Consuelo Morgado-Valle

Sodium and Calcium Current-Mediated Pacemaker Neurons and Respiratory Rhythm Generation

The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBötzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (INaP)-mediated bursting in ∼5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. INaP-mediated bursting was voltage dependent and blocked by 20 μm riluzole (RIL). We found Ca2+ current (ICa)-dependent bursting in 7.5% of inspiratory neurons in P8-P10 slices, but in P0-P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 μm Cd2+ or flufenamic acid (FFA) (10-200 μm), which suggests that a Ca2+-activated inward cationic current (ICAN) underlies burst generation. These data substantiate our observation that P0-P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nm TTX or coapplication of 20 μm RIL + FFA (100-200 μm) stops the respiratory rhythm, but that adding 2 μm substance P restarts it. We conclude that INaP and ICAN enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When INaP and ICAN are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBötzinger complex.

Respiratory rhythm: an emergent network property?

We tested the hypothesis that pacemaker neurons generate breathing rhythm in mammals. We monitored respiratory-related motor nerve rhythm in neonatal rodent slice preparations. Blockade of the persistent sodium current (I(NaP)), which was postulated to underlie voltage-dependent bursting in respiratory pacemaker neurons, with riluzole (< or =200 microM) did not alter the frequency of respiratory-related motor output. Yet, in every pacemaker neuron recorded (50/50), bursting was abolished at much lower concentrations of riluzole (< or =20 microM). Thus, eliminating the pacemaker population (our statistics confirm that this population is reduced at least 94%, p < 0.05) does not affect respiratory rhythm. These results suggest that voltage-dependent bursting in pacemaker neurons is not essential for respiratory rhythmogenesis, which may instead be an emergent network property.

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

Abstract The ion Ca2+ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca2+ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca2+ channel blocker was used. In contrast, none of the Ca2+ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca2+ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [3H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca2+ channels in neurotransmitter release. Total high voltage Ca2+ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca2+ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.

Glycinergic Pacemaker Neurons in PreBötzinger Complex of Neonatal Mouse

The preBötzinger complex (preBötC) is essential for normal respiratory rhythm generation in rodents, for which the underlying mechanisms remain unknown. Excitatory preBötC pacemaker neurons are proposed to be necessary for rhythm generation. Here we report the presence of a population of preBötC glycinergic pacemaker neurons. We used rhythmic in vitro transverse slice preparations from transgenic mice where neurons expressing the glycine transporter 2 (GlyT2) gene coexpress enhanced green fluorescent protein (EGFP). We combined epifluorescence and whole-cell patch-clamp recording to study preBötC EGFP-labeled, i.e., glycinergic, inspiratory-modulated neurons with pacemaker properties. We defined glycinergic pacemaker neurons as those preBötC EGFP neurons that exhibited the following: (1) ectopic bursting in rhythmic slices when depolarized during their normally silent period and (2) bursting when depolarized in nonrhythmic slices (following AMPA receptor blockade). Forty-two percent of EGFP-labeled neurons were inspiratory (n = 48 of 115), of which 23% (n = 11 of 48 inspiratory; 10% of the total recorded) were pacemakers. We conclude that there is a population of preBötC inspiratory-modulated glycinergic, presumably inhibitory, pacemaker neurons that constitute a substantial fraction of all preBötC pacemaker neurons. These findings challenge contemporary models for respiratory rhythmogenesis that assume the excitatory nature of preBötC pacemaker neurons. Testable and nontrivial predictions of the functional role of excitatory and inhibitory pacemaker neurons need to be proposed and the necessary experiments performed.

Depletion of substance P and glutamate by capsaicin blocks respiratory rhythm in neonatal rat in vitro

The specific role of the neuromodulator substance P (SP) and its target, the neurokinin 1 receptor (NK1R), in the generation and regulation of respiratory activity is not known. The preBötzinger complex (preBötC), an essential site for respiratory rhythm generation, contains glutamatergic NK1R‐expressing neurones that are strongly modulated by exogenously applied SP or acute pharmacological blockade of NK1Rs. We investigated the effects of capsaicin, which depletes neuropeptides (including SP) and glutamate from presynaptic terminals, on respiratory motor output in medullary slice preparations of neonatal rat that generate respiratory‐related activity. Bath application of capsaicin slowed respiratory motor output in a dose‐ and time‐dependent manner. Respiratory rhythm could be restored by bath application of SP or glutamate transporter blockers. Capsaicin also evoked dose‐dependent glutamate release and depleted SP in fibres within the preBötC. Our results suggest that depletion of SP (or other peptides) and/or glutamate by capsaicin causes a cessation of respiratory rhythm in neonatal rat slices.

Comparison between Low Frequency Magnetic Field Stimulation and Nerve Growth Factor Treatment of Cultured Chromaffin Cells, on Neurite Growth, Noradrenaline Release, Excitable Properties, and Grafting in Nigrostriatal Lesioned Rats

Adrenal chromaffin cells in vitro respond to nerve growth factor (NGF) by expressing neuronal traits. Low frequency magnetic (LFM) field stimulation, while inducing a variety of effects on several cell types, has never been studies as to its effects on chromaffin cell cultures. The purpose of this study was to compare the effects of LFM field stimulation with that of NGF on the morphological phenotype, on noradrenaline (NA) release, and on membrane excitability of cultured chromaffin cells. We also tested the effects of grafting LFM and NGF-treated chromaffin cells into the caudate nucleus of rats with 6-hydroxydopamine lesions of the nigrostriatal pathway. The results of this study showed that LFM field stimulation produced neurite growth of cultured chromaffin cells in a manner similar to that of NGF exposure. The combination of the two procedures did not induce changes above those observed by NGF alone. Both NGF- and LFM-treated chromaffin cells released [3H]NA equally in response to a depolarizing concentration of KCl. On the other, Na+ current density of LFM field stimulation increased, but to a lesser extent than that seen in NGF-treated cells. In addition both types of cells when transplanted into nigrostriatal-lesioned animals induced a similar decrease in the motor asymmetries produced by the lesion. When NGF- or LFM-treated chromaffin cells where compared to untreated control cells, no significant differences were observed in [3H]NA release, on Na+ current densities, or on postgraft motor asymmetries. The results are discussed in terms of the fact that LFM-stimulated cells can be differentiated in a manner similar to NGF-treated cells, by acquiring sympathetic like traits which in turn can diminish motor asymmetries when grafted into nigrostriatal-lesioned rats.

NMDA receptors in preBötzinger complex neurons can drive respiratory rhythm independent of AMPA receptors

The role of AMPA receptors (AMPARs) in generation and propagation of respiratory rhythm is well documented both in vivo and in vitro, whereas the functional significance of NMDA receptors (NMDARs) in preBötzinger complex (preBötC) neurons has not been explored. Here we examined the interactions between AMPARs and NMDARs during spontaneous respiratory rhythm generation in slices from neonatal rats in vitro. We tested the hypothesis that activation of NMDARs can drive respiratory rhythm in the absence of other excitatory drives. Blockade of NMDARs with dizocilpine hydrogen maleate (MK‐801, 20 μm) had a negligible effect on respiratory rhythm and pattern under standard conditions in vitro, whereas blockade of AMPARs with NBQX (0.5 μm) completely abolished respiratory activity. Removal of extracellular Mg2+ to relieve the voltage‐dependent block of NMDARs maintained respiratory rhythm without a significant effect on period, even in the presence of high NBQX concentrations (≤ 100 μm). Removal of Mg2+ increased inspiratory‐modulated inward current peak (II) and charge (QI) in preBötC neurons voltage‐clamped at −60 mV by 245% and 309%, respectively, with respect to basal values. We conclude that the normal AMPAR‐mediated postsynaptic current underlying respiratory drive can be replaced by NMDAR‐mediated postsynaptic current when the voltage‐dependent Mg2+ block is removed. Under this condition, respiratory‐related frequency is unaffected by changes in II, suggesting that the two can be independently regulated.

Transplant of cultured neuron-like differentiated chromaffin cells in a parkinson's disease patient. A preliminary report

BACKGROUND Treatment of Parkinsons Disease (PD) has been attempted by others by transplanting either the patients own adrenal medullary tissue or fetal substantia nigra into caudate or putamen areas. However, the difficulties inherent in using the patients own adrenal gland, or the difficulty in obtaining human fetal tissue, has generated the need to find alternative methods. METHODS We report here of an alternative to both procedures by using as transplant material cultured human adrenal chromaffin cells differentiated into neuron-like cells by extremely low frequency magnetic fields (ELF MF). RESULTS The results of this study show that human differentiated chromaffin cells can be grafted into the caudate nucleus of a PD patient, generating substantial clinical improvement, as measured by the Unified Rating Scale for PD, which correlated with glucose metabolism and D2 DA receptor increases as seen in a PET scan, while allowing a 70% decrease in L-Dopa medication. DISCUSSION This is the first preliminary report showing that transplants of cultured differentiated neuron-like cells can be successfully used to treat a PD patient.

Somatic Ca2+ transients do not contribute to inspiratory drive in preBötzinger Complex neurons

PreBötzinger Complex (preBötC) neurons are postulated to underlie respiratory rhythm generation. The inspiratory phase of the respiratory cycle in vitro results from preBötC neurons firing synchronous bursts of action potentials (APs) on top of 10–20 mV, 0.3–0.8 s inspiratory drive potentials. Is the inspiratory drive in individual neurons simply the result of the passive integration of inspiratory‐modulated synaptic currents or do active processes modulate these currents? As somatic Ca2+ is known to increase during inspiration, we hypothesized that it affects inspiratory drive. We combined whole cell recording in an in vitro slice preparation with Ca2+ microfluorometry to detect single inspiratory neuron somatic Ca2+ transients with high temporal resolution (∼μs). In neurons loaded with either Fluo‐4 or Oregon Green BAPTA 5 N, we observed Ca2+ transients associated with each AP. During inspiration, significant somatic Ca2+ influx was a direct consequence of activation of voltage‐gated Ca2+ channels by APs. However, when we isolated the inspiratory drive potential in active preBötC neurons (by blocking APs with intracellular QX‐314 or by hyperpolarization), we did not detect somatic Ca2+ transients; yet, the parameters of inspiratory drive were the same with or without APs. We conclude that, in the absence of APs, somatic Ca2+ transients do not shape the somatic inspiratory drive potential. This suggests that in preBötC neurons, substantial and widespread somatic Ca2+ influx is a consequence of APs during the inspiratory phase and does not contribute substantively to the inspiratory drive potential. Given evidence that the Ca2+ buffer BAPTA can significantly reduce inspiratory drive, we hypothesize that dendritic Ca2+ transients amplify inspiratory‐modulated synaptic currents.

The effect of Parkinson's disease and Huntington's disease on human visuomotor learning.

Visuomotor adaptation is often driven by error‐based (EB) learning in which signed errors update motor commands. There are, however, visuomotor tasks where signed errors are unavailable or cannot be mapped onto appropriate motor command changes, rendering EB learning ineffective; and yet, healthy subjects can learn in these EB learning‐free conditions. While EB learning depends on cerebellar integrity, the neural bases of EB‐independent learning are poorly understood. As basal ganglia are involved in learning mechanisms that are independent of signed error feedback, here we tested whether patients with basal ganglia lesions, including those with Huntingtons disease and Parkinsons disease, would show impairments in a visuomotor learning task that prevents the use of EB learning. We employed two visuomotor throwing tasks that were similar, but were profoundly different in the resulting visual feedback. This difference was implemented through the introduction of either a lateral displacement of the visual field via a wedge prism (EB learning) or a horizontal reversal of the visual field via a dove prism (non‐EB learning). Our results show that patients with basal ganglia degeneration had normal EB learning in the wedge prism task, but were profoundly impaired in the reversing prism task that does not depend on the signed error signal feedback. These results represent the first evidence that human visuomotor learning in the absence of EB feedback depends on the integrity of the basal ganglia.