RESUMEN
The effects of the anesthetic ketamine on properties of inspiratory bursts (I-bursts) in mouse hypoglossal nerve activity were studied in vivo and in vitro. In urethane anesthetized mice we observed rhythmic I-phase activity in only one of eight pups at P9 days. In contrast in older mice rhythmic I-phase hypoglossal activity was almost always observed. Ketamine caused a reduction in I-burst frequency and an increase in peak integrated hypoglossal nerve activity in all three age groups studied (P10-P13, P15-P20 and adult mice). In these mice I-phase oscillations, due to hypoglossal motoneurons firing clusters of action potentials at a particular frequency, were observed in control and after ketamine. Ketamine did not change the frequency of the dominant spectral peak determined from power spectra examined from 0 to 200 Hz. The effects of ketamine were also studied in vitro in the mouse rhythmic medullary slice preparation. Ketamine reduced hypoglossal I-burst frequency and I-burst peak integrated amplitude. Oscillations were observed in I-phase activity, and as in the in vivo studies ketamine did not shift the dominant spectral peak frequency. These results demonstrate that in vivo and in vitro ketamine results in significant changes in I-burst frequency and peak integrated hypoglossal nerve activity, but changes in the oscillation frequency are minimal.
Asunto(s)
Anestésicos Disociativos/farmacología , Nervio Hipogloso/efectos de los fármacos , Nervio Hipogloso/crecimiento & desarrollo , Inhalación/efectos de los fármacos , Ketamina/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Nervio Hipogloso/citología , Técnicas In Vitro , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiologíaRESUMEN
We investigated whether 5-HT(1B) receptor-mediated inhibition of evoked glycinergic inhibitory postsynaptic currents (eIPSCs) in hypoglossal motoneurons (HMs) changed postnatally. In HMs from postnatal days 2-3 (P2-3, neonate) and P10-11 (juvenile) rats bath application of 5-HT (10 microM) caused a not significantly different large reduction in eIPSC amplitude to 35.0+/-22.5% (mean+/-S.D.) and 35.4+/-10.6% of control; respectively. The dose-response relationship for the 5-HT(1B) receptor agonist, CP-93,129, revealed that the mean agonist concentration at half-maximal inhibition (IC(50)) was similar, 1.6 and 2.0 nM, respectively. Additionally, strong antibody labeling of 5-HT(1B) receptors in the hypoglossal motor nucleus was observed in neonates, juveniles and adults. These results demonstrate that over the postnatal period studied, 5-HT(1B) receptor-mediated inhibition of glycinergic eIPSCs is not age dependent.
Asunto(s)
Glicina/metabolismo , Nervio Hipogloso/crecimiento & desarrollo , Neuronas Motoras/fisiología , Inhibición Neural , Receptores de Serotonina/fisiología , Transmisión Sináptica , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Glicinérgicos/farmacología , Nervio Hipogloso/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Inhibición Neural/efectos de los fármacos , Técnicas de Placa-Clamp , Piridinas/farmacología , Pirroles/farmacología , Ratas , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Estricnina/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.
Asunto(s)
Nervio Hipogloso/metabolismo , Inhibición Neural/fisiología , Receptores de GABA-A/metabolismo , Receptores de GABA-B/fisiología , Receptores de Glicina/metabolismo , Transmisión Sináptica/fisiología , Animales , Baclofeno/farmacología , Tronco Encefálico/citología , Tronco Encefálico/fisiología , GABAérgicos/farmacología , Agonistas de Receptores GABA-B , Nervio Hipogloso/citología , Nervio Hipogloso/crecimiento & desarrollo , Neuronas Motoras/metabolismo , Ácidos Nipecóticos/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Mecánica Respiratoria/fisiologíaRESUMEN
Respiratory motoneurons provide the exclusive drive to respiratory muscles and therefore are a key relay between brainstem neural circuits that generate respiratory rhythm and respiratory muscles that control moment of gases into and out of the airways and lungs. This review is focused on postnatal development of fast ionotropic synaptic transmission to respiratory motoneurons, with a focus on hypoglossal motoneurons (HMs). Glutamatergic synaptic transmission to HMs involves activation of both non-NMDA and NMDA receptors and during the postnatal period co-activation of these receptors located at the same synapse may occur. Further, the relative role of each receptor type in inspiratory-phase motoneuron depolarization is dependent on the type of preparation used (in vitro versus in vivo; neonatal versus adult). Respiratory motoneurons receive both glycinergic and GABAergic inhibitory synaptic inputs. During inspiration phrenic and HMs receive concurrent excitatory and inhibitory synaptic inputs. During postnatal development in HMs GABAergic and glycinergic synaptic inputs have slow kinetics and are depolarizing and with postnatal development they become faster and hyperpolarizing. Additionally shunting inhibition may play an important role in synaptic processing by respiratory motoneurons.
Asunto(s)
Neuronas Motoras/fisiología , Mecánica Respiratoria/fisiología , Músculos Respiratorios/crecimiento & desarrollo , Transmisión Sináptica/fisiología , Animales , Humanos , Músculos Respiratorios/inervaciónRESUMEN
During an inspiration the output of hypoglossal (XII) motoneurons (HMs) in vitro is characterized by synchronous oscillatory firing in the 20- to 40-Hz range. To maintain synchronicity it is important that the cells fire with high reliability and precision. It is not known whether the intrinsic properties of HMs are tuned to maintain synchronicity when stimulated with time-varying inputs. We intracellularly recorded from HMs in an in vitro brain stem slice preparation from juvenile mice. Cells were held at or near spike threshold and were stimulated with steady or swept sine-wave current functions (10-s duration; 0- to 40-Hz range). Peristimulus time histograms were constructed from spike times based on threshold crossings. Synaptic transmission was suppressed by including blockers of GABAergic, glycinergic, and glutamatergic neurotransmission in the bath solution. Cells responded to sine-wave stimulation with bursts of action potentials at low (<3- to 5-Hz) sine-wave frequency, whereas they phase-locked 1:1 to the stimulus at intermediate frequencies (3-25 Hz). Beyond the 1:1 frequency range cells were able to phase-lock to subharmonics (1:2, 1:3, or 1:4) of the input frequency. The 1:1 phase-locking range increased with increasing stimulus amplitude and membrane depolarization. Reliability and spike-timing precision were highest when the cells phase-locked 1:1 to the stimulus. Our findings suggest that the coding of time-varying inspiratory synaptic inputs by individual HMs is most reliable and precise at frequencies that are generally lower than the frequency of the synchronous inspiratory oscillatory activity recorded from the XII nerve.
Asunto(s)
Potenciales de Acción/fisiología , Nervio Hipogloso/fisiología , Neuronas Motoras/fisiología , Reclutamiento Neurofisiológico/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Tronco Encefálico/citología , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Nervio Hipogloso/efectos de los fármacos , Técnicas In Vitro , Ratones , Neuronas Motoras/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Reclutamiento Neurofisiológico/efectos de la radiación , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de la radiaciónRESUMEN
Neurons within the intact respiratory network produce bursts of action potentials that cause inspiration or expiration. Within inspiratory bursts, activity is synchronized on a shorter timescale to generate clusters of action potentials that occur in a set frequency range and are called synchronous oscillations. We investigated how GABA and glycine modulate synchronous oscillations and respiratory rhythm during postnatal development. We recorded inspiratory activity from hypoglossal nerves using the in vitro rhythmically active mouse medullary slice preparation from P0-P11 mice. Average oscillation frequency increased with postnatal development, from 17 +/- 12 Hz in P0-P6 mice (n = 15) to 38 +/- 7 Hz in P7-P11 mice (n = 37) (P < 0.0001). Bath application of GABAA and GlyR antagonists significantly reduced oscillation power in neonates (P0-P6) and juveniles (P7-P10) and increased peak integrated activity in both age groups. To test whether elevating slice excitability is sufficient to reduce oscillation power, Substance P was bath applied alone. Substance P, although increasing peak integrated activity, had no significant effect on oscillation power. Prolonging the time course of GABAergic synaptic currents with zolpidem decreased the median oscillation frequency in P9-P10 mouse slices. These data demonstrate that oscillation frequency increases with postnatal development and that both GABAergic and glycinergic transmission contribute to synchronization of activity. Further, the time course of synaptic GABAergic currents is a determinant of oscillation frequency.
Asunto(s)
Glicina/fisiología , Inhalación/fisiología , Neuronas Motoras/fisiología , Periodicidad , Mecánica Respiratoria/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos/fisiología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Nervio Hipogloso/fisiología , Ratones , Ratones Endogámicos , Neurotransmisores/farmacología , Piridinas/farmacología , Receptores de Glicina/antagonistas & inhibidores , Estricnina/farmacología , Sustancia P/farmacología , ZolpidemRESUMEN
Previously, we showed that ethanol increases synaptic glycine currents, an effect that depends on ethanol concentration and developmental age of the preparation. Glycine receptor (GlyR) subunits undergo a shift from alpha2/beta to alpha1/beta from neonate to juvenile ages, with synaptic glycine currents from neonate hypoglossal motoneurons (HMs) being less sensitive to ethanol than those from juvenile HMs. Here we investigate whether these dose and developmental effects are also present in excised membrane patches containing GlyRs and if ethanol changes response kinetics. We excised outside-out patches from rat HM somata and applied glycine using either a picospritzer or piezo stack translator. Ethanol (100 mM) increased the response to glycine (200 microM) of patches from neonate and juvenile HMs. However, 30 mM ethanol increased the response from only juvenile HM patches. Using a lower concentration of glycine (30 microM) to observe single channel openings, we found that 100 mM ethanol increased the number of GlyRs that open in response to glycine and decreased first latency to channel opening. To investigate GlyR kinetic properties, we rapidly applied 1 mM glycine for 1 ms and found that glycine currents were increased by ethanol (100 mM) at both ages. For patches from juvenile HMs, ethanol consistently decreased response rise-time and increased response decay time. Using kinetic modeling, we determined that ethanol's potentiation of the glycine response arises from an increase in the glycine association (k(on)) and a decrease in the dissociation (k(off)) rate constants, resulting in increased glycine affinity of the GlyR.
Asunto(s)
Etanol/farmacología , Tiempo de Reacción/efectos de los fármacos , Receptores de Glicina/fisiología , Factores de Edad , Animales , Relación Dosis-Respuesta a Droga , Glicina/farmacología , Bulbo Raquídeo/efectos de los fármacos , Bulbo Raquídeo/fisiología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/fisiología , Receptores de Glicina/agonistasRESUMEN
NMDA receptor (NMDAR)-mediated spontaneous miniature excitatory postsynaptic currents (mEPSCs) are potentiated by exogenously applied glycine. In this study, we have investigated the effect of blocking glycine uptake on NMDAR-mediated responses from hypoglossal motorneurons (HMs) of rats. We have used N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine (NFPS; 500 nM), an antagonist of glycine transporter-1 (GLYT1), to study the effect of blocking endogenous glycine uptake on NMDAR-mediated synaptic transmission. We show that the charge transfer of NMDAR-mediated mEPSCs was enhanced after NFPS application in neonate (P2-4) and juvenile rats (P8-11), but this enhancement was statistically significant only in the former group. Spontaneous and evoked EPSCs showed a significant increase in NMDAR-mediated charge transfer in both neonates and juveniles. The greater increase observed in spontaneous EPSCs may be due to increased release of glycine from glycinergic terminals in the absence of tetrodotoxin (TTX). Brief application of NMDA onto HMs showed that extrasynaptic NMDARs may be potentiated by NFPS only in the presence of extracellularly applied glycine. Immunohistochemistry of GLYT1 and -2 shows labeling throughout the hypoglossal nucleus. GLYT1 labeling is diffuse and becomes more intense and uniform during development consistent with its glial localization. In contrast, GLYT2 labeling is intense throughout the nucleus and increases in intensity with age. Our results demonstrate the glycine binding site of the NMDAR is not saturated in the brain stem slice during the first 2 wk of development. We suggest that modulation of glycine concentration by GLYT1 is an important mechanism to regulate NMDAR-mediated synaptic transmission.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Nervio Hipogloso/fisiología , Neuronas Motoras/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Sarcosina/análogos & derivados , Transmisión Sináptica/efectos de los fármacos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animales , Animales Recién Nacidos , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática , Nervio Hipogloso/citología , Nervio Hipogloso/efectos de los fármacos , Inmunohistoquímica , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Sarcosina/farmacologíaRESUMEN
Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9-13) are more sensitive to ethanol than neonate HMs (P1-3). We have now extended our previous study to investigate ethanol modulation of synaptic GABA(A) receptors (GABA(A)Rs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 +/- 3%, P < 0.05) and juvenile (16 +/- 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 +/- 17%, P < 0.05) and juvenile (40 +/- 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 +/- 3%, P < 0.0001) and juvenile (41 +/- 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABA(A) receptors and that 100 mM ethanol has the opposite effect on GABA(A)R-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.