Highlights ► We focus on several biological systems which are impaired with age in grey mouse lemurs, as in humans. ►Similarities between human and mouse lemur are demonstrated in both normal and ...pathological ageing process. ►This smaller, rapidly developed and shorter-lived non-human primate could be a suitable model for human ageing research. ►This non-human primate model offer predictive biomarkers of longevity and neuropathological ageing.
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 α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor antagonist protects the cochlea against l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid effect.
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.
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 μM), 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 μM) 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.
Le vieillissement est un facteur majeur de risque dans les maladies neurodégénératives de type Alzheimer (AD). Cependant, les processus du vieillissement cérébral physiologique et de l’AD sont ...différents. Ils s’accompagnent de modifications cellulaires et moléculaires complexes, encore mal connues. L’approche transcriptomique permet de suivre les variations d’expression d’un grand nombre de gènes. Des études récentes ont montré que les profils transcriptionnels du cortex variaient en fonction de l’âge chez l’homme et que ceux-ci présentaient de grandes similitudes avec ceux observé chez les primates. Sur un modèle lémurien pertinent, Microcebus murinu (Mim), nous avons recherché les gènes dont l’expression était modifiée par l’âge et par la pathologie dans le cortex temporal puisque certains animaux présentent au cours du vieillissement des plaques amyloïdes.
Notre étude a été menée avec 18 animaux : 6 jeunes et 12 âgés, parmi ceux-ci 2 étaient porteurs de plaques amyloïdes. L’ARN a été hybridé sur des puces humaines, HG-Affymetrix-133plus2 contenant 55000 sondes. Les données transcriptomiques ont été analysées par différentes méthodes statistiques et informatiques.
Environ 12000 transcrits ont été détectés soit 17% par comparaison avec les tissus humains 30%. Ce résultat confirme la possibilité d’utiliser des puces humaines pour étudier les transcriptomes de primates. A partir de ces 12000 gènes, 350 ont été identifiés comme différentiellement exprimés chez les âgés vs jeunes, et 427 chez les AD-like vs âgés. Parmi ces derniers, 167 transcrits étaient surexprimés chez les AD-like et sous-exprimés chez les âgés. Inversement, 142 transcrits étaient sous-exprimés chez les chez les AD-like et surexprimés chez les âgés. Les profils transcriptionnels étaient différents et spécifiques pour chaque groupe d’animaux. Enfin, les gènes ont été classés en fonction de leurs implications biologiques en 4 grands modules : transmission synaptique, voies de signalisation, métabolisme et facteurs nucléaires.
Nous avons identifié des gènes dont l’expression est spécifiquement modifiée par l’âge ou par la pathologie chez Mim. Ces modifications d’expression géniques reflètent les processus de plasticité synaptique liée au vieillissement et les processus pathologiques liés à l’AD. Ces données constituent les bases d’une signature spécifique du vieillissement et de la pathologie de type Alzheimer dans le cortex temporal de primate.
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.
The postnatal developmental expression and the distribution of the glutamate transporters (GLAST, GLT-1 and EAAC1) were analyzed in rat vestibular nuclei (VN), at birth and during the following 4 ...weeks. Analyses were performed using reverse transcriptase-polymerase chain reaction and immunoblotting of GLAST, GLT-1 and EAAC1 mRNA and protein during the postnatal development of the VN neurons and their afferent connections. We also studied the distribution of each glutamate transporter in the medial and lateral VN by use of immunocytochemistry and confocal microscopy. GLAST, GLT-1 and EAAC1 mRNA and protein were present in the VN at each developmental stage. GLAST was highly expressed mainly in glia from birth to the adult stage, its distribution pattern was heterogeneous depending on the region of the medial and lateral VN. GLT-1 expression increased dramatically during the second and third postnatal weeks. At least during the first postnatal week, GLT-1 was expressed in the soma of neurons. EAAC1 was detected in neurons and decreased from the third week. These temporal and regional patterns of GLAST, GLT-1 and EAAC1 suggest that they play different roles in the maturation of glutamatergic synaptic transmission in the medial and lateral VN during postnatal development.
The presence and the activity of α‐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+,) by spectrofluorimetry. Cultures of dissociated vestibular ganglia from 18 gestation day mouse embryos were grown in vitro for 3–4 days. lmmunocytochemical 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 glutarnate/aspartate‐induced intracellular calcium response.
