The grey mouse lemur: a non-human primate model for ageing studies.

The use of non-human primate models is required to understand the ageing process and evaluate new therapies against age-associated pathologies. The present article summarizes all the contributions of the grey mouse lemur Microcebus murinus, a small nocturnal prosimian primate, to the understanding of the mechanisms of ageing. Results from studies of both healthy and pathological ageing research on the grey mouse lemur demonstrated that this animal is a unique model to study age-dependent changes in endocrine systems, biological rhythms, thermoregulation, sensorial, cerebral and cognitive functions.

Glutamate transporters in the guinea-pig cochlea: partial mRNA sequences, cellular expression and functional implications.

In the cochlea, glutamate plays a major role in synaptic transmission between the inner hair cell and the primary auditory neurons. Extracellular glutamate concentration must be regulated to prevent excitotoxicity. This regulation is mediated by excitatory amino acid transporters, membrane proteins that remove glutamate from the synaptic cleft. In this study, we investigated the distribution and activity of three excitatory amino acid transporters subtypes in the guinea-pig cochlea: glutamate aspartate transporter, glutamate transporter and excitatory amino acid carrier. A partial messenger ribonucleic acid sequence was determined for each of these transporters, by polymerase chain reaction with degenerate primers, using guinea-pig brain complementary deoxyribonucleic acid as the template. Primers specific for each transporter were then designed and used to screen a dissected organ of Corti complementary deoxyribonucleic acid library. The cellular distribution of each transporter was examined by immunocytochemistry. We investigated the functional consequences of inhibiting glutamate uptake by recording cochlear potentials during intracochlear perfusion with either l-trans-pyrrolidine-2,4-dicarboxylic acid or dihydrokainate. At the end of the electrophysiological session, cochleas were processed for electron microscopy. Only the glutamate aspartate transporter messenger ribonucleic acid was detected in the organ of Corti. Consistently, glutamate aspartate transporter protein was detected in the inner hair cell-supporting cells and in the ganglion of Corti satellite cells. Glutamate transporter and excitatory amino acid carrier were found in the afferent auditory neurons. Only intracochlear perfusions with l-trans-pyrrolidine-2,4-dicarboxylic acid resulted in a dose-dependent decrease in the amplitude of the cochlear compound action potential, leaving cochlear microphonic potential unaffected. After l-trans-pyrrolidine-2,4-dicarboxylic acid perfusion, cochleas displayed a swelling of the afferent endings typical of excitotoxicity. [(-)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamyl-2,3-benzodiazepine], a selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist protects the cochlea against l-trans-pyrrolidine-2,4-dicarboxylic acid effect.

Glycine induced calcium concentration changes in vestibular type I sensory cells.

Glutamate is the neurotransmitter of the synapse between vestibular type I hair cells and the afferent nerve calyx. This calyx may also be involved in local feedback, which may modify sensory cell activity via N-methyl-D-aspartate (NMDA) receptors. Glycine is the co-agonist of glutamate in NMDA receptor activation. Both agents have been detected by immunocytochemistry in the nerve calyx. Glutamate and NMDA stimulations cause changes in the intracellular calcium concentration ([Ca(2+)](i)) of isolated type I sensory cells. We investigated the effect of glycine stimulation on [Ca(2+)](i) in guinea pig type I sensory cells by spectrofluorimetry with fura-2. Glycine application to isolated type I sensory cells induced a rapid and transient increase in [Ca(2+)](i). The fluorescence ratio increased by 55% above the resting level. The peak was reached in 9 s and the return to basal level took about 20 s. A specific antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 microM), decreased the calcium response to glycine by 60%. Glycine may activate NMDA receptors. Glycine may also activate the strychnine-sensitive glycine receptor-gated channel. Strychnine (50 microM) decreased the calcium response to glycine by 60%. Thus, glycine probably induces calcium concentration changes in type I vestibular sensory cells via NMDA receptors and/or glycine receptors.

AMPA receptors in cultured vestibular ganglion neurons: detection and activation.

The presence and the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors were investigated in mouse cultured vestibular ganglion neurons using immunocytochemistry and measurement of intracellular calcium concentration ([Ca2+]i) by spectrofluorimetry. Cultures of dissociated vestibular ganglia from 18 gestation day mouse embryos were grown in vitro for 3-4 days. Immunocytochemical labelling of AMPA receptor subunits GluR2/R3 and GluR4 was detected in neuron cell bodies and proximal neurites and more lightly in glial cells. There was no clear selective subcellular localization of the different subunits. For the GluR1 subunit a signal was observed only in some neurons and neurites and was weak. Vestibular ganglion neurons responded to fast application of 1 mM glutamate and 10 mM aspartate through unknown receptors by a transient increase in [Ca2+]i. The mean amplitude of this rapid increase was about nine times the resting level and recovery was complete within 30-45 s after the application. If separated by an interval of at least 10 min, consecutive applications produced similar calcium responses. AMPA (1 mM) application induced the same type of responses. Five minutes prior to the AMPA exposure, the application of a specific AMPA antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1.5 mM), in the external medium inhibited the response to AMPA. Chelation of external calcium by EGTA (1.5 mM) abolished the responses to drug applications, indicating that an influx of external calcium is involved in the [Ca2+]i increase. These observations suggest that heteromeric AMPA receptors are expressed in vestibular ganglion neurons in culture and play a functional role in their glutamate-induced depolarization. Experiments are in progress using specific AMPA and NMDA antagonists to characterize the participation of the two types of ionotropic glutamate receptors in the glutamate/aspartate-induced intracellular calcium response.

Cholinergic agonists increase intracellular calcium concentration in frog vestibular hair cells.

Acetylcholine (ACh) is usually considered to be the neurotransmitter of the efferent vestibular system. The nature and the localization of cholinergic receptors have been investigated on frog isolated vestibular hair cells (VHCs), by measuring variations of intracellular calcium concentration ([Ca2+]i), using calcium sensitive dye fura-2. Focal iontophoretic ACh (1 M, 300 nA.40 ms) application induced a rapid increase in [Ca2+]i, reaching a peak in 20 s and representing about 5-fold the resting level (from 61 +/- 6 to 320 +/- 26 nM). Applications of muscarinic agonists as methacholine and carbachol induced weaker calcium responses (from 78 +/- 25 to 238 +/- 53 nM) than the one obtained with ACh applications. These muscarinic agonists were efficient only in precise zones. Desensitization of muscarinic receptors to successive stimulations was significant. Perfusion of nicotine or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic agonist, induced an increase in [Ca2+]i only in some cells (4/28 with DMPP). These results indicated the presence of cholinergic receptors on frog VHCs: muscarinic receptors were more responsive than nicotinic receptors. Presence of muscarinic and nicotinic receptors in the membrane of VHCs could indicate different modulations of VHCs activity mediated by [Ca2+]i and involving an efferent control which represents a central regulation of the vestibular afferent message.

Glutamate receptors on type I vestibular hair cells of guinea-pig.

Afferent nerve calyces which surround type I vestibular hair cells (VHCI) have recently been shown to contain synaptic-like vesicles and to be immunoreactive to glutamate antibodies. In order to understand the physiological significance of these observations, the presence of glutamate receptors on type I vestibular sensory cells has been investigated. The effect of excitatory amino acids applied by iontophoresis was examined by spectrofluorimetry using fura-2 sensitive dye. Glutamate application caused a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), in a dose-dependent manner. The ionotropic glutamate receptors agonists N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualic acid (QA) induced an increase of [Ca2+]i. The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid and the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione partially blocked the glutamate response, by 39 +/- 10 and 53 +/- 11% respectively. Metabotropic receptors were also revealed by the specific agonist trans-1-amino-cyclopentyl-1,3-dicarboxylate. The presence of different glutamate receptors on the VHCI membrane suggests two kinds of feedback. (i) At the base of the sensory cell, autoreceptors may locally control the synaptic transmission. (ii) At the apex, postsynaptic receptors may modulate sensory transduction from glutamate release at the upper part of the afferent nerve calyx. These feedbacks suggest presynaptic modulation of the vestibular hair cell response which could affect its sensitivity.

Intracellular free calcium in isolated vestibular hair cells and potassium iontophoresis.

The resting free calcium level was measured in 128 isolated mammalian vestibular sensory cells using the calcium-sensitive dye fura-2. Iontophoresis was used to apply short, localised and limited pulses of K+ which evoked dynamic changes in intracellular free calcium concentration. While most of the type I hair cells tested showed brief reversible and specific calcium responses, some were unresponsive. The changes in intracellular free calcium were also measured by videomicroscopic analysis. Iontophoretic application of K+ ions is shown to be a suitable method for inducing fast, transient changes in intracellular free calcium in vestibular hair cells. This technique could be useful for applying several ions and charged molecules such as amino acids in in-vitro cellular methods.

Vestibular hair cells isolated from guinea pig labyrinth.

Living sensory cells were isolated from the cristae ampullaris and macula utriculi of the guinea pig. Enzymatic and mechanical dissociation were used to obtain different populations of hair cells, the most predominant being type I cells. Their form varied: cell body of variable roundness, and neck and cilia of different lengths. The observation of many tilted cuticular plates supports the hypothesis of active mechanisms regulating mechanotransduction at the apex of these cells. Cell viability was verified by double fluorescent labeling (FDA-PI), which indicated that under correct conditions about 90% of the sensory cells could be maintained in vitro for several hours after dissociation. The detection of actin in the cuticular plate and cilia shows that the technique has various potential applications in morphological studies, and can contribute to investigations on the physiology of mammalian vestibular cells.

Inhibition of tyrosine hydroxylase activity by serotonin in explants of newborn rat locus ceruleus.

The long-term regulation of tyrosine hydroxylase (TH) by serotonin has been studied with cultures of newborn rat locus ceruleus explants. The presence of serotonin in the culture medium for a 24-h period was followed by an inhibition of TH activity in the explants. This effect lasted several days, with a maximal effect 2 days after treatment. Moreover, the decrease was reversible and dependent on the concentration of serotonin used (from 1 microM to 1 mM). The mechanisms of this regulation have been studied using drugs such as those known to act specifically on serotoninergic receptors and those known to interfere with protein synthesis. Thus, the action of serotonin (10(-5) M) on TH activity was suppressed with equimolar concentrations of serotoninergic antagonists such as metergoline or methiothepin. It was reproduced by quipazine, a drug capable of acting as a serotoninergic agonist. Inhibitors of protein synthesis acting either at the transcriptional or the translational levels can reproduce the inhibition of TH activity by serotonin alone. Furthermore, the effects of one or the other of these compounds and that of serotonin were not additive. This study confirms the hypothesis of an inhibitory control by serotonin on TH activity in the noradrenergic neurons of the locus ceruleus. Serotonin could regulate the synthesis of the enzyme through specific serotoninergic receptors.

Culture of rat locus coeruleus explants: a model to study transneuronal regulation of tyrosine hydroxylase in central noradrenergic neurons.

A culture method is described to maintain explants of rat locus coeruleus in vitro for several days. Both anatomical and biochemical methods were used to control the evolution of the cultures with time. Explants were first examined 7 days after culture by immunohistochemistry with specific antisera directed against enzymes specific for non-neuronal or neuronal cells. Non-neuronal enolase immunoreactive cells were observed throughout the explants except at their edge where outgrowth zones were observed. Neuronal cells which were separately stained by three markers (neuron-specific enolase, tyrosine hydroxylase and dopamine-?-hydroxylase antisera) share common morphological features: they are grouped in clusters; their soma have similar shape and size and the staining was always seen inside the cytoplasm of the cell bodies and their proximal processes. These results show that noradrenergic neurons are still present in the explants 7 days after culture. Biochemical characteristics of the explants were followed throughout culture. Non neuronal enolase activity doubled during the first week in vitro. In contrast neuron specific enolase activity decreased to about half its initial value. Similarly the amount of 1-[(3)H]noradrenaline taken up in the explants decreased abruptly during the two first days in culture to reach a stable value. Tyrosine hydroxylase activity decreased significantly to about a third its initial value. All biochemical markers became stable after 7 days in culture. The cholinergic control of tyrosine hydroxylase in the noradrenergic cells has been investigated using 1-week old cultures. Tyrosine hydroxylase activity in locus coeruleus explants incubated in the presence of oxotremorine (10(?5) M, during 24 h) is significantly increased (+108%) as compared to non treated tissues. This increase was abolished if atropine sulfate (10(?5) M) was present together with oxotremorine. Immunotitrations of tyrosine hydroxylase in homogenates of the explants treated with the cholinergic agonist revealed that the increased enzyme activity is due to a change in its catalytic properties without variation in the number of enzyme molecules (delayed activation). This experiment indicates that an activation of tyrosine hydroxylase can result from the stimulation of cholinergic receptors located in the locus coeruleus region. The present investigation shows that central noradrenergic neurons of the locus coeruleus region can survive in culture conditions. These neurons retain most of their in situ morphological and biochemical characteristics. This in vitro approach appears to be an interesting means to study tyrosine hydroxylase regulation in central noradrenergic neurons.