fg-9041 and Ischemia

We have developed an approach to directly probe neuronal excitability during the period beginning with induction of global ischemia and extending after reperfusion using transgenic mice expressing channelrhodopsin-2 (ChR2) to activate deep layer cortical neurons independent of synaptic or sensory stimulation. Spontaneous, ChR2, or forepaw stimulation-evoked electroencephalogram (EEG) or local field potential (LFP) records were collected from the somatosensory cortex. Within 20 s of ischemia, a >90% depression of spontaneous 0.3-3 Hz EEG and LFP power was detected. Ischemic depolarization followed EEG depression with a ∼2 min delay. Surprisingly, neuronal excitability, as assessed by the ChR2-mediated EEG response, was intact during the period of strong spontaneous EEG suppression and actually increased before ischemic depolarization. In contrast, a decrease in the somatosensory-evoked potential (forepaw-evoked potential, reflecting cortical synaptic transmission) was coincident with the EEG suppression. After 5 min of ischemia, the animal was reperfused, and the ChR2-mediated response mostly recovered within 30 min (>80% of preischemia value). However, the recovery of the somatosensory-evoked potential was significantly delayed compared with the ChR2-mediated response (<40% of preischemia value at 60 min). By assessing intrinsic optical signals in combination with EEG, we found that neuronal excitability approached minimal values when the spreading ischemic depolarization wave propagated to the ChR2-stimulated cortex. Our results indicate that the ChR2-mediated EEG/LFP response recovers much faster than sensory-evoked EEG/LFP activity in vivo following ischemia and reperfusion, defining a period where excitable but synaptically silent neurons are present.

Topics: Anesthetics, Local; Animals; Bacterial Proteins; Carrier Proteins; Channelrhodopsins; Disease Models, Animal; Electroencephalography; Evoked Potentials; Excitatory Amino Acid Antagonists; Forelimb; Hyperalgesia; In Vitro Techniques; Ischemia; Luminescent Proteins; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Optogenetics; Physical Stimulation; Quinoxalines; Reperfusion Injury; Tetrodotoxin; Valine

We investigated the effect of glutamate receptor antagonists on progressive inner hair cell (IHC) loss following transient cochlear ischemia in gerbils. Transient cochlear ischemia was induced by 15-min bilateral vertebral artery occlusion. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), or an N-methyl-D-aspartate (NMDA)-type receptor antagonist, MK-801, was administered 10 min before the ischemic insult. Hearing was assessed by sequentially recording compound action potentials (CAPs) before, during, and after the ischemia. The degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day after ischemia, the increases in the CAP threshold and the progressive IHC loss were significantly reduced in cochleae treated with DNQX, while MK-801 was ineffective. These results suggest that the AMPA receptor plays a critical role in the development of the progressive IHC loss induced by ischemia/reperfusion injury in the cochlea.

Topics: Acoustic Stimulation; Action Potentials; Animals; Cochlea; Cochlear Nerve; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Gerbillinae; Hair Cells, Auditory, Inner; Hearing; Immunohistochemistry; Ischemia; Male; Microscopy, Electron; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses

The synapses between the inner hair cells (IHCs) and the radial auditory dendrites are thought to be glutamatergic. Besides its fast excitatory properties, glutamate is known to be neurotoxic when released in excess or incompletely recycled. In the cochlea, this may occur in two pathological conditions: ischemia and noise trauma. We have further investigated the acute excitotoxicity (i.e. the swelling of type I afferent dendrites) by electron microscopy processing on guinea pig cochleas after an ischemic exposure lasting 5 to 40 min. The radial auditory dendrites reacted to ischemia in a time-dependent manner, with the swelling extending when the duration of ischemia increased. The type and the specificity of swelling were comparable to what acutely occurs after an exposure to glutamate analogs such as kainic acid or AMPA. A protection against this swelling was obtained by perfusing the cochlea with glutamate antagonists prior to ischemia. DNQX, an antagonist at AMPA/kainate receptors, had a powerful protective effect, and almost complete protection was obtained by perfusing both DNQX and D-AP5 (a NMDA antagonist). The latter results indicate that the two classes of glutamate receptors (AMPA/kainate and NMDA), both found to be electrophysiologically active at the IHC-auditory nerve synapse, are also involved in the excitotoxic processes. In addition, we also report data involving dopamine (its D2 agonist piribedil) a putative neurotransmitter at the lateral efferent synapses, in a postsynaptic protection of primary auditory neurons during transient ischemia. Altogether, these findings constitute a promising pharmacological approach of cochlear pathologies such as neural presbycusis.

Topics: Animals; Cochlea; Dendrites; Dopamine; Glutamates; Glutamic Acid; Guinea Pigs; Hair Cells, Auditory, Inner; Ischemia; Piribedil; Quinoxalines; Receptors, Glutamate; Synapses; Vestibulocochlear Nerve

We have investigated the hypothesis that the acute ischemic swelling of the radial dendrites connected to the inner hair cells (IHCs) is mediated by glutamatergic receptors. In control cochleas, after 20 min ischemia all the dendrites were dramatically swollen. Conversely, after a perfusion of 50 microM 6-7-dinitroquinoxaline-2,3-dione (DNQX) before ischemia, most dendrites were protected although those contacting the IHCs on their modiolar side frequently swelled. After 50 microM of D-2-amino-5-phosphonopentanoate (D-AP5), no dendrite protection could be obtained. Finally, after DNQX and D-AP5, no dendrite swelling occurred. These results suggest that, in the cochlea, the acute ischemic swelling of dendrites primarily occurs via non-NMDA receptors. However, in radial dendrites contacting the IHCs on their modiolar side, NMDA receptors may contribute to excitotoxicity.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cochlea; Dendrites; Guinea Pigs; Hair Cells, Auditory; Ischemia; Microscopy, Electron; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Reperfusion