A Patterned Architecture of Monoaminergic Afferents in the Cerebellar Cortex: Noradrenergic and Serotonergic Fibre Distributions within Lobules and Parasagittal Zones.

The geometry of the glutamatergic mossy-parallel fibre and climbing fibre inputs to cerebellar cortical Purkinje cells has powerfully influenced thinking about cerebellar functions. The compartmentation of the cerebellum into parasagittal zones, identifiable in olivo-cortico-nuclear projections, and the trajectories of the parallel fibres, transverse to these zones and following the long axes of the cortical folia, are particularly important. Two monoaminergic afferent systems, the serotonergic and noradrenergic, are major inputs to the cerebellar cortex but their architecture and relationship with the cortical geometry are poorly understood. Immunohistochemistry for the serotonin transporter (SERT) and for the noradrenaline transporter (NET) revealed strong anisotropy of these afferent fibres in the molecular layer of rat cerebellar cortex. Individual serotonergic fibres travel predominantly medial-lateral, along the long axes of the cortical folia, similar to parallel fibres and Zebrin II immunohistochemistry revealed that they can influence multiple zones. In contrast, individual noradrenergic fibres run predominantly parasagittally with rostral-caudal extents significantly longer than their medial-lateral deviations. Their local area of influence has similarities in form and size to those of identified microzones. Within the molecular layer, the orthogonal trajectories of these two afferent systems suggest different information processing. An individual serotonergic fibre must influence all zones and microzones within its medial-lateral trajectory. In contrast, noradrenergic fibres can influence smaller cortical territories, potentially as limited as a microzone. Evidence is emerging that these monoaminergic systems may not supply a global signal to all of their targets and their potential for cerebellar cortical functions is discussed.

Activation of a Temporal Memory in Purkinje Cells by the mGluR7 Receptor.

Cerebellar Purkinje cells can learn to respond to a conditioned stimulus with an adaptively timed pause in firing. This response was usually ascribed to long-term depression of parallel fiber to Purkinje cell synapses but has recently been shown to be due to a previously unknown form of learning involving an intrinsic cellular timing mechanism. Here, we investigate how these responses are elicited. They are resistant to blockade of GABAergic inhibition, suggesting that they are caused by glutamate release rather than by a changed balance between GABA and glutamate. We show that the responses are abolished by antagonists of the mGlu7 receptor but not significantly affected by other glutamate antagonists. These results support the existence of a distinct learning mechanism, different from changes in synaptic strength. They also demonstrate in vivo post-synaptic inhibition mediated by glutamate and show that the mGlu7 receptor is involved in activating intrinsic temporal memory.

Distribution of neural plasticity in cerebellum-dependent motor learning.

The cerebellum is essential for some forms of motor learning. Two examples that provide useful experimental models are modification of the vestibulo-ocular reflex and classical conditioning of the nictitating membrane response (NMR) in the rabbit. There has been considerable analysis of these behavioral models and of conditioning of the eyelid blink reflex, which is similar in several respects to NMR conditioning but with some key differences in its control circuitry. The evidence is consistent with the suggestion that storage of these motor memories is to be found within the cerebellum and its associated brainstem circuitry. The cerebellum presents many advantages as a model system to characterize the cellular and molecular mechanisms underpinning behavioral learning. And yet, localizing the essential synaptic changes has proven to be difficult. A major problem has been to establish to what extent these neural changes are distributed through the cerebellar cortex, cerebellar nuclei, and associated brainstem nuclei. Inspired by recent theoretical work, here we review evidence that the distribution of plasticity across cortical and cerebellar nuclear (or brainstem vestibular system) levels for different learning tasks may be different and distinct. Our primary focus is on classical conditioning of the NMR and eyelid blink, and we offer comparisons with mechanisms for modifications of the vestibulo-ocular reflex. We describe a view of cerebellar learning that satisfies theoretical and empirical analysis.

Cerebellar theta burst stimulation impairs eyeblink classical conditioning.

Theta burst stimulation (TBS) protocols of repetitive transcranial magnetic stimulation (rTMS) have after-effects on excitability of motor areas thought to be due to LTP- and LTD-like processes at cortical synapses. The present experiments ask whether, despite the low intensities of stimulation used and the anatomy of the posterior fossa, TBS can also influence the cerebellum. Acquisition and retention of eyeblink classical conditioning (EBCC) was examined in 30 healthy volunteers after continuous theta burst stimulation (cTBS) over the right cerebellar hemisphere. In subjects who received cerebellar cTBS, conditioned responses were fewer and their onsets were earlier (in the last half of the acquisition blocks) than those from control subjects. There was, however, no effect of cerebellar cTBS on the re-acquisition of EBCC in another session of EBCC 7­10 days later. There was also no effect of cerebellar cTBS on the re-acquisition of EBCC in subjects not naïve to EBCC when the stimulation was delivered immediately before a re-acquisition session. Control experiments verified that suppressive effects of cTBS on EBCC were not due to changes in motor cortical excitability or sensory disturbance caused by cTBS. Based on previous EBCC studies in various cerebellar pathologies, our data are compatible with the hypothesis that cerebellar cTBS has a focal cerebellar cortical effect, and are broadly in line with data from studies of EBCC in various animal models. These results confirm that cerebellar TBS has measurable effects on the function of the cerebellum, and indicate it is a useful non-invasive technique with which to explore cerebellar physiology and function in humans.

Sensory prediction or motor control? Application of marr-albus type models of cerebellar function to classical conditioning.

Marr-Albus adaptive filter models of the cerebellum have been applied successfully to a range of sensory and motor control problems. Here we analyze their properties when applied to classical conditioning of the nictitating membrane response in rabbits. We consider a system-level model of eyeblink conditioning based on the anatomy of the eyeblink circuitry, comprising an adaptive filter model of the cerebellum, a comparator model of the inferior olive and a linear dynamic model of the nictitating membrane plant. To our knowledge, this is the first model that explicitly includes all these principal components, in particular the motor plant that is vital for shaping and timing the behavioral response. Model assumptions and parameters were systematically investigated to disambiguate basic computational capacities of the model from features requiring tuning of properties and parameter values. Without such tuning, the model robustly reproduced a range of behaviors related to sensory prediction, by displaying appropriate trial-level associative learning effects for both single and multiple stimuli, including blocking and conditioned inhibition. In contrast, successful reproduction of the real-time motor behavior depended on appropriate specification of the plant, cerebellum and comparator models. Although some of these properties appear consistent with the system biology, fundamental questions remain about how the biological parameters are chosen if the cerebellar microcircuit applies a common computation to many distinct behavioral tasks. It is possible that the response profiles in classical conditioning of the eyeblink depend upon operant contingencies that have previously prevailed, for example in naturally occurring avoidance movements.

Memory consolidation in the cerebellar cortex.

Several forms of learning, including classical conditioning of the eyeblink, depend upon the cerebellum. In examining mechanisms of eyeblink conditioning in rabbits, reversible inactivations of the control circuitry have begun to dissociate aspects of cerebellar cortical and nuclear function in memory consolidation. It was previously shown that post-training cerebellar cortical, but not nuclear, inactivations with the GABAA agonist muscimol prevented consolidation but these findings left open the question as to how final memory storage was partitioned across cortical and nuclear levels. Memory consolidation might be essentially cortical and directly disturbed by actions of the muscimol, or it might be nuclear, and sensitive to the raised excitability of the nuclear neurons following the loss of cortical inhibition. To resolve this question, we simultaneously inactivated cerebellar cortical lobule HVI and the anterior interpositus nucleus of rabbits during the post-training period, so protecting the nuclei from disinhibitory effects of cortical inactivation. Consolidation was impaired by these simultaneous inactivations. Because direct application of muscimol to the nuclei alone has no impact upon consolidation, we can conclude that post-training, consolidation processes and memory storage for eyeblink conditioning have critical cerebellar cortical components. The findings are consistent with a recent model that suggests the distribution of learning-related plasticity across cortical and nuclear levels is task-dependent. There can be transfer to nuclear or brainstem levels for control of high-frequency responses but learning with lower frequency response components, such as in eyeblink conditioning, remains mainly dependent upon cortical memory storage.

Electrophysiological localization of eyeblink-related microzones in rabbit cerebellar cortex.

The classically conditioned eyeblink response in the rabbit is one of the best-characterized behavioral models of associative learning. It is cerebellum dependent, with many studies indicating that the hemispheral part of Larsell's cerebellar cortical lobule VI (HVI) is critical for the acquisition and performance of learned responses. However, there remain uncertainties about the distribution of the critical regions within and around HVI. In this learning, the unconditional stimulus is thought to be carried by periocular-activated climbing fibers. Here, we have used a microelectrode array to perform systematic, high-resolution, electrophysiological mapping of lobule HVI and surrounding folia in rabbits, to identify regions with periocular-evoked climbing fiber activity. Climbing fiber local field potentials and single-unit action potentials were recorded, and electrode locations were reconstructed from histological examination of brain sections. Much of the sampled cerebellar cortex, including large parts of lobule HVI, was unresponsive to periocular input. However, short-latency ipsilateral periocular-evoked climbing fiber responses were reliably found within a region in the ventral part of the medial wall of lobule HVI, extending to the base of the primary fissure. Small infusions of the AMPA/kainate receptor antagonist CNQX into this electrophysiologically defined region in awake rabbits diminished or abolished conditioned responses. The known parasagittal zonation of the cerebellum, supported by zebrin immunohistochemistry, indicates that these areas have connections consistent with an essential role in eyeblink conditioning. These small eyeblink-related areas provide cerebellar cortical targets for analysis of eyeblink conditioning at a neuronal level but need to be localized with electrophysiological identification in individual animals.

Response linearity determined by recruitment strategy in detailed model of nictitating membrane control.

Many models of eyeblink conditioning assume that there is a simple linear relationship between the firing patterns of neurons in the interpositus nucleus and the time course of the conditioned response (CR). However, the complexities of muscle behaviour and plant dynamics call this assumption into question. We investigated the issue by implementing the most detailed model available of the rabbit nictitating membrane response (Bartha and Thompson in Biol Cybern 68:135-143, 1992a and in Biol Cybern 68:145-154, 1992b), in which each motor unit of the retractor bulbi muscle is represented by a Hill-type model, driven by a non-linear activation mechanism designed to reproduce the isometric force measurements of Lennerstrand (J Physiol 236:43-55, 1974). Globe retraction and NM extension are modelled as linked second order systems. We derived versions of the model that used a consistent set of SI units, were based on a physically realisable version of calcium kinetics, and used simulated muscle cross-bridges to produce force. All versions showed similar non-linear responses to two basic control strategies. (1) Rate-coding with no recruitment gave a sigmoidal relation between control signal and amplitude of CR, reflecting the measured relation between isometric muscle force and stimulation frequency. (2) Recruitment of similar strength motor units with no rate coding gave a sublinear relation between control signal and amplitude of CR, reflecting the increase in muscle stiffness produced by recruitment. However, the system response could be linearised by either a suitable combination of rate-coding and recruitment, or by simple recruitment of motor units in order of (exponentially) increasing strength. These plausible control strategies, either alone or in combination, would in effect present the cerebellum with the simplified virtual plant that is assumed in many models of eyeblink conditioning. Future work is therefore needed to determine the extent to which motor neuron firing is in fact linearly related to the nictitating membrane response.

The mGlu1 antagonist CPCCOEt enhances the climbing fibre response in Purkinje neurones independently of glutamate receptors.

CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) is frequently used to test for the involvement of mGlu1 receptors. Using whole-cell voltage recording from Purkinje cells in slices of rat cerebellum we find that CPCCOEt, at concentrations used to block mGlu1 receptors, causes an enhancement of the climbing fibre response. Application of alternative antagonists with activity at mGlu1 neither mimicked nor occluded the effects of CPCCOEt. Receptor antagonists demonstrated that this non-mGlu1 action of CPCCOEt was not mediated by other mGlu receptors or GABA(B) receptors. Voltage-clamped climbing fibre EPSCs are unaffected by CPCCOEt whilst application of a glutamate transport blocker did not occlude the CPCCOEt effect. This suggests that a postsynaptic voltage-dependent component of the complex climbing fibre response is the target. We have found no evidence for the involvement of the hyperpolarisation-activated current, I(h), and calcium-activated conductances. Voltage-gated sodium, calcium and potassium channels are possible targets with inhibition of a potassium channel the most likely. Awareness of this non-mGlu-mediated effect of CPCCOEt is likely to be important for the correct interpretation of its actions.

Time and tide in cerebellar memory formation.

The notion that the olivocerebellar system is crucial for motor learning is well established. In recent years, it has become evident that there can be many forms of both synaptic and non-synaptic plasticity within this system and that each might have a different role in developing and maintaining motor learning across a wide range of tasks. There are several possible molecular and cellular mechanisms that could underlie adaptation of the vestibulo-ocular reflex and eyeblink conditioning. Although causal relationships between particular cellular processes and individual components of a learned behaviour have not been demonstrated unequivocally, an overall picture is emerging that the different types and sites of cellular plasticity relate importantly to the stage of learning and/or its temporal specifics.

Temporal properties of cerebellar-dependent memory consolidation.

Classical conditioning of the nictitating membrane response in rabbits is a well defined model of cerebellar-dependent motor memory. This memory undergoes a period of consolidation after the training session, when it is sensitive to reversible inactivations of the cerebellar cortex, but not of the cerebellar nuclei, with the GABA(A) receptor agonist muscimol. Here, the temporal properties of this cerebellar cortex-dependent consolidation were examined using delayed infusions of muscimol in cortical lobule HVI. Cortical infusions delayed by 5 or 45 min after a conditioning session produced significant and very similar impairments of consolidation, but infusions delayed by 90 min produced little or no impairment. Behavioral measures indicate that the muscimol infusions produced significant effects after approximately 30 min and they lasted for a few hours. So, over a time window beginning approximately 1 hr after the end of the training session and closing 1 hr after that, intracortical activity is critical for consolidation of this motor memory.

Contextual fear conditioning regulates the expression of brain-specific small nucleolar RNAs in hippocampus.

Some small nucleolar RNAs (snoRNAs) are exclusively expressed in the brain but they have no known role in higher brain function. We analysed the expression pattern of four brain-specific snoRNAs: MBI-36, MBII-48, MBII-52 and MBII-85, in mouse brain using in situ hybridization. All of these genes were expressed in the hippocampus and, except for MBII-85, their levels in ventral parts were higher than those in dorsal parts. Using quantitative real-time polymer chain reaction we determined hippocampal expression changes after contextual fear conditioning in mice. Ninety minutes, but not 25 h, after conditioning, we observed significant downregulation of MBII-48 and upregulation of MBII-52. Our finding that the expression of MBII-48 and MBII-52 is regulated during learning suggests that these snoRNAs have an important role in higher brain function.

Cerebellar function in consolidation of a motor memory.

Several forms of motor learning, including classical conditioning of the eyeblink and nictitating membrane response (NMR), are dependent upon the cerebellum, but it is not known how motor memories are stored within the cerebellar circuitry. Localized infusions of the GABA(A) agonist muscimol were used to target putative consolidation processes by producing reversible inactivations after NMR conditioning sessions. Posttraining inactivations of eyeblink control regions in cerebellar cortical lobule HVI completely prevented conditioning from developing over four sessions. In contrast, similar inactivations of eyeblink control regions in the cerebellar nuclei allowed conditioning to develop normally. These findings provide evidence that there are critical posttraining memory consolidation processes for eyeblink conditioning mediated by the cerebellar cortex.

Compartmentation of the rabbit cerebellar cortex.

The cytoarchitecture of the adult rabbit cerebellum is revealed by using zebrin II/aldolase c immunocytochemistry in both wholemount and sectioned material. Zebrin II is expressed by approximately half of the Purkinje cells of the cerebellar cortex. In most regions these form a symmetrical array of zebrin II positive and negative parasagittal bands. Four transverse expression domains are identified in the vermis: (1) an anterior zone, comprising four narrow bands, one at the midline and three laterally to either side, extending throughout the anterior lobe to the primary fissure; (2) a central zone with broad immunoreactive bands separated by narrow zebrin II negative bands that disappear caudally to leave no apparent compartmentation; (3) a posterior zone with prominent alternating zebrin II positive and negative bands; and (4) a nodular zone in which all Purkinje cells express zebrin II. In the hemispheres a striped topography is found in lobules HVI, HVII, and crus I, and all Purkinje cells are zebrin II+ in the flocculus and paraflocculus. Because of its importance for the classical conditioning of the eyeblink response, we made a detailed analysis of lobule HVI of the hemisphere. The immunocytochemical data show a complex substructure within HVI with three prominent zebrin II positive bands (probably homologous with P4a+, P4b+, and P5+ of rodents) separated by two zebrin II negative regions (P4- and P4b-). Thus, the organization of the rabbit cerebellum is consistent with the patterns described previously for rat, mouse, and opossum and suggests that there may be a common ground plan for the mammalian cerebellum.

Cerebellar mechanisms in eyeblink conditioning.

A recent model of cerebellar learning in eyeblink conditioning predicts two sites of plasticity, the cerebellar cortex and cerebellar nuclei, which store information relating to timing and driving the movement, respectively. Consistent with this idea, lesions of the cortex or reversible "disconnections" of Purkinje cell output to the nuclei have been shown to disrupt response timing to produce short-latency conditioned eyeblinks. To better characterize potential cortical and nuclear plasticities, we analyzed the effects upon nictitating membrane (NM) and eyeblink conditioned responses (CRs) of different drugs administered to the cortex and to the nuclei. When either excitatory or inhibitory inputs to the cerebellar cortical lobule HVI were blocked by infusions of the AMPA receptor antagonist CNQX or the GABA-A receptor antagonists picrotoxin or SR95531, CRs were abolished. Similarly GABA-A receptor antagonists in the cerebellar nuclei abolished CRs. CR latencies were never shortened. However, blockade of AMPA/kainate receptor-mediated excitatory transmission to the nuclei had no effect upon CR frequencies or latencies. These results suggest that normal cortical and nuclear function is required for performance of NM and eyeblink CRs. We saw no evidence that CRs can be driven by AMPA/kainate receptor-mediated transmission from mossy fiber afferents to the cerebellar nuclei. So, although plasticity in the cerebellar nuclei is not ruled out, it is unlikely that a long-term change in AMPA receptor-mediated transmission from mossy fiber inputs to the nuclei is an essential mechanism in eyeblink conditioning. Our findings indicate that a fully functional olivo-cortico-nuclear loop is required to express all characteristics of associatively conditioned responses.

Somatosensory Trigeminal Projections to the Inferior Olive, Cerebellum and other Precerebellar Nuclei in Rabbits.

We have analysed the pathways through which somatosensory information from the face reaches the inferior olive and the cerebellum in rabbits. We used wheatgerm agglutinin - horseradish peroxidase (WGA - HRP) to trace projections from all parts of the somatosensory trigeminal system to the olive, cerebellar cortex, the cerebellar deep nuclei and the pontine nuclei. Projections to the cerebellar cortex and inferior olive were verified using retrograde transport of WGA - HRP. Two regions of the inferior olive-the medial dorsal accessory olive and the ventral leaf of the principal olive-receive inputs from pars interpolaris (Vi) and rostral pars caudalis (Vc) of the spinal trigeminal nucleus and from the principal trigeminal nucleus (Vp). Another area in the caudal medial accessory olive receives inputs from rostral Vo (pars oralis of the spinal trigeminal nucleus), caudal Vi and Vc. There are trigemino-olivo-cortical inputs to lobule HVI via all these olivary areas and to the paramedian lobe via the principal olive only. Cerebellar cortex-lobules HVI, crus I and II, paramedian lobe and IX-receives direct mossy fibre inputs from Vp, Vo and rostral Vi. The pontine nuclei receive an input only from rostral Vi. We saw no trigeminal projections to other precerebellar nuclei or to the deep cerebellar nuclei. The concentration of face somatosensory cortical inputs, via several pathways, upon lobule HVI may underlie its important role in the regulation of learned and unlearned eyeblinks.