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Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10-8 M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93, 129), and the 5-HT1D/1B agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionamide dihydrochloride quaternary-hydrate (WAY 100135) (10-7 M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-piperazinyl]-1H-indole-2-carboxylate (SDZ 216-525) (10-7 M), and that of CP-93, 129 was blocked by methiothepin (10-7 M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10-6 M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development. [ABSTRACT FROM AUTHOR]

Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABA A (GABA A R) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABA A R and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABA A and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABA A /glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABA A R, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI., Competing Interests: Declaration of Competing Interest No competing financial interest exist, (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT 2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT 2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Ageing and alteration of the functions of the retinal pigment epithelium (RPE) are at the origin of lost of vision seen in age-related macular degeneration (AMD). The RPE is known to be vulnerable to high-energy blue light. The white light-emitting diodes (LED) commercially available have relatively high content of blue light, a feature that suggest that they could be deleterious for this retinal cell layer. The aim of our study was to investigate the effects of "white LED" exposure on RPE. For this, commercially available white LEDs were used for exposure experiments on Wistar rats. Immunohistochemical stain on RPE flat mount, transmission electron microscopy and Western blot were used to exam the RPE. LED-induced RPE damage was evaluated by studying oxidative stress, stress response pathways and cell death pathways as well as the integrity of the outer blood-retinal barrier (BRB). We show that white LED light caused structural alterations leading to the disruption of the outer blood-retinal barrier. We observed an increase in oxidized molecules, disturbance of basal autophagy and cell death by necrosis. We conclude that white LEDs induced strong damages in rat RPE characterized by the breakdown of the BRB and the induction of necrotic cell death., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Upregulation of the persistent sodium current (I(NaP)) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate I(NaP) and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of I(NaP) in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of I(NaP). The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, I(NaP) in motoneurons and spasticity in rats with SCI. Similarly, the blockade of I(NaP) by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of I(NaP) and spasticity.

Spectra of "white LEDs" are characterized by an intense emission in the blue region of the visible spectrum, absent in daylight spectra. This blue component and the high intensity of emission are the main sources of concern about the health risks of LEDs with respect to their toxicity to the eye and the retina. The aim of our study was to elucidate the role of blue light from LEDs in retinal damage. Commercially available white LEDs and four different blue LEDs (507, 473, 467, and 449nm) were used for exposure experiments on Wistar rats. Immunohistochemical stain, transmission electron microscopy, and Western blot were used to exam the retinas. We evaluated LED-induced retinal cell damage by studying oxidative stress, stress response pathways, and the identification of cell death pathways. LED light caused a state of suffering of the retina with oxidative damage and retinal injury. We observed a loss of photoreceptors and the activation of caspase-independent apoptosis, necroptosis, and necrosis. A wavelength dependence of the effects was observed. Phototoxicity of LEDs on the retina is characterized by a strong damage of photoreceptors and by the induction of necrosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In healthy adults, activation of γ-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)), which is regulated by proteins in the plasma membrane: the K(+)-Cl(-) cotransporter KCC2 and the Na(+)-K(+)-Cl(-) cotransporter NKCC1, which extrude and intrude Cl(-) ions, respectively. A high [Cl(-)](i) leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of gamma-aminobutyric acid(A) (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl-) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl- equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.

This review presents recent advances in the development of strategies to restore posture and locomotion after spinal cord injury (SCI). A set of strategies focusing on the lesion site includes prevention of secondary damages, promotion of axonal sprouting/regeneration, and replacement of lost cells. Other strategies focus on spinal central pattern generators (CPGs). Training promotes functional recovery by enhancing the plasticity of CPGs and these sublesional networks can be reactivated by means of pharmacological or electrical stimulation. It is now clear that substantial functional recovery will require a combination of strategies adapted to each phase following SCI. Finally, improvements in the understanding of the mechanisms underlying spasticity may lead to new treatments of this disabling complication affecting patients with SCI., (Copyright 2009 Elsevier Ltd. All rights reserved.)

After disruption of the descending respiratory pathways induced by unilateral cervical spinal cord injury (SCI) in rats, the inactivated ipsilateral (ipsi) phrenic nerve (PN) discharge may partially recover following some specific experimental procedures [such as contralateral (contra) phrenicotomy (Phx)]. This phrenic reactivation involves normally silent contra pathways decussating at the level of the phrenic nucleus, but the mechanisms of this crossed phrenic activation are still poorly understood. The present study investigates the contribution of sensory phrenic afferents to this process by comparing the acute effects of ipsi and contra Phx. We show that the phrenic discharge (recorded on intact PNs) was almost completely suppressed 0 h and 3 h after a lateral cervical SCI, but was already spontaneously reactivated after 1 week. This ipsi phrenic activity was enhanced immediately after contra Phx and was completely suppressed by an acute contra cervical section, indicating that it is triggered by crossed phrenic pathways located laterally in the contra spinal cord. Ipsi phrenic activity was also abolished immediately after ipsi Phx that interrupts phrenic sensory afferents, an effect prevented by prior acute ablation of the cervical dorsal root ganglia, indicating that crossed phrenic activation depends on excitatory phrenic sensory afferents but also putatively on inhibitory non-phrenic afferents. On the basis of these data, we propose a new model for crossed phrenic activation after partial cervical injury, with an essential role played by ipsi-activating phrenic sensory afferents.

Lateral hemisection of the cervical (C2) spinal cord in the rat interrupts ipsilateral bulbospinal respiratory pathways arising mainly from the rostral ventral respiratory group (rVRG) and Bötzinger complex and projecting to phrenic motoneurons in C3-C5. Deafferented phrenic motoneurons can be reactivated via previously silent contralateral pathways, a process called the crossed phrenic phenomenon (CPP). In order to further characterise the neuronal bases of the CPP and quantify the neurons involved, respiratory neurons projecting to the ipsilateral phrenic nucleus in hemisected rats were labelled by injection of the monosynaptic retrograde tracer fluorogold (FG) in ipsilateral C3-C4 metamers. Respectively 36% and 23% neurons were labelled in the contralateral and ipsilateral rVRG in hemisected rats compared to controls, and respectively 26% and 2% in the contralateral and ipsilateral Bötzinger complex. This shows that phrenic motoneurons located under the C2 hemisection may still be activated by axons or collaterals of contralateral respiratory premotoneurons located in the rVRG and Bötzinger complex which cross the spinal cord midline at the level of the phrenic nuclei, and also by axon collaterals of ipsilateral rVRG premotoneurons which cross the midline both in the brainstem and in the spinal cord. Neurons with double crossing axons were twice as many in the caudal part of the rVRG (38%) compared to the part located rostrally to the area postrema (20%), which further argues in favour of a subdivision of this nucleus. These pathways may be involved in the CPP and could be differentially activated in acute or chronic lesioned rats.

The use of fluorescent dyes has been a major improvement for paths tracing studies. However, these tracers present different properties and have to be chosen carefully. The present study compares the ability of different tracers to specifically label phrenic motoneurons (PMNs) innervating the rat diaphragm. The administration of fluorogold (FG) from the transected phrenic nerve specifically labeled PMNs in the ipsilateral spinal cord. However, when FG was injected into one hemidiaphragm, in addition with ipsilateral PMNs, a less intense artifactual labeling was observed in the spinal cord (mainly in contralateral PMNs) and in the medulla oblongata (mainly in the area postrema and cranial motor nuclei). Similar results were observed using horseradish peroxidase, while no labeling was observed after injection of nuclear yellow or diamidino yellow into the diaphragm. By contrast, the dextran amine fluororuby (FR) and the carbocyanine DiAsp selectively and exclusively labeled ipsilateral PMNs 2 or 3 weeks after injection into the diaphragm, respectively. The lipophilic properties of DiAsp and the high molecular weight of FR may prevent their diffusion to adjacent tissues and into the blood stream which seems to account for the artifactual labeling observed with the other tracers. The higher homogeneity and quality of the labeling observed with FR compared to DiAsp make it the most appropriate tracer for the specific monosynaptic fluorescent labeling of PMNs after injection into the diaphragm.

Transgenic (TG) mice deficient in glucocorticoid receptors (GR) were used in order to study the effects of a reduced GR function on adrenocorticotropin hormone and corticosterone plasma levels and on serotonin metabolism in different brain areas under basal resting conditions, after a 30-min restraint stress and 60 min after the end of the restraint stress. There was no difference in basal or stress-induced levels of either adrenocorticotropin hormone or corticosterone in control and TG mice, but the return of adrenocorticotropin hormone to basal values after the end of the stress was delayed in TG mice. Under basal conditions, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine was decreased only in the hippocampus of TG mice compared to controls. In the brain stem, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased compared to basal values after a 30-min restraint stress and values were still high 60 min after the end of the restraint stress in both control and TG mice. In the hippocampus, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased at the end of the stress and returned to basal levels 60 min later in control mice, whereas there was no change at the end of the stress but an increase 60 min later in TG mice. Finally there was no change in serotonin metabolism in the cortex, striatum or hypothalamus in either group or situation. Our results support the hypothesis of a tonic activation of serotonin turnover by corticosterone through GR in the mouse hippocampus. Moreover, stress-induced stimulation of serotonin metabolism in the brain stem and hippocampus appears to be delayed in TG mice compared to control mice. These results are particularly relevant for mood disorders such as depression where alterations of serotoninergic transmission might be secondary to an impairment of GR functions., (Copyright 1999 S. Karger AG, Basel.)

Previous works have shown the potential location of 5-HT1B binding sites on the retinal afferents to the superior colliculus in the rat. In order to confirm this hypothesis, the distribution of 5-HT1B binding sites, labelled by S-CM-G[125I]TNH2, was analysed by quantitative autoradiography at both light and electron microscopic levels in the upper stratum griseum superficiale (SGS) of the superior colliculus, 5 days after unilateral eye ablation. At the light microscopic level, no 5-HT1B sites were found to be associated with capillaries, glial, or neuronal cell bodies, but the fine neuropile was specifically labelled. At the electron microscopic level, the quantitative analysis performed with 50% probability circles showed that classical dendritic processes, presynaptic dendritic processes, and processes containing flat synaptic vesicles were not labelled. In the SGS ipsilateral to the eye ablation, silver grains were specifically associated with processes containing round and pleomorphic vesicles and with non-synaptic contacts between those processes and dendritic or other non identified neuronal processes. In the deafferented contralateral SGS, 5-HT1B receptors were associated with degenerating retinal terminals, with processes containing round vesicles and with non-synaptic contacts between those two tissue compartments. This is the first direct demonstration of the existence of 5-HT1B receptors in non-synaptic contacts and in non-serotonergic terminals. The existence of 5-HT1B terminal heteroreceptors localised on primary visual afferents shows that serotonin might modulate the transmission of visual messages to the superior colliculus.

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.

The affinities of several 5-hydroxy-indole derivatives for serotonin-1 (5-HT1) binding site subtypes, labeled with 2 nM [3H]5-HT, were assessed by quantitative autoradiography on rat brain sections. The results obtained with known ligands, namely 5-hydroxytryptamine (5-HT), 5-methoxytryptamine (5-Me-OT), 5-methoxy-N,N- dimethyl-tryptamine (5-Me-ODMT), 5-hydroxy-N,N-dimethyl-tryptamine (bufotenine) and 8-hydroxy-2-[di-N-propylamino]tetralin (8-OH-DPAT) demonstrate the reliability and the advantages of this technique for pharmacological studies. Novel serotonin derivatives were synthesized by carboxymethylation of the hydroxyl group. One of those new ligands, serotonin-O-carboxy-methyl- glycyl-tyrosinamide (S-CM-GTNH2), inhibited 2 nM [3H]5-HT binding to the substantia nigra with an IC50 of 22.4 nM, a value which is 22 times lower than that found in the dentate gyrus and choroid plexus. This demonstrates the preferential affinity of S-CM-GTNH2 for 5-HT1B versus 5-HT1A and 5-HT1C binding sites. S-CM-GTNH2 contains a tyrosine residue, which may be useful for the synthesis of a radioactive iodinated molecule and for the preparation of 'long-lasting ligands' linked through peptide bonds with a protein. These derivatives could be of great interest for ultrastructural and behavioral studies relevant to 5-HT1B sites.

We describe here the synthesis of a new serotonin conjugate, S-CM-GTNH2, and its radioiodinated derivative. Quantitative autoradiographic studies on rat and guinea pig brain sections incubated with 2 nM [3H]5-HT showed a preferential affinity of S-CM-GTNH2 for 5-HT1B and 5-HT1D sites. Autoradiograms from brain sections incubated with 0.02 nM S-CM-G[125I]TNH2 showed a heterogeneous anatomical distribution of the labelling with high densities in regions rich in 5-HT1B or 5-HT1D binding sites, and with no labelling of those rich in 5-HT1A or 5-HT1C sites. The pharmacological profiles of the binding sites corresponded to those of 5-HT1B and 5-HT1D receptor subtypes. The radioligand S-CM-G[125I]TNH2 is a good probe for the study of these sites and will be used for their subcellular localization in electron microscopy.

We have developed a computer-assisted analytic system for quantifying autoradiograms. The system was tested by studying the characteristics of high affinity serotonin binding sites (5-HT1) in rat brain sections, using [3H]5-HT as a radioligand. Autoradiographic data are digitized and transferred to a Macintosh II by means of a high speed, high resolution solid state camera. The physical characteristics of the device (uniformity, temporal stability, linearity) are such that the accuracy of the measurements obtained is highly satisfactory. The autoradiogram grey levels were measured and converted into radioligand concentrations (fmol/mg tissue) with the "BIOLAB" program which was specially written at our department in Macintosh Programming Workshop (MPW). The consistency of the measurements performed on small anatomical structures confirms the reliability of the system. The greatest discrepancies were due to the processing of the sections and also to the biological variability from one animal to another. The low cost of the device described, the high picture definition, the speed with which measurements can be obtained, the reliability of the system, and the original character of the program make it a valuable means to easier analyse quantitative autoradiography in pharmacological and physiological research.