Storage of a naturally acquired conditioned response is impaired in patients with cerebellar degeneration.

Previous findings suggested that the human cerebellum is involved in the acquisition but not the long-term storage of motor associations. The finding of preserved retention in cerebellar patients was fundamentally different from animal studies which show that both acquisition and retention depends on the integrity of the cerebellum. The present study investigated whether retention had been preserved because critical regions of the cerebellum were spared. Visual threat eye-blink responses, that is, the anticipatory closure of the eyes to visual threats, have previously been found to be naturally acquired conditioned responses. Because acquisition is known to take place in very early childhood, visual threat eye-blink responses can be used to test retention in patients with adult onset cerebellar disease. Visual threat eye-blink responses were tested in 19 adult patients with cerebellar degeneration, 27 adult patients with focal cerebellar lesions due to stroke, 24 age-matched control subjects, and 31 younger control subjects. High-resolution structural magnetic resonance images were acquired in patients to perform lesion-symptom mapping. Voxel-based morphometry was performed in patients with cerebellar degeneration, and voxel-based lesion-symptom mapping in patients with focal disease. Visual threat eye-blink responses were found to be significantly reduced in patients with cerebellar degeneration. Visual threat eye-blink responses were also reduced in patients with focal disease, but to a lesser extent. Visual threat eye-blink responses declined with age. In patients with cerebellar degeneration the degree of cerebellar atrophy was positively correlated with the reduction of conditioned responses. Voxel-based morphometry showed that two main regions within the superior and inferior parts of the posterior cerebellar cortex contributed to expression of visual threat eye-blink responses bilaterally. Involvement of the more inferior parts of the posterior lobe was further supported by voxel-based lesion symptom mapping in focal cerebellar patients. The present findings show that the human cerebellar cortex is involved in long-term storage of learned responses.

Blocking glutamate-mediated inferior olivary signals abolishes expression of conditioned eyeblinks but does not prevent their acquisition.

The inferior olive (IO) is considered a crucial component of the eyeblink conditioning network. The cerebellar learning hypothesis proposes that the IO provides the cerebellum with a teaching signal that is required for the acquisition and maintenance of conditioned eyeblinks. Supporting this concept, previous experiments showed that lesions or inactivation of the IO blocked CR acquisition. However, these studies were not conclusive. The drawback of the methods used by those studies is that they not only blocked task-related signals, but also completely shut down the spontaneous activity within the IO, which affects the rest of the eyeblink circuits in a nonspecific manner. We hypothesized that more selective blocking of task-related IO signals could be achieved by using injections of glutamate antagonists, which reduce, but do not eliminate, the spontaneous activity in the IO. We expected that if glutamate-mediated IO signals are required for learning, then blocking these signals during training sessions should prevent conditioned response (CR) acquisition. To test this prediction, rabbits were trained to acquire conditioned eyeblinks to a mild vibrissal airpuff as the conditioned stimulus while injections of the glutamate antagonist γ-d-glutamylglycine were administered to the IO. Remarkably, even though this treatment suppressed CRs during training sessions, the postacquisition retention test revealed that CR acquisition had not been abolished. The ability to acquire CRs with IO unconditioned stimulus signals that were blocked or severely suppressed suggests that mechanisms responsible for CR acquisition are extremely resilient and probably less dependent on IO-task-related signals than previously thought.

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.

Assessing the role of inferior olivary sensory signaling in the expression of conditioned eyeblinks using a combined glutamate/GABAA receptor antagonist protocol.

The inferior olive (IO) is a major component of the eyeblink conditioning neural network. The cerebellar learning hypothesis assumes that the IO supplies the cerebellum with a "teaching" unconditioned stimulus input required for the acquisition of the conditioned response (CR) and predicts that inactivating this input leads to the extinction of CRs. Previous tests of this prediction attempted to block the teaching input by blocking glutamatergic sensory inputs in the IO. These tests were inconclusive because blocking glutamate neurotransmission in the IO produces a nonspecific tonic malfunction of cerebellar circuits. The purpose of the present experiment was to examine whether the behavioral outcomes of blocking glutamate receptors in the IO could be counterbalanced by reducing GABA-mediated inhibition in the IO. We found that injecting the IO with the glutamate antagonist γ-d-glutamylglycine (DGG) abolished previously learned CRs, whereas injecting the GABA(A) receptor antagonist gabazine at the same site did not affect CR incidence but shortened CR latencies and produced tonic eyelid closure. To test whether the glutamate antagonist-induced behavioral deficit could be offset by elevating IO activity with GABA(A) antagonists, rabbits were first injected with DGG and then with gabazine in the same training session. While DGG abolished CRs, follow-up injections of gabazine accelerated their recovery. These findings suggest that the level of IO neuronal activity is critical for the performance of CRs, and that combined pharmacological approaches that maintain spontaneous activity at near normal levels hold tremendous potential for unveiling the role of IO-mediated signals in eyeblink conditioning.

A trigeminal conditioned stimulus yields fast acquisition of cerebellum-dependent conditioned eyeblinks.

Classical conditioning of the eyeblink response in the rabbit is a form of motor learning whereby the animal learns to respond to an initially irrelevant conditioned stimulus (CS). It is thought that acquired conditioned responses (CRs) are adaptive because they protect the eye in anticipation of potentially harmful events. This protective mechanism is surprisingly inefficient because the acquisition of CRs requires extensive training - a condition that is unlikely to occur in nature. We hypothesized that the rate of conditioning in rabbits could depend on CS modality and that stimulating mystacial vibrissae as the CS could produce CR acquisition faster than the traditional auditory or visual stimulation. We tested this hypothesis by conditioning naïve rabbits in the delay paradigm using a weak airpuff CS (vCS) directed to the ipsilateral mystacial vibrissae. We found that the trigeminal vCS yields significantly faster CR acquisition. We next examined if vCS-evoked CRs are dependent on the intermediate cerebellum in the same fashion as CRs evoked by the traditional auditory CS. We found that vibrissal CRs could be abolished by inactivating the cerebellar interposed nuclei (IN) with muscimol. In addition, injections of picrotoxin in the IN shortened the onset latency of vibrissal CRs. These findings suggest that the tone and vCS-evoked CRs share similar cerebellar dependency.

Inactivating the middle cerebellar peduncle abolishes the expression of short-latency conditioned eyeblinks.

The interposed nuclei (IN) of the cerebellum play a crucial role in the classically conditioned eyeblink circuit. It has previously been shown in well-trained animals that injecting the IN with GABA(A) antagonists produces short-latency conditioned responses (SLRs). The mechanism underlying SLR generation is not clear. According to one concept, SLRs originate in cerebellar nuclei in response to direct inputs from collaterals of mossy fibers. An alternate explanation is that SLRs are produced by extra-cerebellar circuits that are excited by increased tonic activity in cerebellar nuclei or by the combined action of inputs to cerebellar nuclei from mossy fiber collaterals and incompletely blocked Purkinje cells. In the present study, we examined whether cerebellar afferent axons in the middle cerebellar peduncle (MCP) participate in SLR expression. We hypothesized that if SLRs are evoked by the sensory mossy fiber input to the IN and cerebellar cortex, then blocking the MCP should abolish these responses. Well-trained animals, which had been implanted with dual injection cannulae in the left IN and the left MCP, were injected with gabazine (GZ) into the IN to produce SLRs followed by an injection of the sodium channel blocker tetrodotoxin (TTX) into the MCP. TTX infusions in the MCP suppressed both CRs and SLRs. These findings suggest that the expression of SLRs depends on both direct and cerebellar cortex-mediated sensory information from the mossy fiber system.

Blocking GABAA neurotransmission in the interposed nuclei: effects on conditioned and unconditioned eyeblinks.

The interposed nuclei (IN) of the intermediate cerebellum are critical components of the circuits that control associative learning of eyeblinks and other defensive reflexes in mammals. The IN, which represent the sole output of the intermediate cerebellum, receive massive GABAergic input from Purkinje cells of the cerebellar cortex and are thought to contribute to the acquisition and performance of classically conditioned eyeblinks. The specific role of deep cerebellar nuclei and the cerebellar cortex in eyeblink conditioning are not well understood. One group of studies reported that blocking GABA(A) neurotransmission in the IN altered the time profile of conditioned responses (CRs), suggesting that the main function of the cerebellar cortex is to shape the timing of CRs. Other studies reported that blocking GABA(A) neurotransmission in the IN abolished CRs, indicating a more fundamental involvement of the cerebellar cortex in CR generation. When examining this controversy, we hypothesized that the behavioral effect of GABA(A) blockers could be dose-dependent. The IN of classically conditioned rabbits were injected with high and low doses of picrotoxin and gabazine. Both GABA(A) blockers produced tonic eyelid closure. A high dose of both drugs abolished CRs, whereas a less complete block of GABA(A)-mediated inputs with substantially smaller drug doses shortened CR latencies. In addition, low doses of picrotoxin facilitated the expression of unconditioned eyeblinks evoked by trigeminal stimulation. These results suggest that the intermediate cerebellum regulates both associative and non-associative components of the eyeblink reflex, and that behavioral effects of blocking Purkinje cell action on IN neurons are related to collective changes in cerebellar signals and in the excitability of extra-cerebellar eyeblink circuits.

Cerebellar dysfunction explains the extinction-like abolition of conditioned eyeblinks after NBQX injections in the inferior olive.

Classical conditioning of the eyeblink response is a form of motor learning that is controlled by the intermediate cerebellum and related brainstem structures. The inferior olive (IO) is commonly thought to provide the cerebellum with a "teaching" unconditioned stimulus (US) signal required for cerebellar learning. Testing this concept has been difficult because the IO, in addition to its putative learning function, also controls tonic activity in the cerebellum. Previously, it was reported that inactivation of AMPA/kainate receptors in the IO produces extinction of conditioned responses (CRs), suggesting that it blocks the transmission of US signals without perturbing the functional state of the cerebellum. However, the electrophysiological support for this critical finding was lacking, mostly because of methodological difficulties in maintaining stable recordings from the same set of single units throughout long drug injection sessions in awake rabbits. To address this critical issue, we used our microwire-based multiple single-unit recording method. The IO in trained rabbits was injected with the AMPA/kainate receptor blocker, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), and its effects on CR expression and neuronal activity in the cerebellar interposed nuclei (IN) were examined. We found that NBQX abolished CR expression and that delayed drug effects were independent of the presentation of the conditioned stimulus and were therefore not related to extinction. In parallel to these behavioral effects, the spontaneous neuronal activity and CR-related neuronal responses in the IN were suppressed, suggesting cerebellar dysfunction. These findings indicate that testing the role of IO in learning requires methods that do not alter the functional state of the cerebellum.

A long-range, wide field-of-view infrared eyeblink detector.

Classical conditioning of the eyeblink response in the rabbit is one of the most advanced models of learning and memory in the mammalian brain. Successful use of the eyeblink conditioning paradigm requires precise measurements of the eyeblink response. One common technique of eyelid movement detection utilizes measurements of infrared (IR) light reflected from the surface of the eye. The performance of current IR sensors, however, is limited by their sensitivity to ambient infrared noise, by their small field-of-view and by short working distances. To address these limitations, we developed an IR eyeblink detector consisting of a pulsing (62.5 kHz) IR light emitting diode (LED) paired with a silicon IR photodiode and circuit that synchronously demodulates the recorded signal and rejects background IR noise. The working distance of the sensor exceeds 20 mm, and the field-of-view is larger than the area of a rabbit's eye. Due to its superior characteristics, the new sensor is ideally suited for both standard eyeblink conditioning and for studies that utilize IR-containing visual stimuli and/or that are conducted in an environment contaminated with IR noise.

Inactivation of the brachium conjunctivum prevents extinction of classically conditioned eyeblinks.

It is well established that the intermediate cerebellum is involved in the acquisition of classically conditioned eyeblink responses (CRs). Recent studies that inactivated the interposed nuclei (IN) demonstrated that blocking the intermediate cerebellum also interrupts CR extinction. Is this extinction deficit related to interrupting the information flow to efferent targets of the IN? To address this question, we inactivated axons of IN neurons in the brachium conjunctivum (BC). This treatment blocked the output of the intermediate cerebellum without directly affecting neurons in the deep cerebellar nuclei. Rabbits were trained in a delay classical conditioning paradigm, using a tone as the conditioned stimulus (CS) and a corneal air puff as the unconditioned stimulus (US). Then, the BC was microinjected with a sodium channel blocker, tetrodotoxin, during 4 extinction sessions in which rabbits were presented only with the CS. Tests performed after the 4-day injection period revealed that CRs did not extinguish in BC inactivation sessions but extinguished at a normal rate in the absence of the drug. CRs were then re-acquired. These data show that the normal flow of information along axons of cerebellar nuclear cells is required for CR extinction.

On-line compensation for perturbations of a reaching movement is cerebellar dependent: support for the task dependency hypothesis.

Although the cerebellum has been shown to be critical for the acquisition and retention of adaptive modifications in certain reflex behaviors, this structure's role in the learning of motor skills required to execute complex voluntary goal-directed movements still is unclear. This study explores this issue by analyzing the effects of inactivating the interposed and dentate cerebellar nuclei on the adaptation required to compensate for an external elastic load applied during a reaching movement. We show that cats with these nuclei inactivated can adapt to predictable perturbations of the forelimb during a goal-directed reach by including a compensatory component in the motor plan prior to movement initiation. In contrast, when comparable compensatory modifications must be triggered on-line because the perturbations are applied in randomized trials (i.e., unpredictably), such adaptive responses cannot be executed or reacquired after the interposed and dentate nuclei are inactivated. These findings provide the first demonstration of the condition-dependent nature of the cerebellum's contribution to the learning of a specific volitional task.

Role of the cerebellum in eyeblink conditioning.

The mammalian cerebellum is thought to participate in motor control and motor learning. The specific cerebellar contribution to these processes is not clear, however. Advances in understanding cerebellar function have been relatively slow, because, at least in most cases, the cerebellum appears to play only an ancillary role in the behaviors studied to date. A remarkable exception is classical conditioning of eyeblink responses in the rabbit. In this model, an intact cerebellum is critical for both the acquisition and expression of conditioned responses. Recent experiments suggest that the cerebellar role in classical conditioning might be similar in all mammals, including the human. Moreover, anticipatory defensive reflexes in other effector systems show a similar dependence on the intermediate cerebellum. Further developments in our understanding of cerebellar function will depend on examination of a wider array of cerebellar-involved neural networks. There is also need for the development of new experimental approaches to associative learning in both the nonhuman primate and the human.

Video recording system for the measurement of eyelid movements during classical conditioning of the eyeblink response in the rabbit.

Classical conditioning of the eyeblink response in the rabbit is a popular model for studying the neural substrates of associative learning. Most of the common eyeblink recording techniques require invasive procedures. To perform experiments in intact animals, a non-invasive, high-speed video recording system was developed to measure eyeblink responses of rabbits during classical conditioning experiments. Besides being non-invasive, this method does not require excessive restraint of the animal. The PC-based system combines a Pulnix video camera with National Instruments video capture and timing hardware to control the experiment and acquire images of the peri-ocular region. The software developed for controlling these experiments also detects the eyeblink by measuring the movement of the upper and lower eyelids, the area of the exposed surface of the eye, and head movements in the sagittal plane. The time resolution of this relatively inexpensive system is 8.33 ms, and at a working distance of 0.8 m, it can detect movements as small as 0.11 mm.