Raudel Sánchez-Campusano

Transcranial direct-current stimulation modulates synaptic mechanisms involved in associative learning in behaving rabbits

Transcranial direct-current stimulation (tDCS) is a noninvasive brain stimulation technique that has been successfully applied for modulation of cortical excitability. tDCS is capable of inducing changes in neuronal membrane potentials in a polarity-dependent manner. When tDCS is of sufficient length, synaptically driven after-effects are induced. The mechanisms underlying these after-effects are largely unknown, and there is a compelling need for animal models to test the immediate effects and after-effects induced by tDCS in different cortical areas and evaluate the implications in complex cerebral processes. Here we show in behaving rabbits that tDCS applied over the somatosensory cortex modulates cortical processes consequent to localized stimulation of the whisker pad or of the corresponding area of the ventroposterior medial (VPM) thalamic nucleus. With longer stimulation periods, poststimulation effects were observed in the somatosensory cortex only after cathodal tDCS. Consistent with the polarity-specific effects, the acquisition of classical eyeblink conditioning was potentiated or depressed by the simultaneous application of anodal or cathodal tDCS, respectively, when stimulation of the whisker pad was used as conditioned stimulus, suggesting that tDCS modulates the sensory perception process necessary for associative learning. We also studied the putative mechanisms underlying immediate effects and after-effects of tDCS observed in the somatosensory cortex. Results when pairs of pulses applied to the thalamic VPM nucleus (mediating sensory input) during anodal and cathodal tDCS suggest that tDCS modifies thalamocortical synapses at presynaptic sites. Finally, we show that blocking the activation of adenosine A1 receptors prevents the long-term depression (LTD) evoked in the somatosensory cortex after cathodal tDCS.

An in vitro and in vivo study of early deficits in associative learning in transgenic mice that over‐express a mutant form of human APP associated with Alzheimer's disease

Transgenic mice over‐expressing a mutated form of the human amyloid precursor protein (APP, 695 isoform) bearing a mutation associated with Alzheimers disease (V642I, so‐called London mutation, hereafter APPLd2) and wild‐type controls were studied at age periods (3 and 10 months) prior to the overt development of neuritic amyloid plaques. Both 3‐ and 10‐month‐old APPLd2 mice had reflex eyelid responses like those of controls, but only younger mice were able to acquire a classical conditioning of eyelid responses in a trace paradigm. In vitro studies on hippocampal slices showed that 10‐month‐old APPLd2 mice also presented deficits in paired‐pulse facilitation and long‐term potentiation, but presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals. It is proposed that definite functional changes may appear well in advance of noticeable structural alterations in this animal model of Alzheimers disease, and that specific learning tasks could have a relevant diagnostic value.

The cerebellar interpositus nucleus and the dynamic control of learned motor responses

The role played by the cerebellum in movement control requires knowledge of interdependent relationships between kinetic neural commands and the performance (kinematics) of learned motor responses. The eyelid motor system is an excellent model for studying how simple motor responses are elaborated and performed. Kinetic variables (n = 24) were determined here by recording the firing activities of orbicularis oculi motoneurons and cerebellar interpositus neurons in alert cats during classical conditioning, using a delay paradigm. Kinematic variables (n = 36) were selected from eyelid position, velocity, and acceleration traces recorded during the conditioned stimulus–unconditioned stimulus interval. Optimized experimental and analytical tools allowed us to determine the evolution of kinetic and kinematic variables, the dynamic correlation functions relating motoneuron and interpositus neuron firing to eyelid conditioning responses, the falling correlation property of the interpositus nucleus across the successive training sessions, the time and significance of the linear relationships between these variables, and finally, the phase-inversion property of interpositus neurons with respect to acquired conditioned responses. Whereas motoneurons encoded eyelid kinematics at every instant of the dynamic correlation range and generated the natural oscillatory properties of the neuromuscular elements involved in eyeblinks, interpositus neurons did not directly encode eyelid performance: namely, their contribution was only slightly significant in the dynamic correlation range, and this regularity caused the integrated neuronal activity to oscillate by progressively inverting phase information. Therefore, interpositus neurons seem to play a modulating role in the dynamic control of learned motor responses, i.e., they could be considered a neuronal phase-modulating device.

Effects of transcranial Direct Current Stimulation (tDCS) on cortical activity: A computational modeling study

Although it is well-admitted that transcranial Direct Current Stimulation (tDCS) allows for interacting with brain endogenous rhythms, the exact mechanisms by which externally-applied fields modulate the activity of neurons remain elusive. In this study a novel computational model (a neural mass model including subpopulations of pyramidal cells and inhibitory interneurons mediating synaptic currents with either slow or fast kinetics) of the cerebral cortex was elaborated to investigate the local effects of tDCS on neuronal populations based on an in-vivo experimental study. Model parameters were adjusted to reproduce evoked potentials (EPs) recorded from the somatosensory cortex of the rabbit in response to air-puffs applied on the whiskers. EPs were simulated under control condition (no tDCS) as well as under anodal and cathodal tDCS fields. Results first revealed that a feed-forward inhibition mechanism must be included in the model for accurate simulation of actual EPs (peaks and latencies). Interestingly, results revealed that externally-applied fields are also likely to affect interneurons. Indeed, when interneurons get polarized then the characteristics of simulated EPs become closer to those of real EPs. In particular, under anodal tDCS condition, more realistic EPs could be obtained when pyramidal cells were depolarized and, simultaneously, slow (resp. fast) interneurons became de- (resp. hyper-) polarized. Geometrical characteristics of interneurons might provide some explanations for this effect.

Behavioral Characteristics, Associative Learning Capabilities, and Dynamic Association Mapping in an Animal Model of Cerebellar Degeneration

Young adult heterozygous Lurcher mice constitute an excellent model for studying the role of the cerebellar cortex in motor performance-including the acquisition of new motor abilities-because of the early postnatal degeneration of almost all of their Purkinje and granular cells. Wild-type and Lurcher mice were classically conditioned for eyelid responses using a delay paradigm with or without an electrolytic lesion in the interpositus nucleus. Although the late component of electrically evoked blink reflexes was smaller in amplitude and had a longer latency in Lurcher mice than that in controls, the two groups of animals presented similar acquisition curves for eyeblink conditioning. The lesion of the interpositus nucleus affected both groups of animals equally for the generation of reflex and conditioned eyelid responses. Furthermore, we recorded the multiunitary activity at the red and interpositus nuclei during the same type of associative learning. In both nuclei, the neural firing activity lagged the beginning of the conditioned response (determined by orbicularis oculi muscle response). Although red nucleus neurons and muscle activities presented a clear functional coupling (strong correlation and low asymmetry) across conditioning, the coupling between interpositus neurons and either red nucleus neurons or muscle activities was slightly significant (weak correlation and high asymmetry). Lurcher mice presented a nonlinear coupling (high asymmetry) between red nucleus neurons and muscle activities, with an evident compensatory adjustment in the correlation of firing between interpositus and red nuclei neurons (a coupling with low asymmetry), aimed probably at compensating the absence of cerebellar cortical neurons.

Dynamic associations in the cerebellar-motoneuron network during motor learning.

We assessed here true causal directionalities in cerebellar–motoneuron (MN) network associations during the classical conditioning of eyelid responses. For this, the firing activities of identified facial MNs and cerebellar interpositus (IP) nucleus neurons were recorded during the acquisition of this type of associative learning in alert behaving cats. Simultaneously, the eyelid conditioned response (CR) and the EMG activity of the orbicularis oculi (OO) muscle were recorded. Nonlinear association analysis and time-dependent causality method allowed us to determine the asymmetry, time delays, direction in coupling, and functional interdependences between neuronal recordings and learned motor responses. We concluded that the functional nonlinear association between the IP neurons and OO muscle activities was bidirectional and asymmetric, and the time delays in the two directions of coupling always lagged the start of the CR. Additionally, the strength of coupling depended inversely on the level of expression of eyeblink CRs, whereas causal inferences were significantly dependent on the phase information status. In contrast, the functional association between OO MNs and OO muscle activities was unidirectional and quasisymmetric, and the time delays in coupling were always of opposed signs. Moreover, information transfer in cerebellar–MN network associations during the learning process required a “driving common source” that induced the mere “modulating coupling” of the IP nucleus with the final common pathway for the eyelid motor system. Thus, it can be proposed that the cerebellum is always looking back and reevaluating its own function, using the information acquired in the process, to play a modulating-reinforcing role in motor learning.

Consensus paper: current views on the role of cerebellar interpositus nucleus in movement control and emotion.

In the present paper, we examine the role of the cerebellar interpositus nucleus (IN) in motor and non-motor domains. Recent findings are considered, and we share the following conclusions: IN as part of the olivo-cortico-nuclear microcircuit is involved in providing powerful timing signals important in coordinating limb movements; IN could participate in the timing and performance of ongoing conditioned responses rather than the generation and/or initiation of such responses; IN is involved in the control of reflexive and voluntary movements in a task- and effector system-dependent fashion, including hand movements and associated upper limb adjustments, for quick effective actions; IN develops internal models for dynamic interactions of the motor system with the external environment for anticipatory control of movement; and IN plays a significant role in the modulation of autonomic and emotional functions.

Differential Contribution of Hippocampal Circuits to Appetitive and Consummatory Behaviors during Operant Conditioning of Behaving Mice

Operant conditioning is a type of associative learning involving different and complex sensorimotor and cognitive processes. Because the hippocampus has been related to some motor and cognitive functions involved in this type of learning (such as object recognition, spatial orientation, and associative learning tasks), we decided to study in behaving mice the putative changes in strength taking place at the hippocampal CA3–CA1 synapses during the acquisition and performance of an operant conditioning task. Mice were chronically implanted with stimulating electrodes in the Schaffer collaterals and with recording electrodes in the hippocampal CA1 area and trained to an operant task using a fixed-ratio (1:1) schedule. We recorded the field EPSPs (fEPSPs) evoked at the CA3–CA1 synapse during the performance of appetitive (going to the lever, lever press) and consummatory (going to the feeder, eating) behaviors. In addition, we recorded the local field potential activity of the CA1 area during similar behavioral displays. fEPSPs evoked at the CA3–CA1 synapse presented larger amplitudes for appetitive than for consummatory behaviors. This differential change in synaptic strength took place in relation to the learning process, depending mainly on the moment in which mice reached the selected criterion. Thus, selective changes in CA3–CA1 synaptic strength were dependent on both the behavior display and the learning stage. In addition, significant changes in theta band power peaks and their corresponding discrete frequencies were noticed during these behaviors across the sequence of events characterizing this type of associative learning but not during the acquisition process.

A Differential and Timed Contribution of Identified Hippocampal Synapses to Associative Learning in Mice

Although it is generally assumed that the hippocampus is involved in associative learning, the specific contribution of the different synapses present in its intrinsic circuit or comprising its afferents and efferents is poorly defined. We studied here activity-dependent changes in synaptic strength of 9 hippocampal synapses (corresponding to the intrinsic hippocampal circuitry and to its main inputs and outputs) during the acquisition of a trace eyeblink conditioning in behaving mice. The timing and intensity of synaptic changes across the acquisition process was determined. The evolution of these timed changes in synaptic strength indicated that their functional organization did not coincide with their sequential distribution according to anatomical criteria and connectivity. Furthermore, we explored the functional relevance of the extrinsic and intrinsic afferents to CA3 and CA1 pyramidal neurons, and evaluated the distinct input patterns to the intrinsic hippocampal circuit. Results confirm that the acquisition of a classical eyeblink conditioning is a multisynaptic process in which the contribution of each synaptic contact is different in strength, and takes place at different moments across learning. Thus, the precise and timed activation of multiple hippocampal synaptic contacts during classical eyeblink conditioning evokes a specific, dynamic map of functional synaptic states in that circuit.

A Variable Oscillator Underlies the Measurement of Time Intervals in the Rostral Medial Prefrontal Cortex during Classical Eyeblink Conditioning in Rabbits

We were interested in determining whether rostral medial prefrontal cortex (rmPFC) neurons participate in the measurement of conditioned stimulus–unconditioned stimulus (CS-US) time intervals during classical eyeblink conditioning. Rabbits were conditioned with a delay paradigm consisting of a tone as CS. The CS started 50, 250, 500, 1000, or 2000 ms before and coterminated with an air puff (100 ms) directed at the cornea as the US. Eyelid movements were recorded with the magnetic search coil technique and the EMG activity of the orbicularis oculi muscle. Firing activities of rmPFC neurons were recorded across conditioning sessions. Reflex and conditioned eyelid responses presented a dominant oscillatory frequency of ≈12 Hz. The firing rate of each recorded neuron presented a single peak of activity with a frequency dependent on the CS-US interval (i.e., ≈12 Hz for 250 ms, ≈6 Hz for 500 ms, and≈3 Hz for 1000 ms). Interestingly, rmPFC neurons presented their dominant firing peaks at three precise times evenly distributed with respect to CS start and also depending on the duration of the CS-US 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) CS-US intervals. rmPFC neurons seem not to encode the oscillatory properties characterizing conditioned eyelid responses in rabbits, but are probably involved in the determination of CS-US intervals of an intermediate range (250–1000 ms). We propose that a variable oscillator underlies the generation of working memories in rabbits. SIGNIFICANCE STATEMENT The way in which brains generate working memories (those used for the transient processing and storage of newly acquired information) is still an intriguing question. Here, we report that the firing activities of neurons located in the rostromedial prefrontal cortex recorded in alert behaving rabbits are controlled by a dynamic oscillator. This oscillator generated firing frequencies in a variable band of 3–12 Hz depending on the conditioned stimulus–unconditioned stimulus intervals (1 s, 500 ms, 250 ms) selected for classical eyeblink conditioning of behaving rabbits. Shorter (50 ms) and longer (2 s) intervals failed to activate the oscillator and prevented the acquisition of conditioned eyelid responses. This is an unexpected mechanism to generate sustained firing activities in neural circuits generating working memories.