Conditioned Eyelid Responses Research Articles

Discrimination training affects stimulus generalization in mice during Pavlovian eyeblink conditioning.

The delicate balance between discrimination and generalization of responses is crucial for survival in our ever-changing environment. In particular, it is important to understand how stimulus discrimination affects the level of stimulus generalization. For example, when we use non-differential training for Pavlovian eyeblink conditioning to investigate generalization of cerebellar-related eyelid motor responses, we find generalization effects on amount, amplitude and timing of the conditioned responses. However, it is unknown what the generalization effects are following differential training. We trained mice to close their eyelids to a 10 kHz tone with an air-puff as the reinforcing stimulus (CS+), while alternatingly exposing them to a tone frequency of either 4 kHz, 9 kHz or 9.5 kHz without the air-puff (CS-) during the training blocks. We tested the generalization effects during the expression of the responses after the training period with tones ranging from 2 kHz to 20 kHz. Our results show that the level of generalization tended to positively correlate with the difference between the CS+ and the CS- training stimuli. These effects of generalization were found for the probability, amplitude but not for the timing of the conditioned eyelid responses. These data indicate the specificity of the generalization effects following differential versus non-differential training, highlighting the relevance of discrimination learning for stimulus generalization.

Noradrenergic projections regulate the acquisition of classically conditioned eyelid responses in wild-type and are impaired in kreisler mice

During embryonic development, heterozygous mutant kreisler mice undergo ectopic expression of the Hoxa3 gene in the rostral hindbrain, affecting the opioid and noradrenergic systems. In this model, we have investigated behavioral and cognitive processes in their adulthood. We confirmed that pontine and locus coeruleus neuronal projections are impaired, by using startle and pain tests and by analyzing immunohistochemical localization of tyrosine hydroxylase. Our results showed that, even if kreisler mice are able to generate eyelid reflex responses, there are differences with wild-types in the first component of the response (R1), modulated by the noradrenergic system. The acquisition of conditioned motor responses is impaired in kreisler mice when using the trace but not the delay paradigm, suggesting a functional impairment in the hippocampus, subsequently confirmed by reduced quantification of alpha2a receptor mRNA expression in this area but not in the cerebellum. Moreover, we demonstrate the involvement of adrenergic projection in eyelid classical conditioning, as clonidine prevents the appearance of eyelid conditioned responses in wild-type mice. In addition, hippocampal motor learning ability was restored in kreisler mice by administration of adrenergic antagonist drugs, and a synergistic effect was observed following simultaneous administration of idazoxan and naloxone.

Functional properties of eyelid conditioned responses and involved brain centers.

For almost a century the classical conditioning of nictitating membrane/eyelid responses has been used as an excellent and feasible experimental model to study how the brain organizes the acquisition, storage, and retrieval of new motor abilities in alert behaving mammals, including humans. Lesional, pharmacological, and electrophysiological approaches, and more recently, genetically manipulated animals have shown the involvement of numerous brain areas in this apparently simple example of associative learning. In this regard, the cerebellum (both cortex and nuclei) has received particular attention as a putative site for the acquisition and storage of eyelid conditioned responses, a proposal not fully accepted by all researchers. Indeed, the acquisition of this type of learning implies the activation of many neural processes dealing with the sensorimotor integration and the kinematics of the acquired ability, as well as with the attentional and cognitive aspects also involved in this process. Here, we address specifically the functional roles of three brain structures (red nucleus, cerebellar interpositus nucleus, and motor cortex) mainly involved in the acquisition and performance of eyelid conditioned responses and three other brain structures (hippocampus, medial prefrontal cortex, and claustrum) related to non-motor aspects of the acquisition process. The main conclusion is that the acquisition of this motor ability results from the contribution of many cortical and subcortical brain structures each one involved in specific (motor and cognitive) aspects of the learning process.

Cerebellum Involvement in Dystonia During Associative Motor Learning: Insights From a Data-Driven Spiking Network Model.

Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements, postures, or both. Although dystonia is traditionally associated with basal ganglia dysfunction, recent evidence has been pointing to a role of the cerebellum, a brain area involved in motor control and learning. Cerebellar abnormalities have been correlated with dystonia but their potential causative role remains elusive. Here, we simulated the cerebellar input-output relationship with high-resolution computational modeling. We used a data-driven cerebellar Spiking Neural Network and simulated a cerebellum-driven associative learning task, Eye-Blink Classical Conditioning (EBCC), which is characteristically altered in relation to cerebellar lesions in several pathologies. In control simulations, input stimuli entrained characteristic network dynamics and induced synaptic plasticity along task repetitions, causing a progressive spike suppression in Purkinje cells with consequent facilitation of deep cerebellar nuclei cells. These neuronal processes caused a progressive acquisition of eyelid Conditioned Responses (CRs). Then, we modified structural or functional local neural features in the network reproducing alterations reported in dystonic mice. Either reduced olivocerebellar input or aberrant Purkinje cell burst-firing resulted in abnormal learning curves imitating the dysfunctional EBCC motor responses (in terms of CR amount and timing) of dystonic mice. These behavioral deficits might be due to altered temporal processing of sensorimotor information and uncoordinated control of muscle contractions. Conversely, an imbalance of excitatory and inhibitory synaptic densities on Purkinje cells did not reflect into significant EBCC deficit. The present work suggests that only certain types of alterations, including reduced olivocerebellar input and aberrant PC burst-firing, are compatible with the EBCC changes observed in dystonia, indicating that some cerebellar lesions can have a causative role in the pathogenesis of symptoms.

Stimulus Generalization in Mice during Pavlovian Eyeblink Conditioning.

Here, we investigate stimulus generalization in a cerebellar learning paradigm, called eyeblink conditioning. Mice were conditioned to close their eyes in response to a 10-kHz tone by repeatedly pairing this tone with an air puff to the eye 250 ms after tone onset. After 10 consecutive days of training, when mice showed reliable conditioned eyelid responses to the 10-kHz tone, we started to expose them to tones with other frequencies, ranging from 2 to 20 kHz. We found that mice had a strong generalization gradient, whereby the probability and amplitude of conditioned eyelid responses gradually decreases depending on the dissimilarity with the 10-kHz tone. Tones with frequencies closest to 10 kHz evoked the most and largest conditioned eyelid responses and each step away from the 10-kHz tone resulted in fewer and smaller conditioned responses (CRs). In addition, we found that tones with lower frequencies resulted in CRs that peaked earlier after tone onset compared with those to tones with higher frequencies. Together, our data show prominent generalization patterns in cerebellar learning. Since the known function of cerebellum is rapidly expanding from pure motor control to domains that include cognition, reward-learning, fear-learning, social function, and even addiction, our data imply generalization controlled by cerebellum in all these domains.

Young Domestic Pigs (Sus scrofa) Can Perform Pavlovian Eyeblink Conditioning.

Introduction: Pigs have been an increasingly popular preclinical model in nutritional neuroscience, as their anatomy, physiology, and nutrition requirements are highly comparable to those of humans. Eyeblink conditioning is one of the most well-validated behavioral paradigms in neuroscience to study underlying mechanisms of learning and memory formation in the cerebellum. Eyeblink conditioning has been performed in many species but has never been done on young pigs. Therefore, our aim here was to develop and validate an eyeblink conditioning paradigm in young pigs.Method: Eighteen intact male pigs were artificially reared from postnatal day 2–30. The eyeblink conditioning setup consisted of a sound-damping box with a hammock that pigs were placed in, which allowed the pig to remain comfortable yet maintain a typical range of head motion. In a delay conditioning paradigm, the conditional stimulus (CS) was a 550 ms blue light-emitting diode (LED), the unconditional stimulus (US) was a 50 ms eye air-puff, the CS-US interval was 500 ms. Starting at postnatal day 14, pigs were habituated for 5 days to the eyeblink conditioning setup, followed by 5 daily sessions of acquisition training (40 paired CS-US trials each day).Results: The group-averaged amplitude of conditioned eyelid responses gradually increased over the course of the 5 days of training, indicating that pigs learned to make the association between the LED light CS and the air-puff US. A similar increase was found for the conditioned response (CR) probability: the group-averaged CR probability on session 1 was about 12% and reached a CR probability of 55% on day 5. The latency to CR peak time lacked a temporal preference in the first session but clearly showed preference from the moment that animals started to show more CRs in session 2 and onwards whereby the eyelid was maximally closed exactly at the moment that the US would be delivered.Conclusion: We concluded that 3-week-old pigs have the capability of performing in a cerebellar classical conditioning task, demonstrating for the first time that eyeblink conditioning in young pigs has the potential to be a valuable behavioral tool to measure neurodevelopment.

Medial Auditory Thalamus Is Necessary for Expression of Auditory Trace Eyelid Conditioning.

Transforming a brief sensory event into a persistent neural response represents a mechanism for linking temporally disparate stimuli together to support learning. The cerebellum requires this type of persistent input during trace conditioning to engage associative plasticity and acquire adaptively timed conditioned responses (CRs). An initial step toward identifying the sites and mechanisms generating and transmitting persistent signals to the cerebellum is to identify the input pathway. The medial auditory thalamic nuclei (MATN) are the necessary and sufficient source of auditory input to the cerebellum for delay conditioning in rodents and a possible input to forebrain sites generating persistent signals. Using pharmacological and computational approaches, we test (1) whether the necessity of MATN during auditory eyelid conditioning is conserved across species, (2) whether the MATN are necessary for the expression of trace eyelid CRs, and if so, (3)whether this relates to the generation of persistent signals. We find that contralateral inactivation of MATN with muscimol largely abolished trace and delay CRs in male rabbits. Residual CRs were decreased in amplitude, but CR timing was unaffected. Results from large-scale cerebellar simulations are consistent with previous experimental demonstrations that silencing only CS-duration inputs does not abolish trace CRs, and instead affects their timing. Together, these results suggest that the MATN are a necessary component of both the direct auditory stimulus pathway to the cerebellum and the pathway generating task-essential persistent signals.SIGNIFICANCE STATEMENT Persistent activity is required for working memory-dependent tasks, such as trace conditioning, and represents a mechanism by which sensory information can be used over time for learning and cognition. This neuronal response entails the transformation of a discrete sensory-evoked response into a signal that extends beyond the stimulus event. Understanding the generation and transmission of this stimulus transformation requires identifying the input sources necessary for task-essential persistent signals. We report that the medial auditory thalamic nuclei are required for the expression of auditory trace conditioning and suggest that these nuclei are a component of the pathway-generating persistent signals. Our study provides a foundation for testing circuit-level mechanisms underlying persistent activity in a cerebellar learning model with identified inputs and well defined behavioral outputs.

Timing correlations between cerebellar interpositus neuronal firing and classically conditioned eyelid responses in wild-type and Lurcher mice

Classical eyeblink conditioning is an experimental model widely used for the study of the neuronal mechanisms underlying the acquisition of new motor and cognitive skills. There are two principal interpretations of the role of the cerebellum in the learning of eyelid conditioned responses (CRs). One considers that the cerebellum is the place where this learning is acquired and stored, while the second suggests that the cerebellum is mostly involved in the proper performance of acquired CRs, implying that there must be other brain areas involved in the learning process. We checked the timing of cerebellar interpositus nucleus (IPN) neurons’ firing rate with eyelid CRs in both wild-type (WT) and Lurcher (a model of cerebellar cortex degeneration) mice. We used delay and trace conditioning paradigms. WT mice presented a better execution for delay vs. trace conditioning and also for these two paradigms than did Lurcher mice. IPN neurons were activated during CRs following the activation of the orbicularis oculi muscle. Firing patterns of IPN neurons were altered in Lurcher mice. In conclusion, the cerebellum seems to be mostly related with the performance of conditioned responses, rather than with their acquisition.

Cerebellar Processing Common to Delay and Trace Eyelid Conditioning

Results from previous lesion studies have been interpreted as evidence that the cerebellar cortex plays different roles for delay and trace conditioning of eyelid responses. However, the cerebellar cortex is organized by parasagittal stripes of Purkinje cells (PCs) that converge onto common deep nucleus neurons and receive common or related climbing fiber inputs. Based on this organization, we hypothesized that cerebellar tasks involving the same response system, such as delay and trace eyelid conditioning, would engage the same PCs and that the relationships between PC activity and expression of behavioral responses would be similar for both tasks. To test these hypotheses, we used tetrode recordings from eyelid PCs in rabbits during expression of delay- and trace-conditioned eyelid responses. Previous recording studies during delay conditioning described a strong relationship between eyelid PC activity and the kinematics of conditioned eyelid responses. The present results replicate these findings for delay conditioning and show that the same relationship exists during trace eyelid conditioning. During transitions from delay to trace responding, the relationship between eyelid PCs and behavioral responses was relatively stable. We found that an inverse firing rate model tuned to predict PC activity during one training paradigm could then predict equally well the PC activity during the other training paradigm. These results provide strong evidence that cerebellar cortex processing is similar for delay and trace eyelid conditioning and that the parasagittal organization of the cerebellum, not the conditioning paradigm, dictate which neurons are engaged to produce adaptively timed conditioned responses.SIGNIFICANCE STATEMENT A variety of evidence from eyelid conditioning and other cerebellar-dependent behaviors indicates that the cerebellar cortex is necessary for learning and proper timing of cerebellar learned responses. Debates exist about whether trace eyelid conditioning data show that fundamentally different mechanisms operate in the cerebellum during tasks when input from the forebrain is necessary for learning. We show here that learning-related changes in a specific population of Purkinje cells control the timing and amplitude of cerebellar responses the same way regardless of the inputs necessary to learn the task. Our results indicate the parasagittal organization of the cerebellar cortex, not the complexity of inputs to the cerebellum, determines which neurons are engaged in the learning and execution of cerebellar-mediated responses.

Learning as a Functional State of the Brain: Studies in Wild-Type and Transgenic Animals.

Contemporary neuroscientists are paying increasing attention to subcellular, molecular, and electrophysiological mechanisms underlying learning and memory processes. Recent studies have examined the development of transgenic mice affected at different stages of the learning process, or have emulated in animals various human pathological conditions involving cognition and motor learning. However, a parallel effort is needed to develop stimulating and recording techniques suitable for use in behaving mice in order to understand activity-dependent synaptic changes taking place during the very moment of the learning process. The in vivo models should incorporate information collected from different molecular and in vitro approaches. Long-term potentiation (LTP) has been proposed as the neural mechanism underlying synaptic plasticity, and NMDA receptors have been proposed as the molecular substrate of LTP. It now seems necessary to study the relationship of both LTP and NMDA receptors to functional changes in synaptic efficiency taking place during actual learning in selected cerebral cortical structures. Here, we review data collected in our laboratory during the past 10years on the involvement of different hippocampal synapses in the acquisition of the classically conditioned eyelid responses in behaving mice. Overall the results indicate a specific contribution of each cortical synapse to the acquisition and storage of new motor and cognitive abilities. Available data show that LTP, evoked by high-frequency stimulation of Schaffer collaterals, disturbs both the acquisition of conditioned eyelid responses and the physiological changes that occur at hippocampal synapses during learning. Moreover, the administration of NMDA-receptor antagonists is able not only to prevent LTP induction in vivo, but also to hinder both the formation of conditioned eyelid responses and functional changes in the strength of the CA3-CA1 synapse. Nevertheless, many other neurotransmitter receptors, intracellular mediators, and transcription factors are also involved in learning and memory processes. In summary, it can be proposed that learning and memory in behaving mammals are the result of the activation of complex and distributed functional states involving many different cerebral cortical synapses, with the participation also of various neurotransmitter systems.

The Motor Cortex Is Involved in the Generation of Classically Conditioned Eyelid Responses in Behaving Rabbits.

Classical blink conditioning is a well known model for studying neural generation of acquired motor responses. The acquisition of this type of associative learning has been related to many cortical, subcortical, and cerebellar structures. However, until now, no one has studied the motor cortex (MC) and its possible role in classical eyeblink conditioning. We recorded in rabbits the activity of MC neurons during blink conditioning using a delay paradigm. Neurons were identified by their antidromic activation from facial nucleus (FN) or red nucleus (RN). For conditioning, we used a tone as a conditioned stimulus (CS) followed by an air puff as an unconditioned stimulus (US) that coterminated with it. Conditioned responses (CRs) were determined from the electromyographic activity of the orbicularis oculi muscle and/or from eyelid position recorded with the search coil technique. Type A neurons increased their discharge rates across conditioning sessions and reached peak firing during the CS-US interval, while type B cells presented a second peak during US presentation. Both of them project to the FN. Type C cells increased their firing across the CS-US interval, reaching peak values at the time of US presentation, and were activated from the RN. These three types of neurons fired well in advance of the beginning of CRs and changed with them. Reversible inactivation of the MC during conditioning evoked a decrease in learning curves and in the amplitude of CRs, while train stimulation of the MC simulated the profile and kinematics of conditioned blinks. In conclusion, MC neurons are involved in the acquisition and expression of CRs. Classical blink conditioning is a popular experimental model for studying neural mechanisms underlying the acquisition of motor skills. The acquisition of this type of associative learning has been related to many cortical, subcortical, and cerebellar structures. However, until now, no one has studied the motor cortex (MC) and its possible role in classical eyeblink conditioning. Here, we report that the firing activities of MC neurons, recorded in behaving rabbits, are related to and preceded the initiation of conditioned blinks. MC neurons were identified as projecting to the red or facial nuclei and encoded the kinematics of conditioned eyelid responses. The timed stimulation of recording sites simulated the profile of conditioned blinks. MC neurons play a role in the acquisition and expression of these acquired motor responses.

Enhancement of delay eyelid conditioning by microcurrent electrical stimulation of the medial prefrontal cortex is triggered by the expression of Fos protein in guinea pigs.

Eyelid conditioning, including delay eyelid conditioning and trace eyelid conditioning, has been used extensively to study neural structures and mechanisms of learning and memory as a form of associative learning. In the present study, microcurrent electrical stimulation was used to stimulate the medial prefrontal cortex (mPFC) to induce delay eyelid conditioning in guinea pigs. The acquisition rate and relative latency of the conditioned eyelid response (CR) and the startle eyelid response (SR) were analyzed. The mPFC sites in the guinea pigs were examined under a light microscope following Nissl staining. In addition, the expression of Fos protein in neurons was detected using immunohistochemistry and western blot analysis. The results indicated that the acquisition rates of CR and SR were increased significantly (P<0.05), whilst the relative latencies of CR and SR were decreased significantly (P<0.05). Lesions were observed in the mPFC regions in the training group when compared with the control group. In addition, immunohistochemistry and western blot analysis revealed that Fos expression was significantly increased in the training group when compared with the sham group for the control and resident-intruder test guinea pigs (P<0.05). Therefore, the enhancement of delay eyelid conditioning by microcurrent electrical stimulation of the mPFC in guinea pigs was triggered by the expression of Fos protein. The observations of the present study further expand the understanding of conditioned reflexes and may aid future investigations into the formation of eyelid conditioning and the mechanisms underlying the circuit in various conditions.

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

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.

Relating cerebellar purkinje cell activity to the timing and amplitude of conditioned eyelid responses.

How Purkinje cell (PC) activity may be altered by learning is central to theories of the cerebellum. Pavlovian eyelid conditioning, because of how directly it engages the cerebellum, has helped reveal many aspects of cerebellar learning and the underlying mechanisms. Theories of cerebellar learning assert that climbing fiber inputs control plasticity at synapses onto PCs, and thus PCs control the expression of learned responses. We tested this assertion by recording 184 eyelid PCs and 240 non-eyelid PCs during the expression of conditioned eyelid responses (CRs) in well trained rabbits. By contrasting the responses of eyelid and non-eyelid PCs and by contrasting the responses of eyelid PCs under conditions that produce differently timed CRs, we test the hypothesis that learning-related changes in eyelid PCs contribute to the learning and adaptive timing of the CRs. We used a variety of analyses to test the quantitative relationships between eyelid PC responses and the kinematic properties of the eyelid CRs. We find that the timing of eyelid PC responses varies systematically with the timing of the behavioral CRs and that there are differences in the magnitude of eyelid PC responses between larger-CR, smaller-CR, and non-CR trials. However, eyelid PC activity does not encode any single kinematic property of the behavioral CRs at a fixed time lag, nor does it linearly encode CR amplitude. Even so, the results are consistent with the hypothesis that learning-dependent changes in PC activity contribute to the adaptively timed expression of conditioned eyelid responses.

Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions.

Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct GAD autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal GAD antibodies. We found that GAD autoantibodies present in patients with stiff person syndrome (n = 7) and cerebellar ataxia (n = 15) recognized an epitope distinct from that recognized by GAD autoantibodies present in patients with type 1 diabetes mellitus (n = 10) or limbic encephalitis (n = 4). We demonstrated that the administration of a monoclonal GAD antibody representing this epitope specificity; (1) disrupted in vitro the association of GAD with γ-Aminobutyric acid containing synaptic vesicles; (2) depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect; (3) significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task; (4) markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm; and (5) induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of GAD by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such GAD antibodies could be envisioned.

Axonal Sprouting and Formation of Terminals in the Adult Cerebellum during Associative Motor Learning

Plastic changes in the efficacy of synapses are widely regarded to represent mechanisms underlying memory formation. So far, evidence for learning-dependent, new neuronal wiring is limited. In this study, we demonstrate that pavlovian eyeblink conditioning in adult mice can induce robust axonal growth and synapse formation in the cerebellar nuclei. This de novo wiring is both condition specific and region specific because it does not occur in pseudoconditioned animals and is particularly observed in those parts of the cerebellar nuclei that have been implicated to be involved in this form of motor learning. Moreover, the number of new mossy fiber varicosities in these parts of the cerebellar nuclei is positively correlated with the amplitude of conditioned eyelid responses. These results indicate that outgrowth of axons and concomitant occurrence of new terminals may, in addition to plasticity of synaptic efficacy, contribute to the formation of memory.

The Rostral Medial Prefrontal Cortex Regulates the Expression of Conditioned Eyelid Responses in Behaving Rabbits

We studied the contribution of the rostral mPFC (rmPFC) to the acquisition and performance of classical eyeblink conditioning in rabbits using a delay paradigm. The rmPFC was determined by its afferent projections from the medial half of the mediodorsal thalamic nucleus. The rmPFC neurons were identified by their antidromic activation from the mediodorsal nucleus and/or by their firing characteristics. The rmPFC neurons increased their firing during the first conditioning sessions, but decreased it when conditioned responses (CRs) reached asymptotic values. Therefore, no significant relationships could be established between neuronal firing rates and the percentage of CRs or the electromyographic (EMG) activity of the orbicularis oculi muscle during conditioning. Electrical train stimulation of the rmPFC produced a significant inhibition of air-puff-evoked blinks and reduced the generation of CRs compared with controls. Inhibition of the rmPFC by the local injection of lidocaine produced an increase in the amplitude of evoked reflex and conditioned eyeblinks and in the percentage of CRs. The rmPFC seems to be a potent inhibitor of reflex and conditioned eyeblinks, controlling the release of newly acquired eyelid responses until advanced stages of the acquisition process--i.e., until the need for the acquired response is fully confirmed. Therefore, the rmPFC seems to act as a "flip-flop" mechanism in controlling behavior.

Using a million cell simulation of the cerebellum: Network scaling and task generality

Several factors combine to make it feasible to build computer simulations of the cerebellum and to test them in biologically realistic ways. These simulations can be used to help understand the computational contributions of various cerebellar components, including the relevance of the enormous number of neurons in the granule cell layer. In previous work we have used a simulation containing 12000 granule cells to develop new predictions and to account for various aspects of eyelid conditioning, a form of motor learning mediated by the cerebellum. Here we demonstrate the feasibility of scaling up this simulation to over one million granule cells using parallel graphics processing unit (GPU) technology. We observe that this increase in number of granule cells requires only twice the execution time of the smaller simulation on the GPU. We demonstrate that this simulation, like its smaller predecessor, can emulate certain basic features of conditioned eyelid responses, with a slight improvement in performance in one measure. We also use this simulation to examine the generality of the computation properties that we have derived from studying eyelid conditioning. We demonstrate that this scaled up simulation can learn a high level of performance in a classic machine learning task, the cart–pole balancing task. These results suggest that this parallel GPU technology can be used to build very large-scale simulations whose connectivity ratios match those of the real cerebellum and that these simulations can be used guide future studies on cerebellar mediated tasks and on machine learning problems.

Red Nucleus Neurons Actively Contribute to the Acquisition of Classically Conditioned Eyelid Responses in Rabbits

The red nucleus (RN) is a midbrain premotor center that has been suggested as being involved in the acquisition and/or performance of classically conditioned nictitating membrane/eyelid responses. We recorded in rabbits the activity of RN and pararubral neurons during classical eyeblink conditioning using a delay paradigm. Neurons were identified by their antidromic activation from contralateral facial and accessory abducens nuclei and by their synaptic activation from the ipsilateral motor cortex (MC) and the contralateral cerebellar interpositus (IP) nucleus. For conditioning, we used a tone as a conditioned stimulus (CS) followed 250 ms later by a 100 ms air puff as an unconditioned stimulus (US) coterminating with it. Conditioned responses (CRs) were determined from the evoked changes in the electromyographic activity of the orbicularis oculi (OO) muscle. Recorded neurons were classified by their antidromic activation and by their changes in firing rate during the CS-US interval. Identified neurons increased their firing rates in relation to the successive conditioning sessions, but their discharge rates were related more to the EMG activity of the OO muscle than to the learning curves. Reversible inactivation of the IP nucleus with lidocaine during conditioning evoked a complete disappearance of both conditioned and unconditioned eyelid responses, and a progressive decrease in CR-related activity of RN neurons. In contrast, MC inactivation evoked a decrease in the acquisition process and an initial disfacilitation of neuronal firing (which was later recovered), together with the late appearance of CRs. Thus, RN neurons presented learning-dependent changes in activity following MC inactivation.

Role of Reuniens Nucleus Projections to the Medial Prefrontal Cortex and to the Hippocampal Pyramidal CA1 Area in Associative Learning

We studied the interactions between short- and long-term plastic changes taking place during the acquisition of a classical eyeblink conditioning and following high-frequency stimulation (HFS) of the reuniens nucleus in behaving mice. Synaptic changes in strength were studied at the reuniens-medial prefrontal cortex (mPFC) and the reuniens-CA1 synapses. Input/output curves and a paired-pulse study enabled determining the functional capabilities of the two synapses and the optimal intensities to be applied at the reuniens nucleus during classical eyeblink conditioning and for HFS applied to the reuniens nucleus. Animals were conditioned using a trace paradigm, with a tone as conditioned stimulus (CS) and an electric shock to the trigeminal nerve as unconditioned stimulus (US). A single pulse was presented to the reuniens nucleus to evoke field EPSPs (fEPSPs) in mPFC and CA1 areas during the CS-US interval. No significant changes in synaptic strength were observed at the reuniens-mPFC and reuniens-CA1 synapses during the acquisition of eyelid conditioned responses (CRs). Two successive HFS sessions carried out during the first two conditioning days decreased the percentage of CRs, without evoking any long-term potentiation (LTP) at the recording sites. HFS of the reuniens nucleus also prevented the proper acquisition of an object discrimination task. A subsequent study revealed that HFS of the reuniens nucleus evoked a significant decrease of paired-pulse facilitation. In conclusion, reuniens nucleus projections to prefrontal and hippocampal circuits seem to participate in the acquisition of associative learning through a mechanism that does not required the development of LTP.