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Aims: Bromocriptine is thought to facilitate left ventricular (LV) recovery in peripartum cardiomyopathy (PPCM) through inhibition of prolactin secretion. However, this potential therapeutic effect remains controversial and was incompletely studied in diverse populations., Methods and Results: Consecutive women with new-onset PPCM (n = 76) between 1994 and 2015 in Quebec, Canada, were classified according to treatment (n = 8, 11%) vs. no treatment (n = 68, 89%) with bromocriptine. We assessed LV functional recovery at mid-term (6 months) and long-term (last follow-up) and compared outcomes among groups. Women treated with bromocriptine experienced better mid-term left ventricular ejection fraction (LVEF) recovery from 23 ± 10% at baseline to 55 ± 12% at 6 months, compared with a change from 30 ± 12% at baseline to 45 ± 13% at 6 months in women treated with standard medical therapy (P interaction < 0.01). At long-term, a similar positive association was found with bromocriptine (9% greater LVEF variation, P interaction < 0.01). In linear regressions adjusted for obstetrical, clinical, echocardiographic, and pharmacological variables, treatment with bromocriptine was associated with a greater improvement in LVEF [β coefficient (standard error), 14.1 (4.4); P = 0.03]. However, there was no significant association between bromocriptine use and the combined occurrence of all-cause death and heart failure events (hazard ratio, 1.18; 95% confidence interval, 0.15 to 9.31), using univariable Cox regressions based over a cumulative follow-up period of 285 patient-years., Conclusions: In women newly diagnosed with PPCM, treatment with bromocriptine was independently associated with greater LV functional recovery., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

A subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT- and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression.

The serotonin (5-HT) hypothesis of depression dates from the 1960s. It originally postulated that a deficit in brain serotonin, corrected by antidepressant drugs, was the origin of the illness. Nowadays, it is generally accepted that recurring mood disorders are brain diseases resulting from the combination, to various degrees, of genetic and other biological as well as environmental factors, evolving through the lifespan. All areas of neuroscience, from genes to behaviour, molecules to mind, and experimental to clinical, are actively engaged in attempts at elucidating the pathophysiology of depression and the mechanisms underlying the efficacy of antidepressant treatments. This first of two special issues of Philosophical Transactions B seeks to provide an overview of current developments in the field, with an emphasis on cellular and molecular mechanisms, and how their unravelling opens new perspectives for future research.

Serotonin (5-HT) 5-HT(1A) autoreceptors (5-HT(1A)autoR) and the plasmalemmal 5-HT transporter (SERT) are key elements in the regulation of central 5-HT function and its responsiveness to antidepressant drugs. Previous immuno-electron microscopic studies in rats have demonstrated an internalization of 5-HT(1A)autoR upon acute administration of the selective agonist 8-OH-DPAT or the selective serotonin reuptake inhibitor antidepressant fluoxetine. Interestingly, it was subsequently shown in cats as well as in humans that this internalization is detectable by positron emission tomography (PET) imaging with the 5-HT(1A) radioligand [(18)F]MPPF. Further immunocytochemical studies also revealed that, after chronic fluoxetine treatment, the 5-HT(1A)autoR, although present in normal density on the plasma membrane of 5-HT cell bodies and dendrites, do not internalize when challenged with 8-OH-DPAT. Resensitization requires several weeks after discontinuation of the chronic fluoxetine treatment. In contrast, the SERT internalizes in both the cell bodies and axon terminals of 5-HT neurons after chronic but not acute fluoxetine treatment. Moreover, the total amount of SERT immunoreactivity is then reduced, suggesting that SERT is not only internalized, but also degraded in the course of the treatment. Ongoing and future investigations prompted by these finding are briefly outlined by way of conclusion.

Melatonin activates two brain G-protein coupled receptors, MT(1) and MT(2), whose differential roles in the sleep-wake cycle remain to be defined. The novel MT(2) receptor partial agonist, N-{2-[(3-methoxyphenyl) phenylamino] ethyl} acetamide (UCM765), is here shown to selectively promote non-rapid eye movement sleep (NREMS) in rats and mice. The enhancement of NREMS by UCM765 is nullified by the pharmacological blockade or genetic deletion of MT(2) receptors. MT(2), but not MT(1), knock-out mice show a decrease in NREMS compared to the wild strain. Immunohistochemical labeling reveals that MT(2) receptors are localized in sleep-related brain regions, and notably the reticular thalamic nucleus (Rt). Microinfusion of UCM765 in the Rt promotes NREMS, and its systemic administration induces an increase in firing and rhythmic burst activity of Rt neurons, which is blocked by the MT(2) antagonist 4-phenyl-2-propionamidotetralin. Since developing hypnotics that increase NREMS without altering sleep architecture remains a medical challenge, MT(2) receptors may represent a novel target for the treatment of sleep disorders.

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.

Cholinergic interneurons are the only known source of acetylcholine in the rat nucleus accumbens (nAcb); yet there is little anatomical data about their mode of innervation and the origin of their excitatory drive. We characterized the cholinergic and thalamic innervations of nAcb with choline acetyltransferase (ChAT) immunocytochemistry and anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L) from the midline/intralaminar/paraventricular thalamic nuclei. The use of a monoclonal ChAT antiserum against whole rat ChAT protein allowed for an optimal visualization of the small dendritic branches and fine varicose axons of cholinergic interneurons. PHA-L-labeled thalamic afferents were heterogeneously distributed throughout the core and shell regions of nAcb, overlapping regionally with cholinergic somata and dendrites. At the ultrastructural level, several hundred single-section profiles of PHA-L and ChAT-labeled axon terminals were analyzed for morphology, synaptic frequency, and the nature of their synaptic targets. The cholinergic profiles were small and apposed to various neuronal elements, but rarely exhibited a synaptic membrane specialization (5% in single ultrathin sections). Stereological extrapolation indicated that less than 15% of these cholinergic varicosities were synaptic. The PHA-L-labeled profiles were comparatively large and often synaptic (37% in single ultrathin sections), making asymmetrical contacts primarily with dendritic spines (>90%). Stereological extrapolation indicated that all PHA-L-labeled terminals were synaptic. In double-labeled material, some PHA-L-labeled terminals were directly apposed to ChAT-labeled somata or dendrites, but synapses were never seen between the two types of elements. These observations demonstrate that the cholinergic innervation of rat nAcb is largely asynaptic. They confirm that the afferents from midline/intralaminar/paraventricular thalamic nuclei to rat nAcb synapse mostly on dendritic spines, presumably of medium spiny neurons, and suggest that the excitatory drive of nAcb cholinergic interneurons from thalamus is indirect, either via substance P release from recurrent collaterals of medium spiny neurons and/or by extrasynaptic diffusion of glutamate.

Because 5-HT1A receptors located on the soma dendrites of serotonin (5-HT) neurons normally mediate an inhibition of 5-HT firing and release, the desensitization of these autoreceptors is essential for obtaining an enhancement of 5-HT transmission after treatment with 5-HT reuptake inhibitors (SSRIs). We have demonstrated previously, using immunoelectron microscopy with specific 5-HT1A antibodies, that an internalization of 5-HT1A autoreceptors is associated with their desensitization in rats given a single dose of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Here, we examined the subcellular distribution of 5-HT1A receptors in dendrites from nucleus raphe dorsalis (NRD) (autoreceptors) and hippocampus (heteroreceptors) after acute treatment with the antidepressant SSRI, fluoxetine (10 mg/kg, i.p.). In parallel experiments, the kinetics of in vivo binding of the 5-HT1A positron emission tomography radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine ([18F]MPPF) was measured in these two brain regions by means of stereotaxically implanted beta microprobes. One hour after treatment, there was a 36% decrease in 5-HT1A immunogold labeling of the plasma membrane of NRD dendrites, and a concomitant increase in their cytoplasmic labeling, without any change in hippocampal dendrites. In vivo binding of [18F]MPPF was reduced by 35% in NRD and unchanged in hippocampus. Both effects were blocked by pretreatment with the 5-HT1A receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide) (1 mg/kg, i.p.). In brain sections of NRD and hippocampus, [18F]MPPF autoradiographic labeling did not differ between fluoxetine- and saline-treated rats. These immunocytochemical results confirmed that internalization of 5-HT1A autoreceptors may account for their desensitization, and the microprobe results suggest that this prerequisite for antidepressant treatment efficacy could be amenable to brain imaging in humans.

Purpose: We previously demonstrated that the anticholinesterase eserine provokes interictal-like discharges in the CA3 area of hippocampal slices from rats in which generalized seizures had been induced by pentylenetetrazol (PTZ) when immature. In this study, we investigated several factors as the possible mechanism for this effect, including age at convulsions., Methods: Rats were injected with PTZ on postnatal day (P) 18-20 or >P60, and neuronal activity was recorded intra- and extracellularly from CA3 5-10 or >40 days later. In additional experiments, convulsions were triggered by kainate or were blocked by pentobarbital. Hippocampal (a) acetylcholine (ACh) innervation density was measured by immunocytochemistry, and ACh and gamma-aminobutyric acid (GABA) contents were determined by high-performance liquid chromatography (HPLC)-electrospray ionization., Results: The excitatory effect of eserine was the most consistent in slices from rats PTZ-treated when immature and after the long interval, whereas the reverse was true in rats treated as adults. This effect was dependent on the occurrence of a seizure and was less prevalent when the seizure had been provoked by kainate. Adult animals PTZ-treated at P20 did not differ from control in (a) poly- or monosynaptic GABAA and GABAB CA3 inhibitory postsynaptic potentials (IPSPs); (b) density of ACh innervation; or (c) tissue content of ACh and GABA., Conclusions: A PTZ-induced generalized seizure in immature rat provokes endogenous ACh-induced interictal-like discharges in adult hippocampal CA3. This effect is only transiently observed if the seizure was induced in adult. It does not appear to be related to a change in GABAergic inhibition, in density of ACh innervation, or in ACh or GABA content.

Serotonin-1A (5-HT(1A)) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT(1A) agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT(1A) receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT(1A) autoreceptors but not heteroreceptors after treatment with 5-HT(1A) receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization.

Unlabelled: Radiolabeled ligands of monoamine transporters have already been used to visualize cerebral monoamine innervation by tissue autoradiography and by PET or SPECT in vivo., Methods: A sampling technique was developed to allow for both the autoradiographic counting of serotonin (5-HT) axonal varicosities, labeled by uptake and storage of [3H]5-HT, and the measurement of the binding of [3H]cyanoimipramine ([3H]CYI), a specific 5-HT transporter ligand, in adjacent slices of adult rat neostriatum. The experiments were conducted in normal, decreased (after 5,7-dihydroxytryptamine lesions in adults) or increased (after 6-hydroxydopamine lesions in neonates) states of neostriatal 5-HT innervation., Results: In normal tissue, the regional density of [3H]CYI binding faithfully reproduced rostrocaudal variations in the number of [3H]5-HT-labeled axonal varicosities. Pairs of values from all three experimental groups showed a highly significant linear correlation (r = 0.93) between the density of [3H]CYI binding and the number of 5-HT varicosities per cubic millimeter of tissue. The intercept of the regression line was close to zero; this confirmed the selectivity of the ligand., Conclusion: Under drug-free conditions, specific [3H]CYI binding is a good quantitative index of 5-HT innervation density in brain tissue and is not significantly up- or downregulated on 5-HT denervation or hyperinnervation. When it is adequately labeled, such a ligand might therefore be appropriate to quantify regional 5-HT innervation in vivo by PET or SPECT. The present approach should also be useful to select ligands to quantify 5-HT and monoamine systems.

Catecholaminergic neurons, which take up and retain exogenous norepinephrine labeled with tritium, were studied by means of high resolution radioautography, in the substantia nigra, the substantia grisea periventricularis, and the locus coeruleus of the rat. Under the conditions required for the radioautographic detection of exogenous norepinephrine-(3)H, it was established that (1) glutaraldehyde was the most suitable fixative for preserving the labeled amine in situ; (2) norepinephrine-(3)H itself, rather than metabolites, accounted for most of the reactions detected in catecholaminergic neurons. At various time intervals after an intraventricular injection of norepinephrine-(3)H, the tracer reached a concentration 15-100 times higher, and disappeared at a slower rate, in presynaptic axons (t(1/2):4 hr) than in nerve cell bodies (t(1/2):0.8-1.3 hr). After pretreatment with a monoamine oxidase inhibitor, the radioautographic reactions increased and persisted longer, especially in the preterminal axons. Within neurons, the labeled amine was ubiquitously distributed in the nerve cell body and concentrated in presynaptic axons and synaptic terminals of various morphological types. Although large granular vesicles were usually present in the labeled axonal bulbs, no structural characteristic could be specifically ascribed to catecholaminergic neurons. It is suggested that exogenous norepinephrine bound to macromolecular complexes is present in all parts of catecholaminergic neurons and mainly concentrated within presynaptic axons.