Quantitative properties of the creation and activation of a cell-intrinsic duration-encoding engram.

The engram encoding the interval between the conditional stimulus (CS) and the unconditional stimulus (US) in eyeblink conditioning resides within a small population of cerebellar Purkinje cells. CSs activate this engram to produce a pause in the spontaneous firing rate of the cell, which times the CS-conditional blink. We developed a Bayesian algorithm that finds pause onsets and offsets in the records from individual CS-alone trials. We find that the pause consists of a single unusually long interspike interval. Its onset and offset latencies and their trial-to-trial variability are proportional to the CS-US interval. The coefficient of variation (CoV = σ/µ) are comparable to the CoVs for the conditional eye blink. The average trial-to-trial correlation between the onset latencies and the offset latencies is close to 0, implying that the onsets and offsets are mediated by two stochastically independent readings of the engram. The onset of the pause is step-like; there is no decline in firing rate between the onset of the CS and the onset of the pause. A single presynaptic spike volley suffices to trigger the reading of the engram; and the pause parameters are unaffected by subsequent volleys. The Fano factors for trial-to-trial variations in the distribution of interspike intervals within the intertrial intervals indicate pronounced non-stationarity in the endogenous spontaneous spiking rate, on which the CS-triggered firing pause supervenes. These properties of the spontaneous firing and of the engram read out may prove useful in finding the cell-intrinsic, molecular-level structure that encodes the CS-US interval.

Kir3 channel blockade in the cerebellar cortex suppresses performance of classically conditioned Purkinje cell responses.

In the eyeblink conditioning paradigm, cerebellar Purkinje cells learn to respond to the conditional stimulus with an adaptively timed pause in its spontaneous firing. Evidence suggests that the pause is elicited by glutamate released from parallel fibers and acting on metabotropic receptors (mGluR7) which initiates a delayed-onset suppression of firing. We suggested that G protein activation of hyperpolarizing Kir3 channels (or 'GIRK', G protein-coupled inwardly-rectifying K+ channels) could be part of such a mechanism. Application of the Kir3 antagonist Tertiapin-LQ locally in the superficial layers of the cerebellar cortex in decerebrate ferrets suppressed normal performance of Purkinje cell pause responses to the conditional stimulus. Importantly, there was no detectable effect on spontaneous firing. These findings suggest that intact functioning of Kir3 channels in the cerebellar cortex is required for normal conditioned Purkinje cell responses.

Internal Clocks, mGluR7 and Microtubules: A Primer for the Molecular Encoding of Target Durations in Cerebellar Purkinje Cells and Striatal Medium Spiny Neurons.

The majority of studies in the field of timing and time perception have generally focused on sub- and supra-second time scales, specific behavioral processes, and/or discrete neuronal circuits. In an attempt to find common elements of interval timing from a broader perspective, we review the literature and highlight the need for cell and molecular studies that can delineate the neural mechanisms underlying temporal processing. Moreover, given the recent attention to the function of microtubule proteins and their potential contributions to learning and memory consolidation/re-consolidation, we propose that these proteins play key roles in coding temporal information in cerebellar Purkinje cells (PCs) and striatal medium spiny neurons (MSNs). The presence of microtubules at relevant neuronal sites, as well as their adaptability, dynamic structure, and longevity, makes them a suitable candidate for neural plasticity at both intra- and inter-cellular levels. As a consequence, microtubules appear capable of maintaining a temporal code or engram and thereby regulate the firing patterns of PCs and MSNs known to be involved in interval timing. This proposed mechanism would control the storage of temporal information triggered by postsynaptic activation of mGluR7. This, in turn, leads to alterations in microtubule dynamics through a "read-write" memory process involving alterations in microtubule dynamics and their hexagonal lattice structures involved in the molecular basis of temporal memory.

Absence of Parallel Fibre to Purkinje Cell LTD During Eyeblink Conditioning.

Long-term depression (LTD) of parallel fibre/Purkinje cell synapses has been the favoured explanation for cerebellar motor learning such as classical eyeblink conditioning. Previous evidence against this interpretation has been contested. Here we wanted to test whether a classical conditioning protocol causes LTD. We applied a conditioning protocol, using a train of electrical pulses to the parallel fibres as the conditional stimulus. In order to rule out indirect effects caused by antidromic granule cell activation or output from Purkinje cells that might produce changes in Purkinje cell responsiveness, we focused the analysis on the first pulse in the conditional stimulus, that is, before any indirect effects would have time to occur. Purkinje cells learned to respond with a firing pause to the conditional stimulus. Yet, there was no depression of parallel fibre excitation after training.

Learned response sequences in cerebellar Purkinje cells.

Associative learning in the cerebellum has previously focused on single movements. In eyeblink conditioning, for instance, a subject learns to blink at the right time in response to a conditional stimulus (CS), such as a tone that is repeatedly followed by an unconditional corneal stimulus (US). During conditioning, the CS and US are transmitted by mossy/parallel fibers and climbing fibers to cerebellar Purkinje cells that acquire a precisely timed pause response that drives the overt blink response. The timing of this conditional Purkinje cell response is determined by the CS-US interval and is independent of temporal patterns in the input signal. In addition to single movements, the cerebellum is also believed to be important for learning complex motor programs that require multiple precisely timed muscle contractions, such as, for example, playing the piano. In the present work, we studied Purkinje cells in decerebrate ferrets that were conditioned using electrical stimulation of mossy fiber and climbing fiber afferents as CS and US, while alternating between short and long interstimulus intervals. We found that Purkinje cells can learn double pause responses, separated by an intermediate excitation, where each pause corresponds to one interstimulus interval. The results show that individual cells can not only learn to time a single response but that they also learn an accurately timed sequential response pattern.

Performance in eyeblink conditioning is age and sex dependent.

A growing body of evidence suggests that the cerebellum is involved in both cognition and language. Abnormal cerebellar development may contribute to neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD), autism, fetal alcohol syndrome, dyslexia, and specific language impairment. Performance in eyeblink conditioning, which depends on the cerebellum, can potentially be used to clarify the neural mechanisms underlying the cerebellar dysfunction in disorders like these. However, we must first understand how the performance develops in children who do not have a disorder. In this study we assessed the performance in eyeblink conditioning in 42 typically developing children between 6 and 11 years old as well as in 26 adults. Older children produced more conditioned eyeblink responses than younger children and adults produced more than children. In addition, females produced more conditioned eyeblink responses than males among both children and adults. These results highlight the importance of considering the influence of age and sex on the performance when studying eyeblink conditioning as a measure of cerebellar development.

Interactive roles of the cerebellum and striatum in sub-second and supra-second timing: Support for an initiation, continuation, adjustment, and termination (ICAT) model of temporal processing.

The contributions of cortico-cerebellar and cortico-striatal circuits to timing and time perception have often been a point of contention. In this review we propose that the cerebellum principally functions to reduce variability, through the detection of stimulus onsets and the sub-division of longer durations, thus contributing to both sub-second and supra-second timing. This sensitivity of the cerebellum to stimulus dynamics and subsequent integration with motor control allows it to accurately measure intervals within a range of 100-2000ms. For intervals in the supra-second range (e.g., >2000ms), we propose that cerebellar output signals from the dentate nucleus pass through thalamic connections to the striatum, where cortico-thalamic-striatal circuits supporting higher-level cognitive functions take over. Moreover, the importance of intrinsic circuit dynamics as well as behavioral, neuroimaging, and lesion studies of the cerebellum and striatum are discussed in terms of a framework positing initiation, continuation, adjustment, and termination phases of temporal processing.

Mechanisms for motor timing in the cerebellar cortex.

In classical eyeblink conditioning a subject learns to blink to a previously neutral stimulus. This conditional response is timed to occur just before an air puff to the eye. The learning is known to depend on the cerebellar cortex where Purkinje cells respond with adaptively timed pauses in their spontaneous firing. The pauses in the inhibitory Purkinje cells cause disinhibition of the cerebellar nuclei, which elicit the overt blinks. The timing of a Purkinje cell response was previously thought to require a temporal code in the input signal but recent work suggests that the Purkinje cells can learn to time their responses through an intrinsic mechanism that is activated by metabotropic glutamate receptors (mGluR7).

Are Purkinje Cell Pauses Drivers of Classically Conditioned Blink Responses?

Several lines of evidence show that classical or Pavlovian conditioning of blink responses depends on the cerebellum. Recordings from cerebellar Purkinje cells that control the eyelid and the conditioned blink show that during training with a conditioning protocol, a Purkinje cell develops a pause response to the conditional stimulus. This conditioned cellular response has many of the properties that characterise the overt blink. The present paper argues that the learned Purkinje cell pause response is the memory trace and main driver of the overt conditioned blink and that it explains many well-known behavioural phenomena.

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.

Purkinje cell activity during classical conditioning with different conditional stimuli explains central tenet of Rescorla­Wagner model [corrected].

A central tenet of Rescorla and Wagner's model of associative learning is that the reinforcement value of a paired trial diminishes as the associative strength between the presented stimuli increases. Despite its fundamental importance to behavioral sciences, the neural mechanisms underlying the model have not been fully explored. Here, we present findings that, taken together, can explain why a stronger association leads to a reduced reinforcement value, within the context of eyeblink conditioning. Specifically, we show that learned pause responses in Purkinje cells, which trigger adaptively timed conditioned eyeblinks, suppress the unconditional stimulus (US) signal in a graded manner. Furthermore, by examining how Purkinje cells respond to two distinct conditional stimuli and to a compound stimulus, we provide evidence that could potentially help explain the somewhat counterintuitive overexpectation phenomenon, which was derived from the Rescorla-Wagner model.

Memory trace and timing mechanism localized to cerebellar Purkinje cells.

The standard view of the mechanisms underlying learning is that they involve strengthening or weakening synaptic connections. Learned response timing is thought to combine such plasticity with temporally patterned inputs to the neuron. We show here that a cerebellar Purkinje cell in a ferret can learn to respond to a specific input with a temporal pattern of activity consisting of temporally specific increases and decreases in firing over hundreds of milliseconds without a temporally patterned input. Training Purkinje cells with direct stimulation of immediate afferents, the parallel fibers, and pharmacological blocking of interneurons shows that the timing mechanism is intrinsic to the cell itself. Purkinje cells can learn to respond not only with increased or decreased firing but also with an adaptively timed activity pattern.

Changes in complex spike activity during classical conditioning.

The cerebellar cortex is necessary for adaptively timed conditioned responses (CRs) in eyeblink conditioning. During conditioning, Purkinje cells acquire pause responses or "Purkinje cell CRs" to the conditioned stimuli (CS), resulting in disinhibition of the cerebellar nuclei (CN), allowing them to activate motor nuclei that control eyeblinks. This disinhibition also causes inhibition of the inferior olive (IO), via the nucleo-olivary pathway (N-O). Activation of the IO, which relays the unconditional stimulus (US) to the cortex, elicits characteristic complex spikes in Purkinje cells. Although Purkinje cell activity, as well as stimulation of the CN, is known to influence IO activity, much remains to be learned about the way that learned changes in simple spike firing affects the IO. In the present study, we analyzed changes in simple and complex spike firing, in extracellular Purkinje cell records, from the C3 zone, in decerebrate ferrets undergoing training in a conditioning paradigm. In agreement with the N-O feedback hypothesis, acquisition resulted in a gradual decrease in complex spike activity during the conditioned stimulus, with a delay that is consistent with the long N-O latency. Also supporting the feedback hypothesis, training with a short interstimulus interval (ISI), which does not lead to acquisition of a Purkinje cell CR, did not cause a suppression of complex spike activity. In contrast, observations that extinction did not lead to a recovery in complex spike activity and the irregular patterns of simple and complex spike activity after the conditioned stimulus are less conclusive.

Feedback control of learning by the cerebello-olivary pathway.

The ability to anticipate future events and to modify erroneous anticipatory actions is crucial for the survival of any organism. Both theoretical and empirical lines of evidence implicate the cerebellum in this ability. It is often suggested that the cerebellum acquires "expectations" or "internal models." However, except in a metaphorical sense, the cerebellum, which consists of a set of interconnected nerve cells, cannot contain "internal models" or "have expectations." In this chapter, we try to untangle these metaphors by translating them back into neurophysiological cause and effect relationships. We approach this task from within the paradigm of classical conditioning, in which a subject, through repeated presentations of a conditional stimulus, followed by an unconditional stimulus, acquires a conditioned response. Importantly, the conditioned response is timed so that it anticipates the unconditioned response. Available neurophysiological evidence suggests that Purkinje cells, in the cerebellar cortex, generate the conditioned response. In addition, Purkinje cells provide negative feedback to the inferior olive, which is a relay for the unconditional stimulus, via the nucleo-olivary pathway. Purkinje cells can therefore regulate the intensity of the signal derived from the unconditional stimulus, which, in turn, decides subsequent plasticity. Hence, as learning progresses, the olivary signal will become weaker and weaker due to increasing negative feedback from Purkinje cells. Thus, in an important sense, learning-induced changes in Purkinje cell activity constitute an "expectation" or "anticipation" of a future event (the unconditional stimulus), and, consistent with theoretical models, future learning depends on the accuracy of this expectation.

Bidirectional plasticity of Purkinje cells matches temporal features of learning.

Many forms of learning require temporally ordered stimuli. In Pavlovian eyeblink conditioning, a conditioned stimulus (CS) must precede the unconditioned stimulus (US) by at least about 100 ms for learning to occur. Conditioned responses are learned and generated by the cerebellum. Recordings from the cerebellar cortex during conditioning have revealed CS-triggered pauses in the firing of Purkinje cells that likely drive the conditioned blinks. The predominant view of the learning mechanism in conditioning is that long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses underlies the Purkinje cell pauses. This raises a serious conceptual challenge because LTD is most effectively induced at short CS-US intervals, which do not support acquisition of eyeblinks. To resolve this discrepancy, we recorded Purkinje cells during conditioning with short or long CS-US intervals. Decerebrated ferrets trained with CS-US intervals ≥150 ms reliably developed Purkinje cell pauses, but training with an interval of 50 ms unexpectedly induced increases in CS-evoked spiking. This bidirectional modulation of Purkinje cell activity offers a basis for the requirement of a minimum CS-US interval for conditioning, but we argue that it cannot be fully explained by LTD, even when previous in vitro studies of stimulus-timing-dependent LTD are taken into account.

Theoretical considerations for understanding a Purkinje cell timing mechanism.

In classical conditioning, cerebellar Purkinje cells learn an adaptively timed pause in spontaneous firing. This pause reaches its maximum near the end of the interstimulus interval. While it was thought that this timing was due to temporal patterns in the input signal and selective engagement of changes in synapse strength, we have shown Purkinje cells learn timed responses even when the conditional stimulus is delivered to its immediate afferents.(1) This shows that Purkinje cells have a cellular timing mechanism. The cellular models of intrinsic timing we are aware of are based on adapting the rise time of the concentration of a given ion. As an alternative, we here propose a selection mechanism in abstract terms for how a Purkinje cell could learn to respond at a particular time after an external trigger.

Number of spikes in climbing fibers determines the direction of cerebellar learning.

Cerebellar learning requires context information from mossy fibers and a teaching signal through the climbing fibers from the inferior olive. Although the inferior olive fires in bursts, virtually all studies have used a teaching signal consisting of a single pulse. Following a number of failed attempts to induce cerebellar learning in decerebrate ferrets with a nonburst signal, we tested the effect of varying the number of pulses in the climbing fiber teaching signal. The results show that training with a single pulse in a conditioning paradigm in vivo does not result in learning, but rather causes extinction of a previously learned response.

Classical conditioning of motor responses: what is the learning mechanism?

According to a widely held assumption, the main mechanism underlying motor learning in the cerebellum, such as eyeblink conditioning, is long-term depression (LTD) of parallel fibre to Purkinje cell synapses. Here we review some recent physiological evidence from Purkinje cell recordings during conditioning with implications for models of conditioning. We argue that these data pose four major challenges to the LTD hypothesis of conditioning. (i) LTD cannot account for the pause in Purkinje cell firing that is believed to drive the conditioned blink. (ii) The temporal conditions conducive to LTD do not match those for eyeblink conditioning. (iii) LTD cannot readily account for the adaptive timing of the conditioned response. (iv) The data suggest that parallel fibre to Purkinje cell synapses are not depressed after learning a Purkinje cell CR. Models based on metabotropic glutamate receptors are also discussed and found to be incompatible with the recording data.

The current status of the simulation theory of cognition.

It is proposed that thinking is simulated interaction with the environment. Three assumptions underlie this 'simulation' theory of cognitive function. Firstly, behaviour can be simulated in the sense that we can activate motor structures, as during a normal overt action, but suppress its execution. Secondly, perception can be simulated by internal activation of sensory cortex in a way that resembles its normal activation during perception of external stimuli. The third assumption ('anticipation') is that both overt and simulated actions can elicit perceptual simulation of their most probable consequences. A large body of evidence, mainly from neuroimaging studies, that supports these assumptions, is reviewed briefly. The theory is ontologically parsimonious and does not rely on standard cognitivist constructs such as internal models or representations. It is argued that the simulation approach can explain the relations between motor, sensory and cognitive functions and the appearance of an inner world. It also unifies and explains important features of a wide variety of cognitive phenomena such as memory and cognitive maps. Novel findings from recent developments in memory research on the similarity of imaging and memory and on the role of both prefrontal cortex and sensory cortex in declarative memory and working memory are predicted by the theory and provide striking support for it. This article is part of a Special Issue entitled "The Cognitive Neuroscience".

Time course of classically conditioned Purkinje cell response is determined by initial part of conditioned stimulus.

Classical conditioning of a motor response such as eyeblink is associated with the development of a pause in cerebellar Purkinje cell firing that is an important driver of the overt response. This conditioned Purkinje cell response is adaptively timed and has a specific temporal profile that probably explains the time course of the overt behavior. It is generally assumed that the temporal properties of the conditioned Purkinje cell response are determined by the temporal pattern of the parallel fiber impulses generated by the conditioned stimulus at the time of the conditioned response. We show here in the decerebrate ferret preparation that a very brief conditioned stimulus, consisting of only one or two impulses in the mossy fibers, can be sufficient to elicit a full conditioned Purkinje cell response with normal time course. The finding suggests that parallel fiber input to the Purkinje cell influences the firing rate several hundred milliseconds later. It poses a serious challenge to the standard view of the role of parallel fiber impulses in response timing.