Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B Synapses in the adult central amygdala

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes

Nature and expression of L and non-L calcium currents by frog crista hair cells

Variations in the electrophysiological properties of hair cells appear to be a general feature of the vertebrate vestibular sensory organs. Here we studied Ca2+ currents in hair cells of frog crista ampullaris using the whole cell variant of the patch clamp technique. Currents were recorded in situ from hair cells in peripheral, intermediate and central regions of the sensory epithelium. Two types of Ca2+ currents were found: a large nifedipine-sensitive current and a small nifedipine insensitive current. The latter was insensitive to R, P/Q and N-type Ca2+ channels antagonists. The amplitude of nifedipine sensitive and insensitive currents varied in cells along the peripheral region. The present results demonstrate the expression of L and non-L Ca2+ channels in frog crista ampullaris that show intraregional variations in magnitude

Action of D-alpha aminoadipic acid on the sensory organs of the inner ear in the frog

Following the suggestions in the literature that glutamate or aspartate may be the transmitter at the primary afferent synapses of acoustico-lateralis organs, we have employed the "selective" excitatory amino acid antagonist. D-alpha amino adipate (DAA) as a tool with which to shed further light on this problem in the labyrinthine organs of the frog. DAA produces a dose-responsive, reversible depression of spontaneous activity in the afferent nerves of the posterior semicircular canal, saccule and basilar papilla. These structures are examples of ampullar, otolithic and auditory organs, respectively. The drug effect seems qualitatively the same throughout the labyrinth. The most interesting finding was that of a presynaptic (hair cell) effect of DAA on the semicircular canal. The means of recording did not permit detection of a presynaptic effect in the other organs examined. All the observed effects of DAA could be explained by a presynaptic action to inhibit transmitter release. Therefore, the ability of DAA to reduce transmission at primary afferent synapses of the frog labyrinth must not necessarily be interpreted to imply that the transmitter is an excitatory amino acid. A presynaptic action to reduce the release of a transmitter (of unknown structure) could explain all our results

Pre- and postsynaptic recovery of semicircular canal activity in the frog following ototoxic insult

Aim: Inner ear hair cells of lower vertebrates regenerate following physical or chemical insults. In the present study we aimed to define the damage and recovery of the pre and postsynaptic activity in the frog crista ampullaris after ototoxic insult with the antibiotic, gentamicin. Methods: Frogs were treated with a single dose of gentamicin sulphate (GM-5mM) administered intraotically. The presynaptic activity was monitored by recording in isolated canal preparations the endoampullar (receptor) potential (Adc). The postsynaptic activity was monitored by recording the resting (R) and evoked spike activity (E) from the whole ampullar nerve and from single canal afferents. Results: We found that Adc was abolished 5 days after GM administration and that it progressively recovered close to control values 20 days after treatment. These results well fitted with the morphological damage and recovery of hair cells in the crista epithelium. Consistently with the presynaptic damage, the canal afferent activity was abolished 5 days after GM treatment, while the recovery of the background and the evoked afferent activity showed a different sequence. Background activity (R) was detected 7-8 days after GM treatment and it was not modulated by mechanical stimulation of the canal. In addition, R reached control values (14/15 days post-treatment), before those of the evoked activity (E-20 days post-treatment). Intracellular recordings confirmed the above mentioned results and revealed that the evoked EPSP discharge, together with the spike activity, reached normal values close to 20 days post-treatment. Conclusion: The present results show that frog semicircular canal rapidly and completely recovers its pre -and postsynaptic function following severe ototoxic insult and that during the recovery, the synaptic function of regenerating hair cells becomes functional before the mechano-transduction function

Differential recovery of background and evoked afferent activity in the frog semicircular canal following ototoxic insult

The present study was aimed to define the damage and recovery of pre- and postsynaptic activity in the frog crista ampullaris after ototoxic insult with gentamicin (GM). We found that the endoampullar potential (receptor potential) recorded from the isolated canal was abolished 6 days after GM administration and that it recovered close to the control 20 days after treatment. Consistently with the presynaptic damage, the afferent activity from the ampullar nerve was abolished 6 days after GM treatment, while the recovery of background and evoked afferent activity showed a different sequence. Background activity reappeared 7-8 days after GM treatment and in this period it was not driven by canal mechanical stimulation. In addition, background activity reached control values around 15 days after the ototoxic insult, while the evoked activity was detected 9-10 days after GM treatment and appeared normal close to 20 days. Intracellular recordings from single canal afferents confirmed the above mentioned results. The present study show that the frog semicircular canal rapidly and completely recovers its pre -and postsynaptic function following severe ototoxic insult and that during the recovery afferent transmission at rest becomes functional before the mechano-transduction function

Gradients of expression of calcium and potassium currents in frog crista ampullaris.

In the present work we studied the intraregional expression of voltage-dependent Ca2+ and K+ currents in hair cells of frog crista ampullaris. The currents were recorded in situ from sensory cells of the peripheral region, the most populated region of the crista, by using the whole-cell variant of the patch-clamp technique. Voltage-clamp recordings revealed that the calcium current (ICa) and the outward potassium currents of IA, IK, IKCa types and the inward rectifier potassium current of IK1 type exhibited a significant gradient of density (pA/pF) along the region. IA density was maximal in cells located at the beginning of the peripheral region and decreased gradually becoming very small at the opposite end. All the other currents showed an opposite gradient of expression. Current-clamp experiments showed that the voltage behaviour of hair cells changed in relation to cell position. Cells located at the beginning of the peripheral region showed large depolarizations from the resting potential (close to –45 mV) which are consistent with the presence of small IK and IKCa, and an IA largely inactivated at rest. These cells also exhibited slowly developing and large hyperpolarizations that approached passive ones, due to the lack of IK1. In contrast, cells located at the opposite side of the region showed smaller depolarizations and hyperpolarizations from the resting potential (close to –65 mV), due to the presence of large IK and IKCa, and IK1, respectively. The possible role of the intraregional variation of Ca2+ and K+ currents in both hair cell function and afferent discharge properties is discussed