Eisuke Haneda

Reversal of hippocampal neuronal maturation by serotonergic antidepressants

Serotonergic antidepressant drugs have been commonly used to treat mood and anxiety disorders, and increasing evidence suggests potential use of these drugs beyond current antidepressant therapeutics. Facilitation of adult neurogenesis in the hippocampal dentate gyrus has been suggested to be a candidate mechanism of action of antidepressant drugs, but this mechanism may be only one of the broad effects of antidepressants. Here we show a distinct unique action of the serotonergic antidepressant fluoxetine in transforming the phenotype of mature dentate granule cells. Chronic treatments of adult mice with fluoxetine strongly reduced expression of the mature granule cell marker calbindin. The fluoxetine treatment induced active somatic membrane properties resembling immature granule cells and markedly reduced synaptic facilitation that characterizes the mature dentate-to-CA3 signal transmission. These changes cannot be explained simply by an increase in newly generated immature neurons, but best characterized as “dematuration” of mature granule cells. This granule cell dematuration developed along with increases in the efficacy of serotonin in 5-HT4 receptor-dependent neuromodulation and was attenuated in mice lacking the 5-HT4 receptor. Our results suggest that serotonergic antidepressants can reverse the established state of neuronal maturation in the adult hippocampus, and up-regulation of 5-HT4 receptor-mediated signaling may play a critical role in this distinct action of antidepressants. Such reversal of neuronal maturation could affect proper functioning of the mature hippocampal circuit, but may also cause some beneficial effects by reinstating neuronal functions that are lost during development.

Phase-dependent roles of reactive microglia and astrocytes in nervous system injury as delineated by imaging of peripheral benzodiazepine receptor.

Elevated levels of peripheral benzodiazepine receptor (PBR) in glia have been documented in diverse nervous system injuries, while the identity and spatiotemporal characteristics of the cells showing upregulation of PBR remain elusive. We examined the astrocytic and microglial expressions of PBR in rat brains during the duration of ethanol-induced neuronal insults in order to clarify the significance of PBR as a biomarker capable of detecting a distinctive subpopulation of these glial cells involved in the impairment and protection of neurons. The levels of PBR, as determined by autoradiographic analysis using a specific radioligand, [11C]DAA1106, began to significantly increase at 3 days after intrastriatal injection of ethanol, and peaked at 7 days. This was consistent with the results of double immunofluorescence staining and high-resolution emulsion autoradiography, which revealed upregulation of PBR in both microglia and astrocytes proliferating in nonoverlapping compartments of the injury site. Notably, increased expression of PBR in astrocytes was transiently observed in a manner parallel to the centripetal migration of these cells to the inflammatory lesion, which may be a response indispensable to the protection of intact tissue. Thereafter, astrocytic PBR was barely detectable, despite the presence of numerous glial fibrillary acidic protein-immunoreactive astrocytes forming glial scarring. By contrast, intense PBR signals were persistently present in microglia localized to the injury epicenter up to 90 days, notwithstanding a gradual reduction in the number of ionized calcium binding adapter molecule-1-positive amoeboid microglia between 7 and 90 days. The long-lasting PBR expression in microglia was finally supported by in vivo positron emission tomography imaging, and suggests that inflammatory tissue damage is potentially expandable unless it is tightly sealed by astrocytic scar. The present findings collectively support the utility of PBR in identifying a unique temporal pattern of astrocytic and microglial activation that conventional glial markers hardly pursue.

Chronic Fluoxetine Bidirectionally Modulates Potentiating Effects of Serotonin on the Hippocampal Mossy Fiber Synaptic Transmission

Selective serotonin reuptake inhibitors (SSRIs) have been used to treat various psychiatric disorders. Although the cellular mechanisms underlying amelioration of particular symptoms are mostly unknown, recent studies have shown critical importance of the dentate gyrus of the hippocampus in behavioral effects of SSRIs in rodents. Here, we show that serotonin potentiates synaptic transmission between mossy fibers, the sole output of the dentate granule cells, and CA3 pyramidal cells in mouse hippocampal slices. This potentiation is mediated by activation of 5-HT4 receptors and intracellular cAMP elevation. A chronic treatment of mice with fluoxetine, a widely used SSRI, bidirectionally modulates the 5-HT-induced potentiation: Fluoxetine enhances the potentiation induced by lower concentrations of serotonin, while attenuates that by the higher concentration, which represents stabilization of synaptic 5-HT action. In contrast to the chronic treatment, an acute application of fluoxetine in slices induces a leftward shift in the dose–response curve of the 5-HT-induced potentiation. Thus, acute and chronic fluoxetine treatments have distinct effects on the serotonergic modulation of the mossy fiber synaptic transmission. Exposure of mice to novel environments induces increases in locomotor activity and hippocampal extracellular 5-HT levels. In mice chronically treated with fluoxetine, the novelty-induced hyperactivity is reduced without significant alterations in home cage activity and motor skills. Our results suggest that the chronic fluoxetine treatment can stabilize the serotonergic modulation of the central synaptic transmission, which may contribute to attenuation of hyperactive behaviors.

Chronic Fluoxetine Selectively Upregulates Dopamine D1-Like Receptors in the Hippocampus

The dentate gyrus of the hippocampus has been implicated in mechanisms of action of selective serotonin reuptake inhibitors (SSRIs). We have recently demonstrated that the SSRI fluoxetine can reverse the state of maturation of the adult dentate granule cells and enhances serotonin 5-HT4 receptor-mediated synaptic potentiation at the synapses formed by their mossy fiber axons. Here, we show that fluoxetine can induce long-lasting enhancement of dopaminergic modulation at the mossy fiber synapse. Synaptic responses arising from the mossy fiber-CA3 pyramidal cell synapse were recorded using acute mouse hippocampal slices. Dopamine potentiates mossy fiber synaptic transmission by activating D1-like receptors. Chronic fluoxetine treatment induced a prominent increase in the magnitude of dopamine-induced synaptic potentiation, and this effect was maintained at least up to 1 month after withdrawal of fluoxetine. Quantitative autoradiography revealed that binding of the D1-like receptor ligand [3H]SCH23390 was selectively increased in the dentate gyrus and along the mossy fiber in fluoxetine-treated mice. However, binding of the 5-HT4 receptor ligand [3H]GR113808 was not significantly changed. These results suggest that chronic fluoxetine enhanced the dopaminergic modulation at least in part by upregulating expression of D1-like receptors, while the enhanced serotonergic modulation may be mediated by modifications of downstream signaling pathways. These enhanced monoaminergic modulations would greatly increase excitatory drive to the hippocampal circuit through the dentate gyrus. The highly localized upregulation of D1-like receptors further supports the importance of the dentate gyrus in the mechanism of action of SSRIs.

Quantification of central substance P receptor occupancy by aprepitant using small animal positron emission tomography.

Background: Central substance P receptors, termed NK-1 receptors, have been considered as therapeutic targets in the development of drugs against diverse conditions, including emesis, overactive bladder, and depression. Methods: Here, we applied small animal positron emission tomography (PET) and a radioligand for NK-1 receptors ([18F]FE-SPA-RQ) for measuring occupancies of these receptors by a selective antagonist (aprepitant) in order to examine the validity of this in vivo imaging system for preclinical characterization of candidate agents acting on NK-1 receptors, and as a tool for predicting optimal doses in humans. Results: PET in gerbils depicted high uptake in the striatum and dose-dependent displacement with increasing doses of aprepitant. Occupancies increased as a function of aprepitant plasma concentrations according to a one-site competition model, which agrees with reported occupancy-concentration relationships in clinical studies after correction for species differences in plasma protein-unbound aprepitant fractions. These occupancy data were further supported by ex vivo autoradiography of brain samples from aprepitant-treated gerbils. In a pilot study of a marmoset, we obtained more accurate determinations of NK-1 receptor occupancy, less affected by spillover of signals from extracranial tissues than in gerbil experiments. Conclusions: These findings support the utility of small animals and quantitative PET in the development of drugs targeting NK-1 receptors.

Distribution and pharmacological characterization of primate NK‐1 and NK‐3 tachykinin receptors in the central nervous system of the rhesus monkey

Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK‐1 and NK‐3 tachykinin receptors (referred to as rmNK‐1 and rmNK‐3) from the rhesus monkey (Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS. The deduced rmNK‐1 amino‐acid sequence differed by only two amino acids from the human NK‐1 (hNK‐1). The deduced rmNK‐3 amino‐acid sequence was two amino acids shorter than human NK‐3 (hNK‐3), with a seven‐amino‐acid difference in sequence. Ligand binding studies revealed that the affinity of rmNK‐1 to substance P (SP) was comparable to that of hNK‐1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK‐1 and hNK‐1. Affinity of rmNK‐3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK‐3. Ca2+ imaging showed that activations of both rmNK‐1 and rmNK‐3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors. The amounts of rmNK‐1 and rmNK‐3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK‐1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK‐1 mRNA in the brain, with less rmNK‐3 mRNA found in the cortex compared to rmNK‐1 mRNA. In the monkey hippocampus and amygdala, rmNK‐1 mRNA was present at markedly higher concentrations than rmNK‐3 mRNA. The present results provide an insight into the distinct physiological nature and significance of the NK‐1 and NK‐3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype‐specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.

Quantitative Analysis of NK1 Receptor in the Human Brain Using PET with 18F-FE-SPA-RQ

18F-fluoroethyl-SPA-RQ (18F-FE-SPA-RQ) was recently developed as a radioligand for the measurement of neurokinin 1 (NK1) receptor with PET. In this study, we used 18F-FE-SPA-RQ with PET to visualize and quantify NK1 receptor in the human brain. Methods: PET scans were performed on 7 healthy men after intravenous injection of 18F-FE-SPA-RQ. Binding potential (BPND) was calculated by the indirect kinetic, simplified reference tissue model (SRTM), and ratio methods. The indirect kinetic method was used as the gold standard method and was compared with the SRTM method, with scan times of 180, 270, and 330 min, and with the ratio method, with time integration intervals of 120–180, 210–270, and 300–330 min. The cerebellum was used as the reference brain region. Results: Regional radioactivity was highest in the caudate head and putamen; mid level in the parahippocampus, cerebral cortex, and thalamus; and lowest in the cerebellum. BPND values by the indirect kinetic method were 3.15 ± 0.36, 3.11 ± 0.66, 1.17 ± 0.25, and 0.46 ± 0.14 in the caudate, putamen, parahippocampal region, and thalamus, respectively. For cerebral cortical regions, BPND values by the indirect kinetic method were 0.94 ± 0.23, 0.82 ± 0.15, 0.76 ± 0.15, and 0.69 ± 0.16 in the occipital, temporal, frontal, and anterior cingulate cortices, respectively. BPND values by the SRTM and ratio methods were in good agreement with those by the indirect kinetic method (r = 0.94–0.98). Conclusion: The regional distribution of 18F-FE-SPA-RQ was in agreement with previous PET studies and postmortem studies of NK1 receptor in the human brain. The ratio method will be useful for clinical research of psychiatric disorders, for the estimation of NK1 receptor without arterial blood sampling and long dynamic PET.