Motoneurons In Slices Research Articles

Staircase Currents in Motoneurons: Insight into the Spatial Arrangement of Calcium Channels in the Dendritic Tree

In spinal motoneurons, activation of dendritically located depolarizing conductances can lead to amplification of synaptic inputs and the production of plateau potentials. Immunohistochemical and computational studies have implicated dendritic CaV1.3 channels in this amplification and suggest that CaV1.3 channels in spinal motoneurons may be organized in clusters in the dendritic tree. Our goal was to provide physiological evidence for the presence of multiple discrete clusters of voltage-gated calcium channels in spinal motoneurons and to explore the spatial arrangement of these clusters in the dendritic tree. We recorded voltage-gated calcium currents from spinal motoneurons in slices of mature mouse spinal cords. We demonstrate that single somatic voltage-clamp steps can elicit multiple inward currents with varying delays to onset, resulting in a current with a "staircase"-like appearance. Recordings from cultured dorsal root ganglion cells at different stages of neurite development provide evidence that these currents arise from the unclamped portions of the dendritic tree. Finally, both voltage- and current-clamp data were used to constrain computer models of a motoneuron. The resultant simulations impose two conditions on the spatial distribution of CaV channels in motoneuron dendrites: one of asymmetry relative to the soma and another of spatial separation between clusters of CaV channels. We propose that this compartmentalization would provide motoneurons with the ability to process multiple sources of input in parallel and integrate this processed information to produce appropriate trains of action potentials for the intended motor behavior.

Roles of ryanodine and inositol triphosphate receptors in regulation of plateau potentials in turtle spinal motoneurons

Generation of plateau potentials in spinal motoneurons depends on activation of voltage sensitive L-type Ca 2+ channels. These channels are facilitated by metabotropic receptors known to promote release of Ca 2+ from intracellular stores. The aim of this study is to determine if Ca 2+-release receptors in the endoplasmic reticulum (ER) that are sensitive to ryanodine (RyRs) and to inositol triphosphate receptors (IP 3Rs) contribute to the generation of plateau potentials. The effects of antagonists to RyRs, IP 3Rs and phospholipase C (PLC) were tested on discharge patterns associated with plateau potentials in motoneurons in slices from the spinal cord of the turtle. Plateau-related discharge patterns, un-facilitated or facilitated by agonists for group I glutamate metabotropic receptors, muscarine-sensitive cholinergic receptors or L-type Ca 2+ channels were inhibited by blockade of RyRs. In contrast, antagonists of IP 3Rs or PLC preferentially inhibited plateau-related discharge patterns when facilitated by activation of metabotropic receptors but in only half of the cells when promoted in the absence of metabotropic facilitators. Our findings show that RyRs and IP 3Rs regulate the generation of plateau potentials in motoneurons and suggest that RyRs may be directly involved with activation of the plateau potential.

Intracellular activity of rat spinal cord motoneurons in slices

Using a modification of Aghajanian and Rasmussen's techniques, we have developed an adult rat cervical spinal cord slice preparation in which motoneurons remain viable. Key factors are replacement of all sodium ions in the perfusion medium with sucrose during cutting and incubation, and gentle manipulation of the tissues to prevent root damage during removal. Intracellular recordings were confirmed as motoneuronal by intracellular injection of Lucifer yellow, allowing visualization of dendrites and cell body, and showing an axonal bleb at the cut end in the ventral root. Over 50 neurons were recorded for periods of between 30 min and 4 h. Cervical motoneurons ( n=10) had an average membrane potential of −62 mV, average input resistance of 24 MΩ, and showed no spontaneous activity. Ionophoresis application of the glutamate agonists, AMPA and NMDA, revealed potent excitation by AMPA but little or no response to NMDA. While NMDA receptors reportedly are prominent in developing rodent motoneurons, these observations indicate otherwise in the adult. Upon prolonged ionophoresis, or bath application, depolarizing responses to AMPA led to depolarization and spike inactivation that was often irreversible. The apparent lack of desensitization of AMPA responses, usually seen in other neurons, may underlie the unique vulnerability of motoneurons to excitotoxic damage.

Calcium dynamics and buffering in motoneurones of the mouse spinal cord.

1. A quantitative analysis of endogenous calcium homeostasis was performed on 65 motoneurones in slices of the lumbar spinal cord from 2- to 8-day-old mice by simultaneous patch-clamp and microfluorometric calcium measurements. 2. Somatic calcium concentrations were monitored with a temporal resolution in the millisecond time domain. Measurements were performed by using a monochromator for excitation and a photomultiplier detection system. 3. Somatic calcium signalling was investigated during defined voltage-clamp protocols. Calcium responses were observed for membrane depolarizations positive to -50 mV. A linear relation between depolarization time and free calcium concentrations ([Ca2+]i) indicated that voltage-dependent calcium influx dominated the response. 4. Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. In the presence of fura-2 and mag-fura-5, calcium transients decayed according to a monoexponential function. Decay-time constants showed a linear dependence on dye concentration and the extrapolated constant in the absence of indicator dye was 371 +/- 120 ms (n = 13 cells, 21 C). 5. For moderate elevations (< 1 microM), recovery kinetics of depolarization-induced calcium transients were characterized by a calcium-independent, 'effective' extrusion rate gamma = 140 +/- 47 s-1 (n = 13 cells, 21 C). 6. The endogenous calcium binding ratio for fixed buffers in spinal motoneurones was kappaB' = 50 +/- 17 (n = 13 cells), indicating that less than 2 % of cytosolic calcium ions contributed to [Ca2+]i. 7. Endogenous binding ratios in spinal motoneurones were small compared to those found in hippocampal or cerebellar Purkinje neurones. From a functional perspective, they provided motoneurones with rapid dynamics of cytosolic [Ca2+]i for a given set of influx, extrusion and uptake mechanisms. 8. With respect to pathophysiological conditions, our measurements are in agreement with a model where the selective vulnerability of spinal motoneurones during excitotoxic conditions and motoneurone disease partially results from low endogenous calcium buffering.

Postsynaptic activity of spinal motoneurons of early postnatal ratsin vitro: Effects of calcium channel blockers

Postsynaptic activity was intracellularly recorded from motoneurons in slices of the spinal cord of early postnatal rats. The amplitude of background EPSP varied within 0.4 to 4.0 mV (a mean of 2.05 ± 0.18 mV); their mean frequency was 11.5 ± 4.9 sec−1. Distributions of the EPSP amplitudes In different cells were unimodal and showed a clear right-side asymmetry. Application of calcium channel blockers (1–2 μM nifedipine, 50–100 μM Cd2+, or 500 μM amyloride) considerably modified the EPSP parameters. Nifedipine (3 cells) somewhat enhanced the amplitude of background EPSP (to 108.3 ± 6.6%, on the average) and significantly Increased their frequency (145.6 ± 14.4%). Cadmium (3 cells) and amyloride (7 cells) decreased both the EPSP amplitude (means of 88.9 ±2.8% and 73.1 ± 3.8%, respectively) and their frequency (69.0 ± 5.6% and 61.4 ± 7.8%). All tested agents evoked no dramatic shifts of the membrane potential of motoneurons. It is concluded that the observed modifications of the EPSP amplitudes and frequencies result from modulation of the activity of both pre- and post-synaptically localized high- and low-threshold Ca2+ channels. The reason for the opposite direction of nifedipine effects, as compared with those of Cd2+ and amyloride, is discussed.

Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse.

1. Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty-five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2- to 6-day-old mice. 2. Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII). 3. Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching tau < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura-2 and mag-fura-5. 4. Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0.7 +/- 0.2 s, suggesting rapid somatic calcium dynamics under physiological conditions. 5. By a process of back-extrapolation, the 'added buffer' method, a calcium binding ratio of 41 +/- 12 (9 cells) was obtained indicating that 98% of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers. 6. Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium-related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.

Contribution of outward currents to spike-frequency adaptation in hypoglossal motoneurons of the rat.

Contribution of outward currents to spike-frequency adaptation in hypoglossal motoneurons of the rat. J. Neurophysiol. 78: 2246-2253, 1997. Spike-frequency adaptation has been attributed to the actions of several different membrane currents. In this study, we assess the contributions of two of these currents: the net outward current generated by the electrogenic Na+-K+ pump and the outward current that flows through Ca2+-activated K+ channels. In recordings made from hypoglossal motoneurons in slices of rat brain stem, we found that bath application of a 4-20 microM ouabain solution produced a partial block of Na+-K+ pump activity as evidenced by a marked reduction in the postdischarge hyperpolarization that follows a period of sustained discharge. However, we observed no significant change in either the initial, early, or late phases of spike-frequency adaptation in the presence of ouabain. Adaptation also has been related to increases in the duration and magnitude of the medium-duration afterhyperpolarization (mAHP) mediated by Ca2+-activated K+ channels. When we replaced the 2 mM Ca2+ in the bathing solution with Mn2+, there was a significant decrease in the amplitude of the mAHP after a spike. The decrease in mAHP amplitude resulted in a decrease in the magnitude of the initial phase of spike-frequency adaptation as has been reported previously by others. However, quite unexpectedly we also found that reducing the mAHP resulted in a dramatic increase in the magnitude of both the early and late phases of adaptation. These changes could be reversed by restoring the normal Ca2+ concentration in the bath. Our results with ouabain indicate that the Na+-K+ pump plays little, if any, role in the three phases of adaptation in rat hypoglossal motoneurons. Our results with Ca2+ channel blockade support the hypothesis that initial adaptation is, in part, controlled by conductances underlying the mAHP. However, our failure to eliminate initial adaptation completely by blocking Ca2+ channels suggests that other membrane mechanisms also contribute. Finally, the increase in both the early and late phases of adaptation in the presence of Mn2+ block of Ca2+ channels lends further support to the hypothesis that the initial and later (i.e., early and late) phases of spike-frequency adaptation are mediated by different cellular mechanisms.

Developmental alterations in NMDA receptor-mediated currents in neonatal rat spinal motoneurons

Postnatal development of N-methyl- d-aspartate (NMDA) receptors expressed in motoneurons was studied by tight-seal whole-cell recordings from identified motoneurons in slices of neonatal rat spinal cord. The magnitude of NMDA-induced currents was large during the early postnatal period, and then it decreased gradually through postnatal day (PND) 15. The pharmacological properties of NMDA-induced currents altered during this period, suggesting that the combination of NMDA receptor subunits changes during development. The magnitude of the NMDA receptor-mediated component of excitatory postsynaptic current evoked by electrical stimulation of an adjacent neuron also decreased from PNDs 1–5 through PNDs 11–15. NMDA receptors appear to underlie the mechanisms of activity-dependent development of spinal motoneurons during early postnatal life.

Adenosine 3':5'-cyclic monophosphate mediates a 5-hydroxytryptamine-induced response in neonatal rat motoneurones.

5-Hydroxytryptamine (5-HT) is present in nerve fibres descending from the brainstem Raphe nuclei to motoneurones and its release is thought to exert excitatory actions. 5-HT, applied from the outside, directly depolarizes spinal and cranial motoneurones in slices. This action of 5-HT is mediated, in part, by an inwardly rectifying cationic current, Ih. In cardiac cells, an equivalent current, if, has been shown to be directly activated by adenosine 3':5'-cyclic monophosphate (cAMP) applied to the inside of the patch membrane. By applying the whole-cell method to thin slices of brainstem and spinal cord, we have shown that intracellularly applied camp and extracellularly applied dibutyryl camp or forskolin mimics the inward current induced by 5-ht. The selective cAMP phosphodiesterase inhibitor, Ro 20-1724, clearly prolonged the 5-HT-induced current. Maximal doses of dibutyryl cAMP or forskolin occluded the 5-HT-induced current. The broad spectrum protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methlypiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) had no effect on the currents induced by 5-HT and forskolin. From these results, we suggest that activation of 5-HT receptors on the motoneuronal membrane stimulates formation of intracellular cAMP, thereby inducing the inward current, possibly by a direct action on Ih.

Ionic mechanisms mediating 5-hydroxytryptamine- and noradrenaline-evoked depolarization of adult rat facial motoneurones.

1. The actions of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) on the membrane properties of facial motoneurones in slices from the adult rat brainstem in vitro were examined using intracellular recording techniques. 2. In voltage clamp recording, hyperpolarizing voltage steps (> 20 mV), from holding potentials at or close to the resting potential, induced a slowly activating, voltage-dependent inward current possessing properties similar to the hyperpolarization-activated current (Ih) seen in other cell types. From tail current analysis two groups of facial motoneurones can be distinguished in terms of the activation range for Ih, one with a half-maximal activation at -81 mV and the other at -94 mV but with similar shapes. 3. 5-HT (120/126) and NA (21/21) depolarized facial motoneurones. The reversal potentials (Em) obtained from peak voltage amplitude I-V plots in varying extracellular potassium concentrations suggested mechanisms involving a decrease in K+ conductance. 4. Under voltage clamp, close to the resting potential, both 5-HT (39/41) and NA (13/13) evoked inward currents. 5. I-V plots and plots of 5-HT-sensitive current at different membrane potentials, obtained from currents evoked by voltage steps and measured before the development of Ih (instantaneous current), indicated that the 5-HT-evoked inward current was predominately associated with a decrease in conductance but with a range of reversal potentials for 5-HT (E5-HT) from close to, to much more negative than the reversal potential for a potassium conductance (EK). In some cases no change or increases in instantaneous conductance were observed. 6. Steady-state I-V relationships and plots of 5-HT-sensitive current, measured after development of Ih, indicated a 5-HT-associated conductance increase with a time and voltage dependence close to that of Ih, which could be abolished by extracellular caesium (2-5 mM). 7. The NA-evoked inward current was always associated with a decrease in conductance. Instantaneous and steady-state I-V relationships as well as plots of NA-sensitive current indicated a reversal potential at EK. 8. The activation curve for Ih was shifted to more positive potentials in the presence of 5-HT. The time constant for activation of Ih showed a similar shift. 9. 5-Carboxamidotryptamine (5-CT), a 5-HT receptor agonist, was selective for the enhancement of Ih and only evoked an inward current when the holding potential was within the activation range of Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of norepinephrine on facial motoneurons in slices of guinea-pig brain stem

Effects of norepinephrine on facial motoneurons in slices of guinea-pig brain stem