Abstract: 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 microM) had a negligible effect on respiratory rhythm and pattern under standard conditions in vitro, whereas blockade of AMPARs with NBQX (0.5 microM) 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 (<or= 100 microM). Removal of Mg2+ increased inspiratory-modulated inward current peak (I(I)) and charge (Q(I)) 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 I(I), suggesting that the two can be independently regulated.

Abstract: 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 preBotzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (I(NaP))-mediated bursting in approximately 5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. I(NaP)-mediated bursting was voltage dependent and blocked by 20 mum riluzole (RIL). We found Ca2+ current (I(Ca))-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 microm Cd2+ or flufenamic acid (FFA) (10-200 microm), which suggests that a Ca2+-activated inward cationic current (I(CAN)) 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 microm RIL + FFA (100-200 microm) stops the respiratory rhythm, but that adding 2 mum substance P restarts it. We conclude that I(NaP) and I(CAN) enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I(NaP) and I(CAN) 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 preBotzinger complex.

Abstract: 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.

Abstract: 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.

Abstract: A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.

Abstract: BACKGROUND: Treatment of Parkinson's Disease (PD) has been attempted by others by transplanting either the patient's own adrenal medullary tissue or fetal substantia nigra into caudate or putamen areas. However, the difficulties inherent in using the patient's 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.

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.

Abstract: Levels of DARP in the cerebrospinal fluid (CSF) of patients having a wide variety of neurological disorders were determined. Neurological disorders were categorized as degenerative, demyelinating, epilepsy, trauma, hydrocephalia, inflammatory, A-V malformation, CNS neoplasia, parasitic and stroke. DARP levels were determined by an enzyme-linked immunoabsorbent assay (ELISA) using monoclonal anti-DARP antibodies. A synthetic peptide corresponding to the first 36 aa of the N-terminal of DARP was used as standard. A total of 7 non-neurological patients and 73 patients with neurological disorders were tested. The relative concentrations of DARP decreased in patients with Parkinson's diseases vs. patients with non-neurological diseases and increased in other neuropathologies such as demyelinating, hydrocephalia and A-V malformations. Data obtained suggest that changes in the percentage and concentration of DARP may correlate with certain neurological disorders, showing particularly low levels in Parkinson's disease patients.

Abstract: Dopamine agonists or grafts compensate impaired motor functions in aged rats. However, there is no evidence showing whether grafting in adulthood retard aging manifestations. Motor performance of 13-month-old rats was tested on 2 meter-long wooden beams which had a 15 degree inclination and whose thickness varied from 3, 6, 12, 18, to 24 mm. Rats at 14 months were randomly assigned to 3 groups: sham graft (Group 1); intrastriatal graft of chromaffin cells cultured with NGF (Group 2); intrastriatal graft of chromaffin cells (Group 3). Motor performance was tested at monthly intervals up until rats were 26 months old. Two more groups were included: 26-month-old naive rats (Group 4); and 3- to 5-month-old naive rats (Group 5) both evaluated only once. At 26 months, the basal activity of ventral mesencephalic dopaminergic neurons was recorded. Results showed in Group 2 delay of motor detriments seen in aged rats, maintenance of basal firing rates of nigral cells compared to those of younger rats, and greater survival of substantia nigra cells. It is suggested that NGF cultured chromaffin cells produce a delay of motor detriments in aged rats, as a result of inducing survival and firing rates of nigral cells comparable to those seen in young rats.

Abstract: 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.