosine hydroxylase messenger RNA in the locus coeruleus after a single dose of nicotine are followed by time-dependent increases in enzyme activity and noradrenaline release.

We have utilized biochemical, molecular biological, and functional neurochemical measurements to investigate the integrated and long-term effects of a single dose of nicotine on the noradrenergic system in the central nervous system of the rat, from enzyme induction to transmitter release. We have found that a single systemic injection of nicotine (0.8 mg/kg) increases messenger RNA for the rate-limiting enzyme in the synthesis of catecholamines, tyrosine hydroxylase, two to six days later in the noradrenergic cell body region, the locus coeruleus (and not in the dopaminergic cell body regions, substantia nigra and ventral tegmental area). This was then followed by a time-dependent increase in enzyme activity, measured in vitro, in terminal regions of the ascending dorsal noradrenergic bundle up to four weeks later. Functionally, the increase in tyrosine hydroxylase activity in the terminals four weeks after a single administration was associated with an increase in the capacity to release noradrenaline in the hippocampus, measured using in vivo microdialysis in freely moving animals. This occurred in response to an acute systemic nicotine injection (0.4 mg/kg) but not to a local, intrahippocampal, challenge with 250 microM nicotine. These experiments have revealed a long-term effect of nicotine on noradrenergic activity in the central nervous system, associated with induction of tyrosine hydroxylase. This is accompanied by a time-dependent increase in terminal tyrosine hydroxylase activity and an increase in noradrenaline release.

Chronic nicotine administration increases tyrosine hydroxylase selectivity in the rat hippocampus.

Acute nicotine treatment increases catecholamine synthesis selectively in the nucleus accumbens, hypothalamus and hippocampus of the rat. In the hippocampus, but not in the other areas mentioned, or in the caudate, chronic pretreatment with nicotine for 28 days potentiates this response. In the present study we have investigated the effects of acute treatment with the same dose of nicotine (0.8 mg/kg s.c.), and of chronic pretreatment with this dose, on the in vitro activity of tyrosine hydroxylase, the rate limiting enzyme for catecholamine synthesis. Tyrosine hydroxylase was not altered by acute nicotine, but was increased by about 2-fold, selectively in the hippocampus, by the chronic pretreatment. These results show that nicotine treatment increases the capacity for noradrenaline synthesis in the hippocampus; taken with our previous results they indicate that this capacity is not manifest until an acute challenge occurs. This effect of nicotine on tyrosine hydroxylase is consistent with nicotinic receptor mediation of central transsynaptic induction.

The effects of cholinergic drugs and cholinergic-rich foetal neural transplants on alcohol-induced deficits in radial maze performance in rats.

Chronic alcohol (20% v/v in drinking water for 28 weeks) impaired acquisition of radial maze spatial and associative tasks by increasing both within-trial working and long-term reference memory errors; animals with high (above the median of 100 mg/100 ml) blood alcohol concentrations (BACs) during treatment were significantly more impaired than those with BACs below the median. Alcohol-treated rats showed improvements in radial maze performance after treatment with cholinergic agonists (arecoline and nicotine) and disruption with antagonists (scopolamine and mecamylamine) at low doses which did not affect controls. These effects were more pronounced for working than reference memory, and not manifest with the peripherally acting antagonists hexamethonium and N-methylscopolamine. Transplants into cortex and hippocampus of cholinergic-rich basal forebrain (BF) and ventral mesencephalon (VM) foetal neural tissue improved radial maze performance of alcohol-treated rats to control level over a period of 9-12 weeks after grafting. Cholinergic-poor foetal hippocampal (HC) grafts were without effect. BF and VM, but not HC, grafts showed dense acetylcholinesterase (AChE) staining, tyrosine-hydroxylase staining was most pronounced in VM sections and dopamine-beta-hydroxylase staining was minimal in all grafts. Choline acetyltransferase (ChAT) activity was significantly reduced in cortex and hippocampus of alcohol-treated rats, except those given cholinergic-rich transplants. Alcohol treatment also significantly reduced AChE-positive cell counts in the nucleus basalis, medial septal and diagonal band brain areas, at the sources of the forebrain cholinergic projection system (FCPS). Cortical levels of noradrenaline were significantly reduced in all alcohol-treated rats, regardless of transplant, whereas cortical dopamine content was significantly elevated in all rats receiving transplants, regardless of behavioural effect, but not in alcohol-treated controls. Forebrain serotonin levels were not significantly altered by grafting or alcohol treatment. These results suggest that damage to the FCPS, as shown by reduced ChAT activity in target areas, and reduced AChE cell counts in projection areas, played an important part in the radial maze deficits displayed by alcohol-treated rats, since these animals were sensitive to cholinergic drug challenge, and cholinergic-rich transplants from two different sites in foetal brain elevated ChAT activity and restored cognitive function. In contrast alcohol- or graft-induced alterations in other transmitter systems did not correlate with the pattern of behavioural deficit and recovery.

LY393558, a 5-hydroxytryptamine reuptake inhibitor and 5-HT(1B/1D) receptor antagonist: effects on extracellular levels of 5-hydroxytryptamine in the guinea pig and rat.

The stimulation of terminal 5-HT(1B/1D) autoreceptors limits the effects of selective serotonin reuptake inhibitors on extracellular levels of 5-hydroxytryptamine (5-HT, serotonin) in vivo. Microdialysis studies show that acute oral administration of LY393558-a 5-HT reuptake inhibitor and antagonist at both the human 5-HT(1B) and 5-HT(1D) receptor-in the dose range 1-20 mg/kg, increases extracellular levels of 5-HT in both the guinea pig hypothalamus and rat frontal cortex. In both species, the levels of 5-HT that were attained were higher than following an acute, maximally effective dose of fluoxetine (20 mg/kg orally), reaching approximately 1500% in the guinea pig hypothalamus and 700% in the rat frontal cortex. In both species, the response to LY393558 (10 mg/kg p.o.) was impulse dependent, being absent in the presence of tetrodotoxin delivered at 1 microM via the microdialysis probe. The sensitivity to tetrodotoxin contrasted with the effects seen with DL-fenfluramine. Studies in rats showed that the microdialysate 5-HT concentration achieved in the frontal cortex after an acute challenge with LY393558 (5 mg/kg p.o.) was significantly greater than following a chronic regime of fluoxetine treatment (10 mg/kg/day orally for 21 days). Moreover, in rats chronically treated with LY393558 (5 mg/kg/day orally for 21 days), the mean basal concentration, 24 h after the final pretreatment dose, was of the same magnitude as that following chronic fluoxetine. However, in contrast to the response seen in fluoxetine-pretreated animals, a challenge dose of LY393558 still elicited a further increase in extracellular 5-HT in LY393558-pretreated animals. LY393558 is a potent 5-HT reuptake inhibitor and 5-HT(1B/1D) receptor antagonist. Microdialysis studies show that acute oral administration increases extracellular levels of 5-HT, by an impulse-dependent mechanism, above those produced by a maximally effective dose of fluoxetine, and in rats to levels only achieved following chronic fluoxetine treatment. Its neurochemical profile in vivo suggests that it may be a more effective antidepressant with the potential for producing an earlier onset of clinical activity than selective serotonin reuptake inhibitors.

Ventral subiculum administration of the somatostatin receptor agonist MK-678 increases dopamine levels in the nucleus accumbens.

Somatostatin (or somatotropin-release inhibitory factor, SRIF) binding and in situ hybridisation studies have indicated a high expression of receptor subtypes throughout the rat brain and, in particular, in subregions of the hippocampus and subiculum. In vitro, somatostatin and related peptides, including seglitide (MK-678), hyperpolarize subicular neurones of the burst firing type-a response, which may have functional consequences for their output. One major projection from the subiculum is to the nucleus accumbens. The functional consequence of somatostatin receptor stimulation in the ventral subiculum has been assessed by measuring extracellular levels of dopamine in the ipsilateral nucleus accumbens. In anaesthetised rats, administration of seglitide (MK-678), a somatostatin analogue with selectivity for the SRIF-1 receptor (comprising somatostatin sst2, sst3 and sst5 subtypes) significantly increased extracellular levels of dopamine in the ipsilateral nucleus accumbens shell. The result suggests that hyperpolarization of subicular neurones by MK-678 may lead to activation of the subiculo-accumbens projection system, and an associated increase in dopaminergic function.

Activation of the retrohippocampal region in the rat causes dopamine release in the nucleus accumbens: disruption by fornix section.

There is a well-described projection from the retrohippocampus (subiculum and entorhinal cortex) to the nucleus accumbens that is involved in the control of psychomotor behaviour, and is implicated in the aetiology of schizophrenia. Cortical abnormalities are widely reported in the brains of schizophrenic patients, but it is unclear whether they are the cause or consequence of those changes in subcortical systems that are treated with neuroleptic drugs. We have, therefore, conducted a series of microdialysis experiments in anaesthetized rats to determine whether infusion of the excitotoxin, N-methyl-D-aspartate, into the retrohippocampus increases mesolimbic dopamine release. We found a clear and reproducible increase in extracellular dopamine in the nucleus accumbens following N-methyl-D-aspartate (2.5 microg), that was abolished when we sectioned the fimbria-fornix. Furthermore, inhibition of gamma-aminobutyric acid receptors following retrohippocampus administration of bicuculline (4 microg), also increased dopamine in the nucleus accumbens. The dopamine response to bicuculline was accompanied by an increase in dopamine metabolism, was long lasting, and also reduced by fornix section.The response to both N-methyl-D-aspartate and bicuculline depends on the integrity of the projection from the retrohippocampus to the nucleus accumbens. The results provide an underlying mechanism whereby a primary insult in the temporal cortex, caused by excessive N-methyl-D-aspartate receptor stimulation, can produce a hyperdopaminergic state. In addition, an increase in subiculo-accumbens activity evoked by bicuculline may also explain why patients with limbic epilepsy can develop a psychosis.

Effect of chronic ritanserin or clorgyline on amine and metabolite levels in rat frontal cortex.

As part of an investigation of ritanserin-induced receptor down-regulation, monoamine and metabolite levels in rat frontal cortex were measured following chronic ritanserin (2 mg/kg per day) or clorgyline (10 mg/kg per day) administration. Clorgyline increased 5-hydroxytryptamine (5-HT) by 83%, noradrenaline (NA) by 54%, and dopamine (DA) by 16% and decreased 5-hydroxyindoleacetic acid (5-HIAA) by 28%, homovanillic acid (HVA) by 57% and 3,4-dihydroxyphenylacetic acid (DOPAC) by 67%. All these changes were statistically significant (P less than 0.001) except for the increase in DA. Ritanserin increased 5-HT by 30%, NA by 33% and DA by 26% and decreased 5-HIAA by 22%, HVA by 23% and DOPAC by 40%; however, only the increases in 5-HT and NA reached statistical significance (P less than 0.05). Monoamine oxidase (MAO) activity in cortical homogenates was also measured following the chronic ritanserin and clorgyline regimens and also following ritanserin administration in vitro. Chronic clorgyline and ritanserin inhibited MAO activity by 60 and 39%, respectively. In vitro, ritanserin administration at concentrations of less than 10(-6) M had no effect on MAO activity but at doses higher than 10(-6) M, MAO activity was inhibited in a dose-dependent manner from 18 +/- 0.5% at 3 x 10(-6) M to 63 +/- 9% at 10(-4) M. Thus, ritanserin appears to act as an MAO inhibitor in addition to being a 5-HT2 antagonist and this may be related to its ability to induce 5-HT2 receptor down-regulation.

Regionally specific effects of acute and chronic nicotine on rates of catecholamine and 5-hydroxytryptamine synthesis in rat brain.

Acute (-)-nicotine administration (0.4 and 0.8 mg/kg s.c.) produced a regionally specific increase in the rate of catecholamine synthesis in the rat nucleus accumbens, hypothalamus and hippocampus but not elsewhere, including the caudate-putamen. In all regions rates of 5-hydroxytryptamine synthesis were unaffected. (-)-Cotinine (0.4 and 0.8 mg/kg), the major metabolite of (-)-nicotine was without effect. (-)-Nicotine-induced increase in catecholamine synthesis occurred by a direct stimulation of central nicotinic receptors, as mecamylamine (5 mg/kg) but not hexamethonium (5 mg/kg) was an effective antagonist. Following repeated daily injections of (-)-nicotine (0.8 mg/kg) for up to 28 days, the induced catecholamine response following a subsequent challenge was unaffected in the nucleus accumbens and hypothalamus, but was increased in the hippocampus. This effect persisted for up to 14 days following withdrawal. Rates of 5-hydroxytryptamine synthesis remained unaltered after chronic pretreatment.

Nicotine-induced catecholamine synthesis after lesions to the dorsal or ventral noradrenergic bundle.

The role of projections from coerulear (A6) and lateral tegmental (A1-A5) noradrenergic cell groups in the induced catecholamine response to (-)-nicotine was studied following lesions to the dorsal (DNAB) or ventral (VNAB) noradrenergic bundle by 6-hydroxydopamine. The lesions produced large reductions in basal noradrenaline levels in hippocampus (after DNAB lesions) and hypothalamus (after VNAB lesions), while not affecting basal levels of dopamine or 5-hydroxytryptamine. Vehicle and sham operated controls showed a significant increase in DOPA accumulation in response to (-)-nicotine (0.8 mg/kg s.c.) following inhibition of amino acid decarboxylase. In DNAB lesioned rats, the response induced by (-)-nicotine in both the hippocampus and hypothalamus was significantly attenuated, whereas in VNAB lesioned rats the induced response was still evident. The effect of (-)-nicotine was also studied in the nucleus accumbens and was found not to be affected by either lesion. These data suggest that increases in catecholamine synthesis in the hippocampus and hypothalamus reflect increased noradrenaline synthesis, and that this effect occurs specifically in noradrenergic projections originating in the locus coeruleus.

Calcium dependence of sensitised dopamine release in rat nucleus accumbens following amphetamine challenge: implications for the disruption of latent inhibition.

Repeated amphetamine treatment results in sensitisation both of its behavioural effects, and of its dopamine (DA)-releasing effects on which the former largely depend. Understanding the nature of the sensitised response may help to explain behaviours which emerge only with repeated treatment, such as particular stereotypies and effects on social behaviour in animals, and links between these effects and the emergence of dependence and psychotic symptoms in humans. We show here that a single pretreatment with amphetamine (1mg/kg) is sufficient to sensitise the locomotor response to amphetamine challenge (1mg/kg) 24h later. We have used in vivo microdialysis in the nucleus accumbens in unrestrained rats to demonstrate a corresponding potentiation in the DA response; the marked increase in accumbens dialysate DA following amphetamine (to 427% of basal) was significantly potentiated (to 675% of basal) by the pretreatment, without any alteration in the basal DA. There was also no change in the expected reduction in DA metabolites. Replacement of perfusate calcium by magnesium left the response to acute amphetamine challenge substantially unaffected, as expected from previous reports; however, the potentiation of the DA response by amphetamine pretreatment was prevented. Similarly the potentiated response was attenuated by administration of ondansetron, a 5HT-3 antagonist, (0.01mg/kg) before each amphetamine treatment. The ability of amphetamine to disrupt latent inhibition (L1), which is also disrupted in acute schizophrenia, has been suggested to provide a model of schizophrenia linking underlying cognitive deficits with the DA theory of the disorder. Since LI is disrupted by two systemic administrations of amphetamine 24h apart, but not by one, the present results are consistent with the concept that it is the calcium, and hence impulse, dependence of increased accumbal DA release, rather than its magnitude, which is critical for the disruption of LI.

Dopamine release in the nucleus accumbens and latent inhibition in the rat following microinjections of a 5-HT1B agonist into the dorsal subiculum: implications for schizophrenia.

Microinjection of a serotonergic 5-HT1B agonist (S-CM-GTNH2, 3 microg/l) into the dorsal subiculum (DS) induced long-lasting increases in dopamine (DA; +58%), dihydroxyphenylacetic acid (DOPAC; +15%) and homovanillic acid (HVA; +31%), without changing extracellular levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), measured by microdialysis in freely moving rats in the shell area of the nucleus accumbens (n. acc). Perfusion of a glutamate-N-methyl-D-aspartate (NMDA) receptor antagonist (MK 801, dizocilpine, 10 microM) through the dialysis probe in the n. acc induced similar long-lasting increases in DA and DOPAC, whereas the glutamate-quisqualate/kainate receptor antagonist (CNQX, 50 microM) had no effect. In the presence of dizocilpine in the n. acc, microinjection of S-CM-GTNH2 into the DS could still increase DOPAC and HVA, but DA levels were not further changed, whereas in the presence of CNQX, microinjection of S-CM-GTNH2 into the DS still increased not only DOPAC and HVA, but also DA levels in a way similar to that in the absence of glutamate antagonist. Therefore, activation of 5-HT1B receptors located in the DS increases the release of DA in the n. acc, presumably via the glutamatergic projection to this structure and acting through NMDA receptors in it. This implies either the suppression of a tonic indirect inhibitory influence and/or stimulation of a phasic excitatory effect of glutamate. Disruption of latent inhibition (LI) has been suggested as a model for a cognitive deficit in schizophrenia (hyperattention to irrelevant stimuli) and is usually associated with an increase in DA release in the n. acc. However, s.c. injection of RU 24 969 (0.5 mg/kg), a mixed 5-HT1A-5-HT1B agonist, which was previously shown to increase DA release in the n. acc, left LI unchanged. Moreover, bilateral microinjections of S-CM-GTNH2 into the rat DS tended to potentiate LI, in spite of the increase in DA in n. acc demonstrated here. It is concluded that not all increases in DA release in the n. acc are functionally equivalent. Sensitization of receptors or impulse-dependent increase in DA release might be necessary to disrupt LI. The possible role of altered serotonergic transmission, through h5-HT1B receptors (human homologue of the rat 5-HT1B receptors) located in the DS, in acute schizophrenia needs to be further investigated.