Livia Carrascal

Phasic and tonic firing properties in rat oculomotor nucleus motoneurons, studied in vitro

Alert‐chronic studies show that ocular motoneurons (Mns) exhibit a phasic and tonic firing correlated with eye saccade‐velocity and position (fixation), respectively. Differences in the phasic and tonic firing among Mns depend on synaptic inputs and/or the intrinsic membrane properties. We have used in vitro slice preparation to investigate the contribution of membrane properties to firing properties of Wistar rat oculomotor nucleus Mns. We recorded different discharge patterns and focused on Mns with sustained discharge (type I) because they were the most abundant, and their firing pattern resembles that reported in alert preparations. Various differences divided these Mns into types IA and IB; the afterhyperpolarization (AHP) phase of the spike was monophasic in IA and biphasic in IB; IA Mns showed tonic or phasic‐tonic firing depending on the current intensity, while IB Mns showed phasic‐tonic discharge; the phasic firing was higher in IB than in IA Mns; IA Mns fired in a narrower range than did IB Mns; and IA Mns showed lower maximum frequency than did IB Mns. In conclusion, IA and IB Mns show different phasic firing properties and dynamic range, supported by intrinsic membrane properties. We suggest that IA and IB Mns innervate fast‐twitch muscle fibres with different contraction speeds, and could contribute to generating a fine phasic signal for a graded muscle contraction. Finally, we have demonstrated an inverse relationship between Mn thresholds and tonic firing gain, concluding that intrinsic membrane properties could not support the covariation between tonic firing gain and recruitment thresholds reported in alert studies.

Muscarinic Modulation of Recruitment Threshold and Firing Rate in Rat Oculomotor Nucleus Motoneurons

Above recruitment threshold, ocular motoneurons (Mns) show a firing rate linearly related with eye position. Current hypothesis suggests that synaptic inputs are determinant for establishing the recruitment threshold and firing rate gain in these Mns. We investigated this proposal by studying the cholinergic modulation in oculomotor nucleus Mns by intracellular recordings in rat brain slice preparation. All recorded Mns were silent at their resting membrane potential. Bath application of carbachol (10 microm) produced a depolarization and a sustained firing that was not silenced on returning membrane potential to the precarbachol value via DC injection. In response to similar membrane depolarization or equal-current steps, carbachol-exposed Mns produced a higher firing rate and a shorter spike afterhyperpolarization phase with lower amplitude. The relationship between injected current and firing rate (I-F) was linear in control and carbachol-exposed Mns. The slope of these relationships (I-F gain) decreased with carbachol exposure. Bath application of agonist and antagonist of nicotinic and muscarinic acetylcholine receptors in addition to immunohistochemical studies support the notion that muscarinic receptors are primarily involved in the preceding responses. We conclude that muscarinic inputs play an important role in determining the recruitment threshold and firing rate gain observed in oculomotor Mns in vivo.

Oxidative stress induced by cumene hydroperoxide evokes changes in neuronal excitability of rat motor cortex neurons.

Oxidative stress and the production of reactive oxygen radicals play a key role in neuronal cell damage. This paper describes an in vitro study that explores the neuronal responses to oxidative stress focusing on changes in neuronal excitability and functional membrane properties. This study was carried out in pyramidal cells of the motor cortex by applying whole-cell patch-clamp techniques on brain slices from young adult rats. Oxygen-derived free radical formation was induced by bath application of 10μM cumene hydroperoxide (CH) for 30min. CH produced marked changes in the electrophysiological properties of neurons (n=30). Resting membrane potential became progressively depolarized, as well as depolarization voltage, with no variations in voltage threshold. Membrane resistance showed a biphasic behavior, increasing after 5min of drug exposure and then it started to decrease, even under control values, after 15 and 30min. At the same time, changes in membrane resistance produced compensatory variations in the rheobase. The amplitude of the action potentials diminished and the duration increased progressively over time. Some of the neurons under study also lost their ability to discharge action potentials in a repetitive way. Most of the neurons, however, kept their repetitive discharge even though their maximum frequency and gain decreased. Furthermore, cancelation of the repetitive firing discharge took place at intensities that decreased with time of exposure to CH, which resulted in a narrower working range. We can conclude that oxidative stress compromises both neuronal excitability and the capability of generating action potentials, and so this type of neuronal functional failure could precede the neuronal death characteristics of many neurodegenerative diseases.

Immunolocalization of human alpha-synuclein in the Thy1-aSyn ("Line 61") transgenic mouse line.

Alpha-synuclein (a-syn) is the major component of the intracytoplasmic inclusions known as Lewy bodies (LB), which constitute the hallmark of Parkinsons disease (PD). Mice overexpressing human a-syn under the Thy-1 promoter (ASO) show slow neurodegeneration and some behavioral deficits similar to those seen in human PD patients. Here, we describe a whole-brain distribution of human a-syn in adult ASO mice. We find that the human a-syn is ubiquitously distributed in the brain including the cerebellar cortex, but the intensity and sub-cellular localization of the staining differed in the various regions of the central nervous system. Among particular CNS areas with human a-syn immunoreactivity, we describe staining patterns in the olfactory bulb, cortex, hippocampus, thalamic region, brainstem nuclei and cerebellar cortex. This immunohistochemical study provides an anatomical map of the human a-syn distribution in ASO mice. Our data show that human a-syn, although not present at levels that were detectable by immunostaining in dopaminergic neurons of substantia nigra or noradrenergic neurons of locus coeruleus, was highly expressed in other PD relevant regions of the brain in different neuronal subtypes. These data will help to relate a-syn expression to the phenotypic manifestations observed in this widely used mouse line.

Diminution of voltage threshold plays a key role in determining recruitment of oculomotor nucleus motoneurons during postnatal development.

The size principle dictates the orderly recruitment of motoneurons (Mns). This principle assumes that Mns of different sizes have a similar voltage threshold, cell size being the crucial property in determining neuronal recruitment. Thus, smaller neurons have higher membrane resistance and require a lower depolarizing current to reach spike threshold. However, the cell size contribution to recruitment in Mns during postnatal development remains unknown. To investigate this subject, rat oculomotor nucleus Mns were intracellularly labeled and their electrophysiological properties recorded in a brain slice preparation. Mns were divided into 2 age groups: neonatal (1–7 postnatal days, n = 14) and adult (20–30 postnatal days, n = 10). The increase in size of Mns led to a decrease in input resistance with a strong linear relationship in both age groups. A well-fitted inverse correlation was also found between input resistance and rheobase in both age groups. However, input resistance versus rheobase did not correlate when data from neonatal and adult Mns were combined in a single group. This lack of correlation is due to the fact that decrease in input resistance of developing Mns did not lead to an increase in rheobase. Indeed, a diminution in rheobase was found, and it was accompanied by an unexpected decrease in voltage threshold. Additionally, the decrease in rheobase co-varied with decrease in voltage threshold in developing Mns. These data support that the size principle governs the recruitment order in neonatal Mns and is maintained in adult Mns of the oculomotor nucleus; but during postnatal development the crucial property in determining recruitment order in these Mns was not the modifications of cell size-input resistance but of voltage threshold.

Changes in Somatodendritic Morphometry of Rat Oculomotor Nucleus Motoneurons During Postnatal Development

This work investigates the somatodendritic shaping of rat oculomotor nucleus motoneurons (Mns) during postnatal development. The Mns were functionally identified in slice preparation, intracellularly injected with neurobiotin, and three‐dimensionally reconstructed. Most of the Mns (∼85%) were multipolar and the rest (∼15%) bipolar. Forty multipolar Mns were studied and grouped as follows: 1–5, 6–10, 11–15, and 21–30 postnatal days. Two phases were distinguished during postnatal development (P1–P10 and P11–P30). During the first phase, there was a progressive increase in the dendritic complexity; e.g., the number of terminals per neuron increased from 26.3 (P1–P5) to 47.7 (P6–P10) and membrane somatodendritic area from 11,289.9 μm2 (P1–P5) to 19,235.8 μm2 (P6–P10). In addition, a few cases of tracer coupling were observed. During the second phase, dendritic elongation took place; e.g., the maximum dendritic length increased from 486.7 μm (P6–P10) to 729.5 μm in adult Mns, with a simplification of dendritic complexity to values near those for the newborn, and a slow, progressive increase in membrane area from 19,235.8 μm2 (P6–P10) to 24,700.3 μm2 (P21–P30), while the somatic area remained constant. In conclusion, the electrophysiological changes reported in these Mns with maturation (Carrascal et al. [ 2006 ] Neuroscience 140:1223–1237) cannot be fully explained by morphometric variations; the dendritic elongation and increase in dendritic area are features shared with other pools of Mns, whereas changes in dendritic complexity depend on each population; the first phase paralleled the establishment of vestibular circuitry and the second paralleled eyelid opening. J. Comp. Neurol. 514:189–202, 2009.

Modulation of the input–output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons

This study deals with synaptic mechanisms involved in the modulation of recruitment threshold and firing rate in ocular motoneurons. In particular, how high and low ambient GABA concentrations modulate these parameters. In a low ambient GABA concentration, ocular motoneurons exhibit a tonic GABAA receptor‐mediated current that modulates input resistance and recruitment threshold. The modulation effect increased with motoneuron size. Tonic inhibition shifts the firing frequency–current relationship without change in gain. Independent of cell size, a high ambient GABA concentration reduced input resistance and increased motoneuron recruitment threshold without a change in firing frequency gain. Our results demonstrate that GABAA mediated tonic inhibition and glutamate mediated current modulate recruitment threshold and firing behaviour of ocular motoneurons depending on motoneuron size. These synaptic mechanisms, dependent on cell size, could be underlying the positive relationship between threshold and eye position sensitivity reported in ocular motoneurons recorded in alert preparation.

Glutamate modulates the firing rate in oculomotor nucleus motoneurons as a function of the recruitment threshold current

•  This study deals with the cellular mechanisms involved in firing rate modulation in vivo in the oculomotor system where there are requirements for high firing rates by motoneurons. •  The study demonstrates that glutamate effects depend on the recruitment threshold and, presumably, motoneuron size. •  Mid‐ and high‐threshold motoneurons in response to glutamate decrease their voltage threshold and strengthened the tonic and phasic components of the firing rate. •  In a functional context, motoneurons could be recruited at lower recruitment threshold and could generate a strong muscle contraction under glutamate modulation to perform saccadic eye movements with different velocities and/or to maintain the eye in different eccentric positions in the orbit. •  Our results suggest that the recruitment and firing behaviour of ocular motoneurons can be modified in vivo by glutamatergic synaptic inputs. It provides a link between cellular function and behavioural motoneuron output.

POSTNATAL DEVELOPMENT ENHANCES THE EFFECTS OF CHOLINERGIC INPUTS ON RECRUITMENT THRESHOLD AND FIRING RATE OF RAT OCULOMOTOR NUCLEUS MOTONEURONS

Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4-7 postnatal days) and adult (20-30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 μM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 μM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction.

Role of Melatonin in the Inflammatory Process and its Therapeutic Potential

Melatonin is an indolamine synthesized and secreted by the pineal gland along with other extrapineal sources including immune system cells, the brain, skin and the gastrointestinal tract. Growing interest in this compound as a potential therapeutic agent in several diseases stems from its pleiotropic effects. Thus, melatonin plays a key role in various physiological activities that include regulation of circadian rhythms, immune responses, the oxidative process, apoptosis or mitochondrial homeostasis. Most of these processes are altered during inflammatory pathologies, among which neurodegenerative and bowel diseases stand out. Therapeutic assays with melatonin indicate that it has a beneficial therapeutic value in the treatment of several inflammatory diseases, such as Alzheimer, Amiotrophic Lateral, Multiple Sclerosis and Huntigton´s disease as well as ulcerative colitis. However, contradictory effects have been demonstrated in Parkinson´s and Chron´s diseases, which, in some cases, the reported effects were beneficial while in others the pathology was exacerbated. These various results may be related to several factors. In the first place, it should be taken into account that at the beginning of the inflammation phase there is a production of reactive oxygen species (ROS) that should not be blocked by exclusively antioxidant molecules, since, on the one hand, it would be interfering with the action of neutrophils and macrophages and, on the other, with the apoptotic signals activated by ROS. It is also important to keep in mind that the end result of an anti-inflammatory molecule will depend on the degree of inflammation or whether or not it has been resolved and has therefore become chronic. In this review we present the use of melatonin in the control of inflammation underlying the above mentioned diseases. These actions are mediated through their receptors but also with their direct antioxidant action and melatonins ability to break the vicious cycle of ROSinflammation. This review is aimed at evaluating the effect of melatonin on activity of the inflammatory process and at its immunomodulator effects.