William L. Nores

Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses

A fundamental tenet of cerebellar learning theories asserts that climbing fibre afferents from the inferior olive provide a teaching signal that promotes the gradual adaptation of movements. Data from several forms of motor learning provide support for this tenet. In pavlovian eyelid conditioning, for example, where a tone is repeatedly paired with a reinforcing unconditioned stimulus like periorbital stimulation, the unconditioned stimulus promotes acquisition of conditioned eyelid responses by activating climbing fibres. Climbing fibre activity elicited by an unconditioned stimulus is inhibited during the expression of conditioned responses—consistent with the inhibitory projection from the cerebellum to inferior olive. Here, we show that inhibition of climbing fibres serves as a teaching signal for extinction, where learning not to respond is signalled by presenting a tone without the unconditioned stimulus. We used reversible infusion of synaptic receptor antagonists to show that blocking inhibitory input to the climbing fibres prevents extinction of the conditioned response, whereas blocking excitatory input induces extinction. These results, combined with analysis of climbing fibre activity in a computer simulation of the cerebellar–olivary system, suggest that transient inhibition of climbing fibres below their background level is the signal that drives extinction.

What the cerebellum computes

The brain is an organ that processes information. Brain systems such as the cerebellum receive inputs from other systems and generate outputs according to their internal rules of information processing. Thus, our understanding of the cerebellum is ultimately best expressed in terms of the information processing it accomplishes and how cerebellar neurons and synapses produce this processing. We review evidence that indicates how Pavlovian eyelid conditioning reveals cerebellar processing to be an example of feedforward control. Eyelid conditioning demonstrates a capacity for learning in the cerebellum that is error driven, associative and temporally specific--as is required for feedforward control. This computation-centered view is consistent with a variety of proposed functions of the cerebellum, including sensory-motor integration, motor coordination, motor learning and timing. Moreover, feedforward processing could be the common link between motor and non-motor functions of the cerebellum.

Cerebellar function: Coordination, learning or timing?

Theories of cerebellar function have largely involved three ideas: movement coordination, motor learning or timing. New evidence indicates these distinctions are not particularly meaningful, as the cerebellum influences movement execution by feedforward use of sensory information via temporally specific learning.

Mechanisms of cerebellar learning suggested by eyelid conditioning.

Classical eyelid conditioning has been used to great advantage in demonstrating that the cerebellum helps to improve movements through experience, and in identifying the underlying mechanisms. Results from recent studies support the hypotheses that learning occurs in both the cerebellar nucleus and cortex, and that these sites make different contributions. Specifically, results indicate that the cerebellar cortex is responsible for temporally specific learning. A combination of experimental and computational studies has been important for arriving at these conclusions, which seem to be applicable to the broad range of movements to which the cerebellum contributes.

Learning-Induced Plasticity in Deep Cerebellar Nucleus

Evidence that cerebellar learning involves more than one site of plasticity comes from, in part, pavlovian eyelid conditioning, where disconnecting the cerebellar cortex abolishes one component of learning, response timing, but spares the expression of abnormally timed short-latency responses (SLRs). Here, we provide evidence that SLRs unmasked by cerebellar cortex lesions are mediated by an associative form of learning-induced plasticity in the anterior interpositus nucleus (AIN) of the cerebellum. We used pharmacological inactivation and/or electrical microstimulation of various sites afferent and efferent to the AIN to systematically eliminate alternative candidate sites of plasticity upstream or downstream from this structure. Collectively, the results suggest that cerebellar learning is mediated in part by plasticity in target nuclei downstream of the cerebellar cortex. These data demonstrate an instance in which an aspect of associative learning, SLRs, can be used as an index of plasticity at a specific site in the brain.

Dissociation of analgesic and rewarding effects of endomorphin-1 in rats

The mu-receptor is the primary mediator of the effects of morphine and the endogenous opiates, endomorphin-1 and endomorphin-2. Here we demonstrate a dissociation of the analgesic and rewarding effects of endomorphin-1 in rats. Tail-flick results revealed that endomorphin-1 produced significant analgesic effects within 10-min after injection. However, it failed to show reward properties in the standard 45- min conditioned place preference (CPP) paradigm or in an abbreviated 10-min pairing which paralleled the time frame of the tail-flick findings. Morphine induced both analgesia and reward. Endomorphin-1 therefore is the first mu opiate shown to produce potent analgesia in the absence of reward behavior, and thus may have significant clinical potential.

Cerebellar cortex contributions to the expression and timing of conditioned eyelid responses.

We used micro-infusions during eyelid conditioning in rabbits to investigate the relative contributions of cerebellar cortex and the underlying deep nuclei (DCN) to the expression of cerebellar learning. These tests were conducted using two forms of cerebellum-dependent eyelid conditioning for which the relative roles of cerebellar cortex and DCN are controversial: delay conditioning, which is largely unaffected by forebrain lesions, and trace conditioning, which involves interactions between forebrain and cerebellum. For rabbits trained with delay conditioning, silencing cerebellar cortex by micro-infusions of the local anesthetic lidocaine unmasked stereotyped short-latency responses. This was also the case after extinction as observed previously with reversible blockade of cerebellar cortex output. Conversely, increasing cerebellar cortex activity by micro-infusions of the GABA(A) antagonist picrotoxin reversibly abolished conditioned responses. Effective cannula placements were clustered around the primary fissure and deeper in lobules hemispheric lobule IV (HIV) and hemispheric lobule V (HV) of anterior lobe. In well-trained trace conditioned rabbits, silencing this same area of cerebellar cortex or reversibly blocking cerebellar cortex output also unmasked short-latency responses. Because Purkinje cells are the sole output of cerebellar cortex, these results provide evidence that the expression of well-timed conditioned responses requires a well-timed decrease in the activity of Purkinje cells in anterior lobe. The parallels between results from delay and trace conditioning suggest similar contributions of plasticity in cerebellar cortex and DCN in both instances.

Trying to understand the cerebellum well enough to build one.

Abstract: The development of an increasingly detailed computer simulation of the cerebellum is briefly described. Specific and relatively direct evaluation of the performance of this simulation is made possible by the straightforward way in which pavlovian eyelid conditioning engages the cerebellum. Inputs to the simulation are based on recordings of mossy fiber and climbing fiber responses to the stimuli used in eyelid conditioning, and the output of the simulation can be evaluated with respect to the extensively characterized behavioral properties of eyelid conditioning. Because construction of the simulation has been guided by a strong aversion to errors of commission, both failures and successes of the simulation have proven informative. The behavior of the simulation related to the inhibitory nucleo‐olivary feedback connection and spontaneous activity of climbing fibers is described. A prediction of the simulation concerning extinction is confirmed by experiment.

Using genetic mutations to study the neural basis of behavior

These examples clearly portend a bright future for the use of genetic mutations to address the function of neural systems. Commenting on his tendency to dream about future scientific technologies, the late Carl Sagan suggested, “Dreams are maps.” Here, in the spirit of Sagans comment, we will ignore limitations or apparent feasibility and will dream about types of mutations that could help revolutionize systems-level neuroscience. We do not imagine that these particular mice comprise a list that is either comprehensive or optimal, nor do we imagine that they are possible in the near future. They are simply our maps.One mouse is inspired by the immunotoxin-mediated cell targeting technique of Watanabe et al. 1998xWatanabe, D., Inokawa, H., Hashimoto, K., Suzuki, N., Kano, M., Shigemoto, R., Hirano, T., Toyama, K., Kaneko, S., Yokoi, M. et al. Cell. 1998; 95: 17–27Abstract | Full Text | Full Text PDF | PubMed | Scopus (135)See all ReferencesWatanabe et al. 1998. The goal is to achieve functional inactivation of a particular cell type with extremely rapid onset and offset. An approach of this type has been employed with great success in certain invertebrate systems where particular cells can be functionally removed from the circuit via direct hyperpolarization through a microelectrode. The cell is still alive, but it cannot communicate with other neurons because the hyperpolarization prevents action potentials. Although this technique affords superb temporal specificity, its application is limited to the number of cells that can be simultaneously impaled with microelectrodes.Perhaps entire classes of cells could be functionally removed from a circuit with cell type–specific expression of a recombinant protein comprised of a known potassium or chloride channel and a receptor for an artificial ligand. If the appropriate artificial receptor–ligand combination could be developed, cells expressing the transgene could be functionally removed from the circuit via hyperpolarization that is activated by injection of the ligand. Combining such a transgene with a cell type–specific promoter could produce a rapid onset and reversible variant of the immunotoxin-mediated technique. Cells could be rapidly removed and, with an artificial competitive antagonist, rapidly reinstated in the circuit. By potentially eliminating compensatory changes such as those characterized in the cerebellum by Watanabe et al. 1998xWatanabe, D., Inokawa, H., Hashimoto, K., Suzuki, N., Kano, M., Shigemoto, R., Hirano, T., Toyama, K., Kaneko, S., Yokoi, M. et al. Cell. 1998; 95: 17–27Abstract | Full Text | Full Text PDF | PubMed | Scopus (135)See all ReferencesWatanabe et al. 1998, such a “hyperpolarization” mouse would provide a tremendous degree of component and temporal specificity.Another type of futuristic mouse might move away from using targeted mutations as lesions (see Siegel and Isacoff 1998xSiegel, M.S. and Isacoff, E.Y. Neuron. 1998; 19: 735–741Abstract | Full Text | Full Text PDF | Scopus (239)See all ReferencesSiegel and Isacoff 1998). Perhaps a recombinant protein could be expressed to record activity of cells. Such a mouse could be used, for example, to determine the cells that are active during a particular form of learning. The recombinant protein might be constructed with three properties: (1) the ability to bind some activity-dependent molecule such as elevated free calcium, (2) the ability to bind an initiator molecule, and (3) when these two binding sites are occupied to undergo a detectable conformational change or to generate a measurable product.The idea would be that the initiator molecule could be injected or otherwise delivered in a temporally discrete manner, and that this would initiate the “recording” process. From that time on, binding at the second, activity-dependent site would generate a measurable product in the cell. The keys would be the temporal specificity of the initiator molecule, as well as the specificity and stringency of the binding site detecting neural activity. If the expression of this molecule could be limited to synapses, the resolution of activity-dependent accumulation of the marker molecule could be at the level of synapses rather than cells. In this way mutant mice could be used not only to identify the neural components responsible for a particular behavior, but also to study how those components interact to produce the input/output properties of the system.Finding synapses that undergo plasticity during learning is a fundamental but technically demanding goal for neurobiologists studying the neural basis of learning. As more is learned about the molecules required for particular forms of synaptic plasticity, it might even be possible to limit the accumulation of marker to synapses that have undergone that form of plasticity since the injection of the initiator molecule. Such a mouse could be used to establish links between forms of plasticity and forms of learning at a level of certainty not currently approachable. A mouse could be injected with the initiator molecule and then trained in a specific task. Subsequent histological analysis could reveal synapses with marker accumulation, and thus synapses that had undergone plasticity during that form of learning could be identified.As systems-level neuroscientists, it is hard to guess whether mice like these are many decades away or just around the corner. Either way, there is no doubt that continuing advances in gene targeting technology, when combined with robust behavioral analysis, will help revolutionize our ability to study and to understand brain systems.

Relative Contributions of the Cerebellar Cortex and Cerebellar Nucleus to Eyelid Conditioning

We have presented data addressing the relative contributions of the cerebellar cortex and nuclei in the acquisition and expression of conditioned eyelid responses. Data from a series of studies support the notion that while plasticity occurs in both the cerebellar cortex and nucleus, the cerebellar cortex is essential for acquisition, extinction, and for the proper expression of conditioned eyelid responses. We have presented arguments that the experiments that support this position were designed in ways to preclude confounds that were present in other attempts to identify the role of the cerebellar cortex in eyelid conditioning. We have also presented evidence that suggests plasticity in both the cerebellar cortex and nucleus is required for the expression of a conditioned response, and that reversing this plasticity only in the cerebellar cortex may be sufficient to produce extinction of conditioned responses. Computer simulations based on the connectivity of the cerebellum and on the sites and rules