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Development of dynorphin-like immunoreactive auditory nerve terminals in the chick.

The novel discovery that auditory nerve terminals in the chick cochlear nucleus magnocellularis (NM) are immunoreactive for the opioid peptide dynorphin (DYN) was recently reported [3]. The present study examines the development of DYN-immunoreactivity (DYN-I) in auditory nerve terminals in NM from embryos through young post-hatch chicks. No DYN-I was observed in NM at embryonic day 13 (E13). DYN-I first appeared at E16 as short flat structures partially surrounding NM cell bodies. Around post-hatch day 1 (P1), these structures had a more rounded, chalice-type of morphology reminiscent of the specialized auditory nerve terminals found in birds, the end-bulbs of Held. At P6, most NM neurons were circumscribed by a prominent DYN-I calyceal-type of ending. By P13, fewer NM cells were ringed by this DYN-I and by the third post-hatch week, there was very little DYN-I in NM. There were no obvious differences in the density of DYN-I terminals across either the rostrocaudal length or the mediolateral width of NM at any age examined. These results suggest that during a restricted time of development, end-bulbs of Held in the chick NM contain DYN.

Animals↗

Cellular generators of the binaural difference potential in cat.

In humans, lateralization and fusion of binaurally presented clicks are correlated with the latency and amplitude of the binaural difference potential (BDP) (e.g., Furst et al., 1985). The BDP is derived by subtracting the brainstem auditory evoked potential (BAEP) for binaural stimulation from the sum of the BAEPs for left and right monaural stimulation. Our aim in this work was to determine the cellular generators of the BDP and thus identify cells that may be crucial for specific types of binaural sound processing. To this end, we injected kainic acid into the superior olivary complex (SOC) or the cochlear nucleus (CN) in cats and examined the effects of the resulting lesions on the click-evoked BDP. Lesions confined to the anterior anteroventral CN (AVCNa) substantially reduced the BDP, while lesions primarily involving more posterior parts of the CN had little or no effect. BDP reductions occurred for lesions involving either high (> 10 kHz) or lower (< 10 kHz) characteristic frequency (CF) regions of the AVCNa (as well as the posterior CN). Lesions involving the SOC reduced the BDP and, in one case, eliminated the high-pass filtered (270 Hz cutoff) BDP. Combining these results with published information about the physiology and anatomy of auditory brainstem cells, we conclude that: (1) spherical cells in the AVCNa are essential for BDP production, (2) the earliest part of the BDP is generated by medial superior olive (MSO) principal cells which receive spherical cell inputs, (3) a later part is probably generated by the cellular targets of MSO principal cells and, (4) the cells involved in BDP generation have CFs above, as well as below, 10 kHz. Since humans, like cats, have a well-developed MSO, we suggest that the MSO may also be essential for BDP production in humans. Thus, perceptual correlates of the BDP, binaural fusion and click lateralization, apparently involve the MSO.

Acoustic Stimulation↗

Activity influences on neuronal connectivity within the auditory pathway.

HYPOTHESIS: Sensorineural hearing loss may be associated with altered patterns of neuronal connections within the central auditory pathway. STUDY DESIGN: The cat auditory system was used to address the relative importance of impulse traffic within the auditory nerve in maintaining central nervous system connections. METHODS: Acute, unilateral deafness was induced by tetrodotoxin intoxication of cochlear hair cells. Analysis focused on the structural patterns of contact between auditory nerve endings called end bulbs of Held and their target neurons. Specifically, end bulb morphology and target cell size within the cochlear nucleus were examined. Highly specialized synaptic contacts at this junction provide a powerful site for study. RESULTS: The principal finding was that abolished activity in the auditory nerve caused nerve endings to assume a different shape, typified by more branching and smaller terminal swellings. The new shape is one typically associated with only a subpopulation of fibers in normal-hearing cats--those exhibiting a high-threshold, low-spontaneous activity profile. This result implies that abolished activity alters patterns of nerve fiber contact with second-order neurons. Tetrodotoxin produced differential effects on subpopulations of target neurons within the brainstem and is interpretable on the basis of "weighing" synaptic inputs. Second-order neurons that receive large axosomatic inputs from their parent fiber were significantly smaller than neurons that receive small, axodendritic terminals. Thus, attenuated auditory activity may produce differential effects across the auditory pathway, thereby disrupting the normal balance of inputs into synaptic stations. CONCLUSIONS: Impulse traffic is a critical factor in the interaction between the ear and central auditory stations and appears necessary for the maintenance of key synapses. As hearing disorders with impaired comprehension may be modeled by studies of auditory deafferentation, these observations extend the possibility that changes in central neuronal connections underlie reduced capabilities for processing restored auditory input.

Animals↗

Effects of congenital deafness in the cochlear nuclei of Shaker-2 mice: an ultrastructural analysis of synapse morphology in the endbulbs of Held.

It is well established that manipulation of the sensory environment can significantly alter central auditory system development. For example, congenitally deaf white cats exhibit synaptic alterations in the cochlear nucleus distinct from age-matched, normal hearing controls. The large, axosomatic endings of auditory nerve fibers, called endbulbs of Held, display reduced size and branching, loss of synaptic vesicles, and a hypertrophy of the associated postsynaptic densities on the target spherical bushy cells. Such alterations, however, could arise from the cat's genetic syndrome rather than from deafness. In order to examine further the role of hearing on synapse development, we have studied endbulbs of Held in the shaker-2 ( sh2 ) mouse. These mice carry a point mutation on chromosome 11, affecting myosin 15 and producing abnormally short stereocilia in hair cells of the inner ear. The homozygous mutant mice are born deaf and develop perpetual circling behavior, although receptor cells and primary neurons remain intact at least for the initial 100 days of postnatal life. Endbulbs of Held in 7-month old, deaf sh2 mice exhibited fewer synaptic vesicles in the presynaptic ending, the loss of intercellular cisternae, and a hypertrophy of associated postsynaptic densities. On average, postsynaptic density area for sh2 endbulbs was 0.23 +/- 0.19 microm(2) compared to 0.07 +/- 0.04 microm(2) ( p < 0.001) for age-matched, hearing littermates. These changes at the endbulb synapse in sh2 mice resemble those of the congenitally deaf white cat and are consistent with the idea that they represent a generalized response to deafness.

Animals↗

Developmental changes in EPSC quantal size and quantal content at a central glutamatergic synapse in rat.

1. Developmental changes in amplitude and time course of single-fibre-evoked and spontaneous EPSCs mediated by AMPA and NMDA receptors at the endbulb-bushy cell synapse of rats from 4 to 22 days of age were recorded using whole-cell patch-clamp methods in in vitro slices of cochlear nucleus. 2. The mean conductance of the AMPA component of evoked EPSCs increased by 66 %, while that of the NMDA component decreased by 61 %, for 12- to 18-day-old rats cf. 4- to 11-day-old rats. 3. The mean AMPA spontaneous EPSC conductance increased by 54 %, while mean NMDA spontaneous EPSC conductance decreased by 83 %, for 12- to 22-day-old rats cf. 4- to 11-day-old rats. The mean number of quanta contributing to peak evoked AMPA conductance also increased by 78 % in the older age group, after correction for the asynchrony of evoked quantal release. 4. The decay time constant of spontaneous AMPA EPSCs showed a small decrease in older animals, while the decay time constant of spontaneous NMDA EPSCs was markedly decreased in older animals. The decay time constants of evoked NMDA EPSCs showed a quantitatively similar decrease to that of spontaneous NMDA EPSCs. This suggests that AMPA receptor subunit composition is unlikely to undergo developmental change, while NMDA receptor subunit composition may be substantially altered during synaptic maturation. 5. These data are consistent with a developmentally increased efficacy of AMPA receptor-mediated synaptic transmission at the endbulb-bushy cell synapse, due to an increase in underlying AMPA-mediated quantal size and content during the same period as a transient co-localization of NMDA receptors.

Animals↗

Quantal size is correlated with receptor cluster area at glycinergic synapses in the rat brainstem.

1. Whole-cell patch electrode recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were obtained in neurons of the rat anteroventral cochlear nucleus (AVCN). Mean mIPSC peak amplitude was found to vary considerably between AVCN neurons (range, -19.1 to -317.9 pA; mean +/- s.d., -159.1 +/- 100.7 pA; 14 cells). 2. Immunolabelling of glycinergic receptor clusters in AVCN neurons was performed using antibodies against the glycine receptor clustering protein gephyrin. Measurements of the area of gephyrin immunoreactive clusters were obtained using confocal fluorescence microscopy. These measurements showed a large variability in cluster area, not only in the same cell (mean coefficient of variation, c.v., 0.66 +/- 0.18; 16 cells), but also in mean cluster area between cells (range, 0.21-0.84 microm2; 16 cells). 3. A possible relationship between mIPSC amplitude and receptor cluster area was investigated in a further series of experiments, in which mIPSCs recordings and immunolabelling of glycine receptor clusters were obtained for the same cells. In these experiments, AVCN neurons were identified using intracellular labelling with neurobiotin. Successful results using a combination of whole-cell recordings, neurobiotin identification and immunolabelling were obtained for a total of 10 AVCN neurons. Analysis of the results revealed a positive, statistically significant correlation between mean receptor cluster size and mean mIPSC amplitude (P < 0.05, 10 cells, Spearman's correlation test). 4. These results provide direct experimental evidence supporting a hypothesis of central glycinergic transmission in which synaptic strength may be regulated by changes in the size of the postsynaptic receptor region.

Animals↗

Glycine receptors in adult guinea pig brain stem auditory nuclei: regulation after unilateral cochlear ablation.

In young adult guinea pigs, the effects of unilateral cochlear ablation were determined on the specific binding of [3H]strychnine measured in subdivisions of the cochlear nucleus (CN), the superior olivary complex, and the auditory midbrain, after 2, 7, 31, 60, and 147 postlesion days. Changes in binding relative to that in age-matched controls were interpreted as altered activity and/or expression of synaptic glycine receptors. Postlesion binding declined ipsilaterally in most of the ventral CN and in the lateral superior olive (LSO). Binding was modestly deficient in the ipsilateral dorsal CN and in the anterior part of the contralateral anteroventral CN. Binding was elevated in the contralateral LSO. Transient changes also occurred. Binding was elevated transiently, between 2 and 31 days, contralaterally in parts of the anteroventral CN, bilaterally in the medial superior olive (MSO), and bilaterally in most of the midbrain nuclei. Binding was deficient transiently, at 60 days, in most of the contralateral CN and bilaterally in the midbrain nuclei. The present findings, together with previously reported postlesion changes in glycine release, were consistent with persistently weakened glycinergic inhibitory transmission ipsilaterally in the ventral CN and the LSO and bilaterally in the dorsal CN. Glycinergic inhibitory transmission was strengthened in the contralateral LSO and transiently strengthened in the MSO bilaterally. A hypothetical model of the findings suggested that glycine receptor regulation may depend on excitatory and glycinergic input to auditory neurons. The present changes in glycine receptor activity may contribute to altered auditory functions, which often accompany hearing loss.

Animals↗

Membrane properties that shape the auditory code in three nuclei of the central nervous system.

OBJECTIVE: We investigated if auditory neurons have an intrinsic ability to radically transform auditory signals. METHOD: We surveyed membrane properties that control coding by neurons, identified with intracellular staining or infrared-DIC videomicroscopy, in three stations of the auditory pathway. We used intracellular and patch-clamp techniques in slices, to study the voltage responses to current pulse injections and distinguished voltage-gated conductances with selective blockers. RESULTS: First order spherical bushy cells in the anteroventral cochlear nucleus responded at a short, stable latency with single spikes, due to a perithreshold interaction of Na+ and Ca2+ conductances. Two K+ conductances suppressed firing after this onset-spike. Second-order principal neurons of the lateral superior olive use unspecified mechanisms to secure stable onset latencies but maintained a very regular tonic firing, resulting in a chopper pattern. Other intrinsic properties induced a marked accommodation in spike rate. When depolarized as during alert states, neurons in the medial geniculate body (MGB) of the thalamus fired with variable latencies in a tonic mode. At negative resting potentials characteristic of sleep states, they responded at the onset of a depolarization and the offset of a hyperpolarization with phasic bursts due to a transient low threshold Ca2+ current. In the phasic, but not tonic mode, MGB neurons produced high-threshold Ca2+ spikes that may couple signal transmission to the neuron's metabolism. The three neuron types exhibit analogue computing abilities that transform the same input into entirely different output patterns. Isoflurane anaesthesia induces a current shunt in MGB neurons, radically changing the properties and preventing normal responses. Thus, thalamocortical auditory codes are compromised under anaesthesia. CONCLUSION: At all investigated stations of the auditory pathway, input signals are transformed by activation of voltage-controlled conductances and other intrinsic membrane properties.

Anesthetics, Inhalation↗

Decreased temporal precision of auditory signaling in Kcna1-null mice: an electrophysiological study in vivo.

The voltage-gated potassium (Kv) channel subunit Kv1.1, encoded by the Kcna1 gene, is expressed strongly in the ventral cochlear nucleus (VCN) and the medial nucleus of the trapezoid body (MNTB) of the auditory pathway. To examine the contribution of the Kv1.1 subunit to the processing of auditory information, in vivo single-unit recordings were made from VCN neurons (bushy cells), axonal endings of bushy cells at MNTB cells (calyces of Held), and MNTB neurons of Kcna1-null (-/-) mice and littermate control (+/+) mice. Thresholds and spontaneous firing rates of VCN and MNTB neurons were not different between genotypes. At higher sound intensities, however, evoked firing rates of VCN and MNTB neurons were significantly lower in -/- mice than +/+ mice. The SD of the first-spike latency (jitter) was increased in VCN neurons, calyces, and MNTB neurons of -/- mice compared with +/+ controls. Comparison along the ascending pathway suggests that the increased jitter found in -/- MNTB responses arises mostly in the axons of VCN bushy cells and/or their calyceal terminals rather than in the MNTB neurons themselves. At high rates of sinusoidal amplitude modulations, -/- MNTB neurons maintained high vector strength values but discharged on significantly fewer cycles of the amplitude-modulated stimulus than +/+ MNTB neurons. These results indicate that in Kcna1-null mice the absence of the Kv1.1 subunit results in a loss of temporal fidelity (increased jitter) and the failure to follow high-frequency amplitude-modulated sound stimulation in vivo.

Acoustic Stimulation↗

Axons and synapses mediating electrically evoked startle: collision tests and latency analysis.

Davis et al. proposed that the primary acoustic startle reflex is mediated by synapses in the ventral cochlear nucleus (VCN), lateral lemniscus (LL) and caudal pontine reticular formation (PnC). The collision test was used here to estimate the locations of synapses mediating startle-like responses in these sites, and the conduction times between and across these synapses. Conditioning (C) and test (T) pulses were delivered to pairs of sites in chloral hydrate anaesthetized rats, and current thresholds for, and latencies of, hindlimb EMG responses were determined. When sites are axonally connected, thresholds increase at the same positive and negative C-T intervals ('symmetric collision'), but when sites are connected by strong synapses, thresholds increase at asymmetric C-T intervals. (i) Asymmetric collisions between VCN and contralateral PnC centered on a C-T interval of + 0.5 ms suggest a monosynaptic connection in ventrolateral pons (VLP), near LL. (ii) Asymmetric collisions between VCN and contralateral medulla centered on a C-T interval of + 0.85 to + 1.0 ms suggest a disynaptic connection. (iii) Asymmetric collisions between VLP and ipsilateral medulla centered on a C-T interval of 0.2-0.4 ms suggest a monosynaptic connection in PnC, as shown previously in freely behaving animals [32]. (4) Symmetric collisions between VLP and rostral PnC, and between caudal PnC and medulla suggest fast axonal connections. Latency differences between electrode pairs were generally similar to collision-derived conduction times. From these, conduction times and transmission times were estimated for each axon bundle and synapse in the circuit.

Animals↗

Deafferentation weakens excitatory synapses in the developing central auditory system.

Decreased excitatory synaptic activity during development often leads to pre- and postsynaptic atrophy, as assessed anatomically. The present study considers the effect of decreased excitatory transmission on the maturation of synaptic strength. Towards this end, cochlear nucleus neurons, which project to the ipsilateral lateral superior olive (LSO), were denervated in gerbils at postnatal day 7, before the onset of hearing. This manipulation was intended to disrupt spontaneous glutamatergic transmission in the LSO while sparing the glycinergic afferents from the medial nucleus of the trapezoid body (MNTB). Afferent-evoked synaptic activity was assessed 1-6 days after ablation in a brain slice preparation using whole-cell current- and voltage-clamp recordings. In control animals, ipsilaterally evoked excitatory postsynaptic potentials (EPSPs) were present in 91% of neurons tested, but were observed in only 60% of neurons following cochlea removal. The maximum EPSP amplitude was significantly smaller in manipulated neurons compared with controls, and this was accompanied by a higher incidence of ipsilaterally evoked inhibitory postsynaptic potentials (IPSPs). To study the efficacy of excitatory synapses in greater detail, voltage-clamp recordings were made in the presence of strychnine and AP-5 [D(O)-2-amino-5-phosphonopentanoic acid]. The minimum excitatory postsynaptic current (EPSC) amplitude, presumed to reflect the efficacy of a single glutamatergic afferent, was approximately 40% smaller in manipulated neurons. In contrast, MNTB-evoked IPSPs were similar in neurons from control and ablated animals. However, manipulated neurons often exhibited a rebound depolarization after a hyperpolarizing current pulse or an afferent-evoked IPSP. In 70% of manipulated neurons, synaptically evoked rebound depolarizations were reduced, but not eliminated, by glutamate receptor antagonists. The glycine receptor antagonist strychnine did eliminate the IPSP-associated depolarization in these neurons. Collectively, these results suggest that functional denervation of excitatory afferents decreases their synaptic efficacy as result of both cell loss as well as decreased strength of individual surviving synapses.

Acoustic Stimulation↗

The source of corticocollicular and corticobulbar projections in area Te1 of the rat.

Cortical area Te1 in the rat commonly is associated with primary auditory cortex. It is the source of direct projections to the inferior colliculus (IC), superior olivary complex (SOC), and the cochlear nucleus (CN). A question that arises is whether these descending pathways derive from a common source or separate populations of cortical neurons. We addressed this question in seven rats by injecting either Diamidino yellow (DiY) or Fast blue (FB) into the IC and injecting the other tracer into the CN (n=4) or SOC (n=3). All injections were made on the left side of the brain. In a sample of sections through area Te1 in both hemispheres, we counted single- and double-labeled cells. We estimate that IC-projecting cells outnumber those projecting to the CN or SOC by at least a factor of ten. The source of corticofugal pathways to the left IC was heavily biased towards the same side of the brain (ipsi/contra ratio 8 +/- 2.5), whereas it was more equally distributed between the two hemispheres for the left CN and SOC (ipsi/contra ratios ranged from 0.7-2.3). Finally, we observed that only 10-20% of those cells filled with a tracer injection in the CN or SOC also contained the tracer injected into the IC. In a previous study, we observed a similarly small percentage of double labeled cells when FB and DiY were injected into the CN and SOC, respectively. Combined with the distinct laminar distribution of IC-, SOC-, and CN-projecting neurons within layer V, the results suggest that these three pathways largely derive from different populations of cortical neurons.

Amidines↗

Development of spontaneous miniature EPSCs in mouse AVCN neurons during a critical period of afferent-dependent neuron survival.

During a critical period prior to hearing onset, cochlea ablation leads to massive neuronal death in the mouse anteroventral cochlear nucleus (AVCN), where cell survival is believed to depend on glutamatergic input. We investigated the development of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in AVCN neurons using whole cell patch-clamp techniques during [postnatal day 7 (P7)] and after (P14, P21) this critical period. We also examined the effects of unilateral cochlea ablation on mEPSC development. The two main AVCN neuron types, bushy and stellate cells, were distinguished electrophysiologically. Bushy cell mEPSCs became more frequent and faster between P7 and P14/P21 but with little change in amplitude. Dendritic filtering of mEPSCs was not detected as indicated by the lack of correlation between 10 and 90% rise times and decay time constants. Seven days after cochlea ablation at P7 or P14, mEPSCs in surviving bushy cells were similar to controls, except that rise and decay times were positively correlated (R = 0.31 and 0.14 for surgery at P7 and P14, respectively). Consistent with this evidence for a shift of synaptic activity from the somata to the dendrites, SV2 staining (a synaptic vesicle marker) forms a ring around somata of control but not experimental bushy cells. In contrast, mEPSCs of stellate cells showed few significant changes over these ages with or without cochlea ablation. Taken together, mEPSCs in mouse AVCN bushy cells show dramatic developmental changes across this critical period, and cochlea ablation may lead to the emergence of excitatory synaptic inputs impinging on bushy cell dendrites.

Action Potentials↗

Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature Lowicryl HM20 resin.

Double labeling is used for localizing two antigens simultaneously in the same tissue. We have used two approaches to post-embedding immunogold labeling to investigate whether nerve terminals in the guinea-pig anteroventral cochlear nucleus (AVCN) that contain gamma-aminobutyric acid (GABA) or glycine are capable of retrieving the other amino acid as part of an investigation of colocalization of these putative neurotransmitters. For this, vibroslices of perfusion-fixed brain stem were freeze-substituted and embedded in the low temperature resin, Lowicryl HM20. Simultaneous labeling of ultrathin sections was then performed with a mixture of a rabbit primary antibody to GABA and a guinea-pig primary antibody to the glycine transporter, GLYT2, followed by labeling with a mixture of secondary antibodies (goat anti-rabbit IgG-30 nm gold, goat anti-guinea pig IgG-15 nm gold). This approach indicated that GLYT2 occurs in the plasma membrane of some terminals that contain GABA. The other approach involved sequential labeling of ultrathin sections with a rabbit primary antibody to the GABA transporter, GAT1, followed by an anti-rabbit secondary antibody conjugated to 15-nm gold particles. Sections were then treated with paraformaldehyde vapor to denature any free anti-IgG binding sites on the first antibody, and labeled with a primary antibody to glycine also raised in rabbit followed by an anti-rabbit secondary antibody conjugated to 30-nm gold particles. This approach indicated that GAT1 occurs in the plasma membrane of some terminals that contain glycine. Thus, these techniques can be used to localize heat-labile multiple antigens in the same tissue.

Acrylic Resins↗

Gene expression differences over a critical period of afferent-dependent neuron survival in the mouse auditory brainstem.

Deprivation of auditory nerve input in young mice results in dramatic neuron death in the anteroventral cochlear nucleus (CN), while the same manipulation performed in older mice does not result in significant neuronal loss. The molecular basis underlying this critical period of susceptibility to loss of afferent input remains largely unknown. One possibility is that developmental differences in baseline mRNA expression of specific genes could predispose CN neurons to either death or survival after deafferentation. We used two microarray platforms to identify differentially expressed genes in the CN during and after this critical period. Results across platforms were also compared to each other. Three ages were examined; during the critical period (postnatal day (P)7), at the closing of the critical period (P14), and 1 week after the critical period (P21). Of all the genes surveyed (22,690 or 15,247), 1,082 were identified as significantly changed in expression during the critical period relative to after. Real-time reverse transcription polymerase chain reaction and immunohistochemistry confirmed and extended the microarray results for a subset of these genes. Further analysis of genes related to apoptotic pathways showed 6 out of 7 differentially expressed known pro-apoptotic genes had higher expression during the critical period. In contrast, 9 out of 11 differentially expressed known pro-survival genes increased after the critical period when CN neurons survive deprivation. This finding supports the concept that multiple neuroprotective mechanisms increase and pro-apoptotic factors decrease over development to protect mature neurons from stressful insults, making them less dependent on afferent input for survival.

Animals↗

Glycinergic mIPSCs in mouse and rat brainstem auditory nuclei: modulation by ruthenium red and the role of calcium stores.

Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in central neurons are usually highly variable in amplitude due to many factors such as intrinsic postsynaptic channel fluctuations at each release site, site-to-site variability between release sites, electrotonic attenuation due to variable dendritic locations of synapses, and the possibility of synchronous multivesicular release. A detailed knowledge of these factors is essential for the interpretation of mIPSC amplitude distributions and mean quantal size. We have studied glycinergic mIPSCs in two auditory brainstem nuclei, the rat anteroventral cochlear nucleus (AVCN) and the mouse medial nucleus of the trapezoid body (MNTB). Our previous results have demonstrated the location of glycinergic synapses on these neurons to be somatic, thus avoiding electrotonic complications. Spontaneous glycinergic mIPSCs were recorded from AVCN and MNTB neurons in brainstem slices, in the presence of TTX to block action potentials, and 6-cyano-7-nitroquinoxaline-2, 3-dione, (+/-)-2-amino-5-phosphonopentanoic acid and bicuculline to block glutamatergic and GABAergic synaptic currents. Ruthenium red (RuR), which was used to increase the frequency of mIPSCs, significantly changed the shape of most (90 %) mIPSC amplitude distributions by increasing the proportion of large-amplitude mIPSCs. The possibility was investigated (following previous evidence at GABAergic synapses) that large-amplitude glycinergic mIPSCs are due to synchronous multivesicular release initiated by presynaptic calcium sparks from ryanodine-sensitive calcium stores. Interval analysis of mIPSCs indicated that the number of potentially undetected (asynchrony < 0.5 ms) multivesicular mIPSCs was low in comparison with the number of large-amplitude mIPSCs. Ryanodine, thapsigargin and calcium-free perfusate did not reduce the frequency of large-amplitude mIPSCs (> 150 pA), arguing against a significant role for presynaptic calcium stores. Our results support previous evidence suggesting that RuR increases miniature postsynaptic current (mSC) frequency by a mechanism that does not involve presynaptic calcium stores. Our results also indicate that at glycinergic synapses in the AVCN and MNTB, site-to-site variability in mIPSC amplitude, rather than multivesicular release, is a major factor underlying the large range of amplitudes of glycinergic mIPSCs.

Animals↗

Serotonin reuptake inhibitors in auditory processing disorders in elderly patients: preliminary results.

OBJECTIVE/HYPOTHESIS: One mechanism associated with degeneration in the elderly is the decrease of neurotransmitters. In the central auditory pathway serotonin, can be found from cochlear nucleus to the auditory cortex, and it constitutes one of the most important neuromodulatory circuits in hearing processing. The present study analyzed the action of citalopram, a selective inhibitor of serotonin reuptake, in aged patients with normal to moderate sensorineural hearing loss (HL) and low performance on auditory processing. STUDY DESIGN/METHOD: Prospective, double-blind, randomized, placebo-controlled study. Thirty-eight selected patients were randomly divided into two groups. Nineteen patients made up group A and received placebo for 60 days. Nineteen patients of Group B received 20 mg per day of citalopram for 60 days. Hearing evaluation was performed initially and after 60 days and included pure-tone audiometry, speech discrimination test (SDT), emittanciometry (acoustic impedance audiometry), identification of synthetic sentences with an ipsilateral competitive message (SSI/ICM), tests of pitch-pattern sequences (PPS), and the staggered spondaic words test (SSW). RESULTS: Comparisons of tests of auditory processing pre- and posttreatment in each group showed a statistical improvement in performance on all tests in group B after 2 months of therapy. Comparisons pre- and posttreatment between groups showed that patients who received citalopram presented statistically significantly better results in the SSI/ICM test (P < .0001) after treatment. The same comparison in results for the PPS test and the SSW test revealed a tendency (P = .09 and 0.058, respectively) toward better performance in the group receiving citalopram. CONCLUSION: These preliminary results suggest that the use of citalopram can have a positive impact on auditory processes in elderly patients with low performance in auditory process.

Aged↗

Phosphorylation regulates spontaneous and evoked transmitter release at a giant terminal in the rat auditory brainstem.

1. The role of phosphorylation in synaptic transmission was investigated at a large glutamatergic terminal, the endbulb of Held, on bushy cells in the rat anteroventral cochlear nucleus (AVCN). 2. Whole-cell recordings of excitatory postsynaptic currents (EPSCs) were used to examine the effects of kinase inhibitors and activators on low-frequency (baseline) evoked release, spontaneous release, paired-pulse facilitation (PPF) or depression (PPD), repetitive stimuli and recovery from depression. 3. Application of the kinase inhibitor H7 (100 microM) reduced low-frequency evoked EPSC amplitude (by 15 %) and simultaneously increased PPF (or reduced PPD), with no significant change in other aspects of transmission. H7 did not affect the amplitude or frequency of spontaneous miniature EPSCs. 4. Phorbol esters increased EPSC amplitude (by 50 %) with a concomitant decrease in PPF (or increase in PPD), and reduced the final EPSC amplitude during repetitive stimuli. The effect of phorbol esters was due exclusively to protein kinase C (PKC) activation, as the specific PKC inhibitor bis-indolylmaleimide (Bis) completely blocked the potentiating effect of phorbol esters on EPSC amplitude. 5. Significantly, phorbol esters did not increase the evoked EPSC amplitude at connections in which release was maximized using high extracellular calcium concentrations (4-6 mM). 6. Phorbol esters increased the frequency of spontaneous miniature EPSCs in physiological calcium (by 275 %), and in high extracellular calcium (by 210 %) when phorbol esters did not increase the evoked EPSC amplitude. 7. Our results are most consistent with the actions of H7 to decrease low-frequency release probability and phorbol esters to increase low-frequency release probability at the endbulb-bushy cell synaptic connection in the AVCN. The effects of H7 and phorbol esters on paired-pulse responses and tetanic depression appear to be largely consequential to these changes in low-frequency release probability.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗