The peptidic antidepressant spadin interacts with prefrontal 5-HT(4) and mGluR(2) receptors in the control of serotonergic function.

This study investigates the mechanism of action of spadin, a putative fast-acting peptidic antidepressant (AD) and a functional blocker of the K(+) TREK-1 channel, in relation with the medial prefrontal cortex (mPFC)-dorsal raphé (DRN) serotonergic (5-HT) neurons connectivity. Spadin increased 5-HT neuron firing rate by 113%, an augmentation abolished after electrolytic lesion of the mPFC. Among the few receptor subtypes known to modulate TREK-1, the stimulation of 5-HT4 receptors and the blockade of mGluR2/3 ones both activated 5-HT impulse flow, effects also suppressed by mPFC lesion. The combination of spadin with the 5-HT4 agonist RS 67333 paradoxically reduced 5-HT firing, an effect reversed by acutely administering the 5-HT1A agonist flesinoxan. It also had a robust synergetic effect on the expression of Zif268 within the DRN. Together, these results strongly suggest that 5-HT neurons underwent a state of depolarization block, and that the mechanisms underlying the influences exerted by spadin and RS 67333 are additive and independent from each other. In contrast, the mGluR2/3 antagonist LY 341495 occluded the effect of spadin, showing that it likely depends on mPFC TREK-1 channels coupled to mGluR2/3 receptors. These in vivo electrophysiological data were confirmed by in vitro Ca(2+) cell imaging performed in cultured cortical neurons. Altogether, our results indicate that spadin, as a natural compound, constitutes a very good candidate to explore the "glutamatergic path" of fast-acting AD research. In addition, they provide the first evidence of 5-HT depolarization block, showing that the combination of 5-HT activators for strategies of AD augmentation should be performed with extreme caution.

Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil.

Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy, a disease caused by the lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying the disease progression are unclear and treatments are limited. Here, we tested the hypothesis of a greater susceptibility to the opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic (mdx) mice (before the development of overt cardiomyopathy) when subjected to a stress protocol and determined whether the prevention of a PTP opening is involved in the cardioprotective effect of sildenafil, which we have previously reported in mdx mice. Using the 2-deoxy-[(3)H]glucose method to quantify the PTP opening in ex vivo perfused hearts, we demonstrate that when compared with those of controls, the hearts from young mdx mice subjected to ischemia-reperfusion (I/R) display an excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage, and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from nonischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and reactive oxygen species handling, but enhanced Ca(2+) uptake velocity and premature opening of the PTP, which may predispose to I/R-induced injury. The administration of a single dose of sildenafil to mdx mice before I/R prevented excessive PTP opening and its downstream consequences and reduced tissue Ca(2+) levels. Furthermore, mitochondrial Ca(2+) uptake velocity was reduced following sildenafil treatment. In conclusion, beyond our documentation that an increased susceptibility to the opening of the mitochondrial PTP in the mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations and is reported safe and well tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca(2+) loading and mitochondrial Ca(2+) uptake.

Increased expression and intramitochondrial translocation of cyclophilin-D associates with increased vulnerability of the permeability transition pore to stress-induced opening during compensated ventricular hypertrophy.

Opening of the permeability transition pore (PTP) of mitochondria is a critical permeation event that compromises cell viability and may constitute a factor that participates to the loss of cardiomyocytes in compromised hearts. Mitochondria from hearts with volume overload-induced compensated hypertrophy are more vulnerable to opening of the PTP opening in response to a Ca2+ stress. Several of the factors known to affect PTP opening, including respiratory function, membrane potential, the rate of mitochondrial Ca2+ uptake and endogenous levels of Ca2+ in the mitochondrial matrix, were not altered by volume overload. In contrast, there was an 80% increase in the abundance of the PTP regulating protein cyclophilin-D and a 3.7 fold enhancement of Cyp-D binding to membrane, which all predispose to PTP opening. Mitochondria from volume overloaded animals also displayed elevated rates of production of reactive oxygen species, which may be causally related to both the intramitochondrial translocation of cyclophilin-D and PTP opening, since incubation of cardiac mitochondria with terbutylhydroperoxyde in vitro increased to binding of cyclophilin-D to mitochondrial membranes in a dose-related fashion, except when cyclosporin A (a ligand of cyclophilin D with a known ability to delay PTP opening) was present prior to the addition of terbutylhydroperoxyde. Taken together, these results constitute the first evidence obtained in a pathophysiologic situation that increased abundance of cyclophilin-D within mitochondrial membranes may increase mitochondrial vulnerability to stress, and thus possibly initiate a vicious cycle of cellular dysfunction that may ultimately lead to activation of cell death.

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles.

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.

Descending GABAergic projections to the mesencephalic locomotor region in the lamprey Petromyzon marinus.

The mesencephalic locomotor region (MLR) plays a significant role in the control of locomotion in all vertebrate species investigated. Forebrain neurons are likely to modulate MLR activity, but little is known about their inputs. Descending GABAergic projections to the MLR were identified by double-labeling neurons using Neurobiotin injected into the MLR combined with immunofluorescence against GABA. Several GABAergic projections to the MLR were identified in the telencephalon and diencephalon. The most abundant GABAergic projection to the MLR came from the caudal portion of the medial pallium, a region that may have similarities with the amygdala of higher vertebrates. A small population of GABAergic cells projecting to the MLR was found in the striatum and the ventral portion of the lateral pallium, which could respectively correspond to the input and output components of the basal ganglia thought to be involved in the selection of motor programs. Other GABAergic projections were found to come from the thalamus and the hypothalamus, which could take part in the motivational aspect of motor behavior in lampreys. Electrophysiological experiments were also carried out to examine the effects of GABA agonists and antagonists injected into the MLR in a semi-intact lamprey preparation. The GABA agonist inhibited locomotion, whereas the GABA antagonist initiated it. These results suggest that the GABAergic projections to the MLR modulate the activity of MLR neurons, which would be inhibited by GABA at rest.

Bimodal locomotion elicited by electrical stimulation of the midbrain in the salamander Notophthalmus viridescens.

The present experiments were designed to identify the mesencephalic locomotor region (MLR) in the salamander. An in vitro semi-intact preparation from a decerebrate adult salamander (Notophthalmus viridescens) was developed in which the locomotor activities were monitored from electromyographic and video recordings. The results show that the two locomotor modes exhibited by salamanders (i.e., stepping and swimming) were evoked by electrical microstimulation (5-15 Hz; 0.1-10 microA; 2 msec pulses) of a circumscribed region in the caudal mesencephalon. At threshold current strength (0.5-3.5 microA at 15 Hz), rhythmic limb movements and intersegmental coordination, such as during stepping, were induced. As the stimulation strength was subsequently increased, the frequency of stepping became more rapid, and, at 2.0-5.5 microA, the limbs were held back against the body wall and swimming movements of the trunk were induced. An additional increase of the stimulation strength induced an increase of the frequency and amplitude of the swimming movements. Anatomical studies conducted in parallel revealed the presence of choline acetyltransferase immunoreactive cells in the functionally identified MLR region. Together, the present results indicate that the MLR is present in salamanders and that its level of activation determines the mode of locomotion. Walking is induced at low activation levels, and swimming, which constitutes a faster mode of locomotion, requires stronger stimulation of the MLR. Furthermore, as in other vertebrates, the MLR contains cholinergic cells.

Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys.

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR-controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose-dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase-immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d-tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi-intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.