An agonist–antagonist cerebellar nuclear system controlling eyelid kinematics during motor learning

The presence of two antagonistic groups of deep cerebellar nuclei neurons has been reported as necessary for a proper dynamic control of learned motor responses. Most models of cerebellar function seem to ignore the biomechanical need for a double activation–deactivation system controlling eyelid kinematics, since most of them accept that, for closing the eyelid, only the activation of the orbicularis oculi (OO) muscle (via the red nucleus to the facial motor nucleus) is necessary, without a simultaneous deactivation of levator palpebrae motoneurons (via unknown pathways projecting to the perioculomotor area). We have analyzed the kinetic neural commands of two antagonistic types of cerebellar posterior interpositus neuron (IPn) (types A and B), the electromyographic (EMG) activity of the OO muscle, and eyelid kinematic variables in alert behaving cats during classical eyeblink conditioning, using a delay paradigm. We addressed the hypothesis that the interpositus nucleus can be considered an agonist–antagonist system controlling eyelid kinematics during motor learning. To carry out a comparative study of the kinetic–kinematic relationships, we applied timing and dispersion pattern analyses. We concluded that, in accordance with a dominant role of cerebellar circuits for the facilitation of flexor responses, type A neurons fire during active eyelid downward displacements—i.e., during the active contraction of the OO muscle. In contrast, type B neurons present a high tonic rate when the eyelids are wide open, and stop firing during any active downward displacement of the upper eyelid. From a functional point of view, it could be suggested that type B neurons play a facilitative role for the antagonistic action of the levator palpebrae muscle. From an anatomical point of view, the possibility that cerebellar nuclear type B neurons project to the perioculomotor area—i.e., more or less directly onto levator palpebrae motoneurons—is highly appealing

Blinking and the Brain: Pathways and Pathology

A blink is a rapid bilateral eyelid closure and co-occurring eye movement. The eyes rotate down towards the tip of the nose and back up again. Seemingly, this generally unnoticed often repeated action is not very spectacular. However, if not for the occasional blink we would all be blind. A blink is an interesting phenomenon worth investigating. Through its role as a protective barrier for the eye and as a distributor of the eye’s tearfi lm, blinking is a necessity for our well-being but is also an important tool for neuroscience research and physicians. It is an extremely useful model to study motor performance, motor control, synaptic plasticity and is an excellent physiological instrument for the assessment of internal networks and nuclei [Nishimura, T. and Mori, K, 1996]. The blink rate, refl ex blink characteristics and learned blinks are the three main parameters that can be studied to this end. The blink rate is the frequency with which spontaneous blinks occur and can give information about the dopaminergic system [Karson 1988] and might even be usable as a measure of fatigue [Stern et al. 1994]. The refl ex blink is a rapid involuntary response evoked by external stimulation of the eye or eyelid. It has a protective function and is for instance used in research and physiological tests that use the blink to provide important information on the integrity of afferent and efferent pathways. However, important gaps remain in the knowledge of pathways underlying blinking, and aberrations in pathways or compensatory mechanisms are not fully understood. The learned blink is acquired during eyeblink or eyelid conditioning in which an involuntary blink-evoking stimulus is repeatedly combined with a neutral stimulus. After training the neutral stimulus can evoke the learned blink

Pattern recognition of neural data: methods and algorithms for spike sorting and their optimal performance in prefrontal cortex recordings

Programa de Doctorado en NeurocienciasPattern recognition of neuronal discharges is the electrophysiological basis of the functional characterization of brain processes, so the implementation of a Spike Sorting algorithm is an essential step for the analysis of neural codes and neural interactions in a network or brain circuit. Extracted information from the neural action potential can be used to characterize neural activity events and correlate them during behavioral and cognitive processes, including different types of associative learning tasks. In particular, feature extraction is a critical step in the spike sorting procedure, which is prior to the clustering step and subsequent to the spike detection-identification step in a Spike Sorting algorithm. In the present doctoral thesis, the implementation of an automatic and unsupervised computational algorithm, called 'Unsupervised Automatic Algorithm', is proposed for the detection, identification and classification of the neural action potentials distributed across the electrophysiological recordings; and for clustering of these potentials in function of the shape, phase and distribution features, which are extracted from the first-order derivative of the potentials under study. For this, an efficient and unsupervised clustering method was developed, which integrate the K-means method with two clustering measures (validity and error indices) to verify both the cohesion-dispersion among neural spike during classification and the misclassification of clustering, respectively. In additions, this algorithm was implemented in a customized spike sorting software called VISSOR (Viability of Integrated Spike Sorting of Real Recordings). On the other hand, a supervised grouping method of neural activity profiles was performed to allow the recognition of specific patterns of neural discharges. Validity and effectiveness of these methods and algorithms were tested in this doctoral thesis by the classification of the detected action potentials from extracellular recordings of the rostro-medial prefrontal cortex of rabbits during the classical eyelid conditioning. After comparing the spike-sorting methods/algorithms proposed in this work with other methods also based on feature extraction of the action potentials, it was observed that this one had a better performance during the classification. That is, the methods/algorithms proposed here allowed obtaining: (1) the optimal number of clusters of neuronal spikes (according to the criterion of the maximum value of the cohesion-dispersion index) and (2) the optimal clustering of these spike-events (according to the criterion of the minimum value of the error index). The analytical implication of these results was that the feature extraction based on the shape, phase and distribution features of the action potential, together with the application of an alternative method of unsupervised classification with validity and error indices; guaranteed an efficient classification of neural events, especially for those detected from extracellular or multi-unitary recordings. Rabbits were conditioned with a delay paradigm consisting of a tone as conditioned stimulus. The conditioned stimulus started 50, 250, 500, 1000, or 2000 ms before and co-terminated with an air puff directed at the cornea as unconditioned stimulus. The results obtained indicated that the firing rate of each recorded neuron presented a single peak of activity with a frequency dependent on the inter-stimulus interval (i.e., ¿ 12 Hz for 250 ms, ¿ 6 Hz for 500 ms, and ¿ 3 Hz for 1000 ms). Interestingly, the recorded neurons from the rostro-medial prefrontal cortex presented their dominant firing peaks at three precise times evenly distributed with respect to conditioned stimulus start, and also depending on the duration of the inter-stimulus interval (only for intervals of 250, 500, and 1000 ms). No significant neural responses were recorded at very short (50 ms) or long (2000 ms) conditioned stimulus-unconditioned stimulus time intervals. Furthermore, the eyelid movements were recorded with the magnetic search coil technique and the electromyographic (EMG) activity of the orbicularis oculi muscle. Reflex and conditioned eyelid responses presented a dominant oscillatory frequency of ¿ 12 Hz. The experimental implication of these results is that the recorded neurons from the rostro-medial prefrontal cortex seem not to encode the oscillatory properties characterizing conditioned eyelid responses in rabbits. As a general experimental conclusion, it could be said that rostro-medial prefrontal cortex neurons are probably involved in the determination of CS-US intervals of an intermediate range (250-1000 ms).Universidad Pablo de Olavide. Departamento de Fisiología, Anatomía y Biología CelularPostprin

The role of the cerebral cortex during classical eyeblink conditioning in the rabbit

Programa de Doctorado en NeurocienciasLearning is defined as the acquisition or modification of specific behaviors that allow the individual to adapt to changes occurring in the surrounding environment. The primary focus of many investigations has been the characterization of neural bases underlying learning. Thus, the motor system of the eyelid and the nictitating membrane has been extensively used as an experimental model to study the mechanisms and neural structures determining associative learning, mainly by the use of different paradigms of classical eyeblink conditioning. Over several decades, the cerebellum and the hippocampus have been proposed as the brain structures responsible for processing the neural codes underlying the generation of learned eyeblink responses. However, some researchers raised the hypothesis of the participation of other brain regions. Specifically, reported changes in neuronal activity in various cortical structures during learning suggest the involvement of sensory and motor pathways in the generation of conditioned responses. The main approach of the experimental work presented in this Doctoral Thesis was to investigate the role of the cerebral cortex during the classical conditioning of eyeblink responses. First, projections from the motor cortex to the facial nucleus were characterized by injecting an anterograde tracer in the cortical region corresponding to the orbicularis oculi muscle. Hence, monosynaptic neural projections from the rabbit¿s motor cortex were identified in the facial nucleus. On the other hand, extracellular unitary activity in the palpebral region of the motor cortex corresponding to the orbicularis oculi was recorded during eyeblink conditioning. As a result, motor cortex neurons activated antidromically from the red nucleus as well as the facial nucleus showed an increase of their firing rates well in advance (¿ 50 ms) with regard to the conditioned response onset. With the aim to verify the importance of the motor cortex during the acquisition process of associative learning it was examined whether modulating excitability of motor cortex neurons by means of transcranial current stimulation would be able to modify the learning process. Similarly, the contribution of the sensory cortex during the acquisition of eyeblink conditioning ¿ using light stimulation as conditioned stimulus ¿ was studied by applying transcranial electrical currents to the primary visual cortex during learning. The induced modulation of neuronal excitability in the motor and visual cortex resulted in a significant impact on learning consisting of a polarity-dependent change in the percentage of learned responses. Specifically, an increase in the number of learned responses was observed when stimulation with anodal polarity was applied to the motor cortex, whereas cathodal polarity decreased the number of conditioned responses when applied to the visual cortex. Regarding the quality of responses as observed for the motor cortex, anodal stimulation promoted increased magnitude, whereas cathodal stimulation reduced magnitude and slightly delayed the beginning of learned responses. Finally, thermal changes of the brain tissue were measured by means of an epidurally implanted thermistor placed under the transcranial current stimulation location to rule out that tissue heating of the stimulated site interfered with the observed effects on motor learning. No significant changes in brain temperature were induced either during or after the application of transcranial stimulation. In brief, this Doctoral Thesis reveals that the participation of the motor cortex during classical eyeblink conditioning is essential for the correct acquisition of learned responses. In addition, the results support the hypothesis of the involvement of the sensory cortex in this type of associative motor learning. Finally, the data presented in this thesis shows for the first time experimental evidence supporting the absence of thermal changes in the brain tissue due to the transcranial current stimulation.Universidad Pablo de Olavide. Departamento de Fisiología, Anatomía y Biología CelularPostprin

Caveats in transneuronal tracing with unmodified rabies virus: An evaluation of aberrant results using a nearly perfect tracing technique

Apart from the genetically engineered, modified, strains of rabies virus (RABV), unmodified ‘fixed’ virus strains of RABV, such as the ‘French’ subtype of CVS11, are used to examine synaptically connected networks in the brain. This technique has been shown to have all the prerequisite characteristics for ideal tracing as it does not metabolically affect infected neurons within the time span of the experiment, it is transferred transneuronally in one direction only and to all types of neurons presynaptic to the infected neuron, number of transneuronal steps can be precisely controlled by survival time and it is easily detectable with a sensitive technique. Here, using the ‘French’ CVS 11 subtype of RABV in Wistar rats, we show that some of these characteristics may not be as perfect as previously indicated. Using injection of RABV in hind limb muscles, we show that RABV-infected spinal motoneurons may already show lysis 1 or 2 days after infection. Using longer survival times we were able to establish that Purkinje cells may succumb approximately 3 days after infection. In addition, some neurons seem to resist infection, as we noted that the number of RABV-infected inferior olivary neurons did not progress in the same rate as other infected neurons. Furthermore, in our hands, we noted that infection of Purkinje cells did not result in expected transneuronal labeling of cell types that are presynaptic to Purkinje cells such as molecular layer interneurons and granule cells. However, these cell types were readily infected when RABV was injected directly in the cerebellar cortex. Conversely, neurons in the cerebellar nuclei that project to the inferior olive did not take up RABV when this was injected in the inferior olive, whereas these cells could be infected with RABV via a transneuronal route. These results suggest that viral entry from the extracellular space depends on other factors or mechanisms than those used for retrograde transneuronal transfer. We conclude that transneuronal tracing with RABV may result in unexpected results, as not all properties of RABV seem to be ubiquitously valid