Eyeblink Conditioning In Mice Research Articles

Climbing fibers provide essential instructive signals for associative learning

Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber (CF) inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. In the present study we used cell-type-specific perturbations of various cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning in mice. Our findings reveal that, although optogenetic stimulation of either CFs or Purkinje cells can drive learning under some conditions, even subtle reductions in CF signaling completely block learning to natural stimuli. We conclude that CFs and corresponding Purkinje cell complex spike events provide essential instructive signals for associative cerebellar learning.

Hippocampal Interneurons are Required for Trace Eyeblink Conditioning in Mice

While the hippocampus has been implicated in supporting the association among time-separated events, the underlying cellular mechanisms have not been fully clarified. Here, we combined in vivo multi-channel recording and optogenetics to investigate the activity of hippocampal interneurons in freely-moving mice performing a trace eyeblink conditioning (tEBC) task. We found that the hippocampal interneurons exhibited conditioned stimulus (CS)-evoked sustained activity, which predicted the performance of conditioned eyeblink responses (CRs) in the early acquisition of the tEBC. Consistent with this, greater proportions of hippocampal pyramidal cells showed CS-evoked decreased activity in the early acquisition of the tEBC. Moreover, optogenetic suppression of the sustained activity in hippocampal interneurons severely impaired acquisition of the tEBC. In contrast, suppression of the sustained activity of hippocampal interneurons had no effect on the performance of well-learned CRs. Our findings highlight the role of hippocampal interneurons in the tEBC, and point to a potential cellular mechanism subserving associative learning.

Blockade of the M1 muscarinic acetylcholine receptors impairs eyeblink serial feature-positive discrimination learning in mice.

The serial feature-positive discrimination task requires the subjects to respond differentially to the identical stimulus depending on the temporal context given by a preceding cue stimulus. In the present study, we examined the involvement of the M1 muscarinic acetylcholine receptors using a selective M1 antagonist VU0255035 in the serial feature-positive discrimination task of eyeblink conditioning in mice. In this task, mice received a 2-s light stimulus as the conditional cue 5 or 6 s before the presentation of a 350-ms tone conditioned stimulus (CS) paired with a 100-ms peri-orbital electrical shock (cued trials), while they did not receive the cue before the presentation of the CS alone (non-cued trials). Each day mice randomly received 30 cued and 30 non-cued trials. We found that VU0255035 impaired acquisition of the conditional discrimination as well as the overall acquisition of the conditioned response (CR) and diminished the difference in onset latency of the CR between the cued and non-cued trials. VU0255035 administration to the control mice after sufficient learning did not impair the pre-acquired conditional discrimination or the CR expression itself. These effects of VU0255035 were almost similar to those with the scopolamine in our previous study, suggesting that among the several types of muscarinic acetylcholine receptors, the M1 receptors may play an important role in the acquisition of the conditional discrimination memory but not in mediating the discrimination itself after the memory had formed in the eyeblink serial feature-positive discrimination learning.

Intrinsic Excitability Increase in Cerebellar Purkinje Cells after Delay Eye-Blink Conditioning in Mice.

Cerebellar-based learning is thought to rely on synaptic plasticity, particularly at synaptic inputs to Purkinje cells. Recently, however, other complementary mechanisms have been identified. Intrinsic plasticity is one such mechanism, and depends in part on the downregulation of calcium-dependent SK-type K+ channels, which contribute to a medium-slow afterhyperpolarization (AHP) after spike bursts, regulating membrane excitability. In the hippocampus, intrinsic plasticity plays a role in trace eye-blink conditioning; however, corresponding excitability changes in the cerebellum in associative learning, such as in trace or delay eye-blink conditioning, are less well studied. Whole-cell patch-clamp recordings were obtained from Purkinje cells in cerebellar slices prepared from male mice ∼48 h after they learned a delay eye-blink conditioning task. Over a period of repeated training sessions, mice received either paired trials of a tone coterminating with a periorbital shock (conditioning) or trials in which these stimuli were randomly presented in an unpaired manner (pseudoconditioning). Purkinje cells from conditioned mice show a significantly reduced AHP after trains of parallel fiber stimuli and after climbing fiber evoked complex spikes. The number of spikelets in the complex spike waveform is increased after conditioning. Moreover, we find that SK-dependent intrinsic plasticity is occluded in conditioned, but not pseudoconditioned mice. These findings show that excitability is enhanced in Purkinje cells after delay eye-blink conditioning, and point toward a downregulation of SK channels as a potential underlying mechanism. The observation that this learning effect lasts at least up to 2 d after training shows that intrinsic plasticity regulates excitability in the long term.SIGNIFICANCE STATEMENT Plasticity of membrane excitability ("intrinsic plasticity") has been observed in invertebrate and vertebrate neurons, coinduced with synaptic plasticity or in isolation. Although the cellular phenomenon per se is well established, it remains unclear what role intrinsic plasticity plays in learning and if it even persists long enough to serve functions in engram physiology beyond aiding synaptic plasticity. Here, we demonstrate that cerebellar Purkinje cells upregulate excitability in delay eye-blink conditioning, a form of motor learning. This plasticity is observed 48 h after training and alters synaptically evoked spike firing and integrative properties of these neurons. These findings show that intrinsic plasticity enhances the spike firing output of Purkinje cells and persists over the course of days.

Bidirectional short-term plasticity during single-trial learning of cerebellar-driven eyelid movements in mice.

The brain is constantly monitoring its own performance, using error signals to trigger mechanisms of plasticity that help improve future behavior. Indeed, adaptive changes in behavior have been observed after a single error trial in many learning tasks, including cerebellum-dependent eyeblink conditioning. Here, we demonstrate that the plasticity underlying single-trial learning during eyeblink conditioning in mice is bidirectionally regulated by positive and negative prediction errors, has an ephemeral effect on behavior (decays in <1 min), and can be triggered in the absence of errors in performance. We suggest that these three properties of single-trial learning may be particularly useful for driving mechanisms of motor adaptation that can achieve optimal performance in the face of environmental disturbances with a fast timescale.

Dynamic modulation of activity in cerebellar nuclei neurons during pavlovian eyeblink conditioning in mice.

While research on the cerebellar cortex is crystallizing our understanding of its function in learning behavior, many questions surrounding its downstream targets remain. Here, we evaluate the dynamics of cerebellar interpositus nucleus (IpN) neurons over the course of Pavlovian eyeblink conditioning. A diverse range of learning-induced neuronal responses was observed, including increases and decreases in activity during the generation of conditioned blinks. Trial-by-trial correlational analysis and optogenetic manipulation demonstrate that facilitation in the IpN drives the eyelid movements. Adaptive facilitatory responses are often preceded by acquired transient inhibition of IpN activity that, based on latency and effect, appear to be driven by complex spikes in cerebellar cortical Purkinje cells. Likewise, during reflexive blinks to periocular stimulation, IpN cells show excitation-suppression patterns that suggest a contribution of climbing fibers and their collaterals. These findings highlight the integrative properties of subcortical neurons at the cerebellar output stage mediating conditioned behavior.

Caffeine has no effect on eyeblink conditioning in mice

Caffeine is one of the most widely used drugs in the world. In the brain, caffeine acts as an antagonist for the adenosine A1 and A2B receptors. Since A1 receptors are highly concentrated in the cortex of the cerebellum, we hypothesized that caffeine could potentially affect learning tasks that require the cerebellar cortex, such as eyeblink conditioning. To test this hypothesis, we examined the effect of low (5mg/kg) and high (50mg/kg) doses of caffeine, injected intraperitoneally before training, on eyeblink conditioning in mice. The results show that, at the dosages we used, caffeine affects neither the rate of acquisition, nor the timing of the onset or peak of the conditioned blink responses. Therefore, we conclude that caffeine neither improves nor worsens performance on eyeblink conditioning.

Cerebellar granule cells acquire a widespread predictive feedback signal during motor learning.

Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.

Role of Muscarinic Acetylcholine Receptors in Serial Feature-Positive Discrimination Task during Eyeblink Conditioning in Mice.

We investigated the role of muscarinic acetylcholine receptors (mAChRs) in eyeblink serial feature-positive discrimination learning in mice using the mAChR antagonist. A 2-s light cue was delivered 5 or 6 s before the presentation of a 350-ms tone paired with a 100-ms periorbital electrical shock (cued trial) but not before the tone-alone presentation (non-cued trial). Mice received 30 cued and 30 non-cued trials each day in a random order. We found that saline-injected control mice were successfully discriminating between cued and non-cued trials within a few days of conditioning. The mice responded more frequently to the tone in cued trials than in non-cued trials. Analysis of conditioned response (CR) dynamics revealed that the CR onset latency was shorter in cued trials than in non-cued trials, despite the CR peak amplitude not differing significantly between the two conditions. In contrast, scopolamine-injected mice developed an equal number of CRs with similar temporal patterns irrespective of the presence of the cue during the 7 days of conditioning, indicating in a failure to acquire conditional discrimination. In addition, the scopolamine administration to the control mice after they had successfully acquired discrimination did not impair the conditional discrimination and expression of pre-acquired CR. These results suggest that mAChRs may play a pivotal role in memory formation in the conditional brain state associated with the feature cue; however they are unlikely to be involved in the development of discrimination after conditional memory had formed in the serial feature-positive discrimination task during eyeblink conditioning.

Climbing fibers encode a temporal-difference prediction error during cerebellar learning in mice.

Climbing fiber inputs to Purkinje cells are thought to play a teaching role by generating the instructive signals that drive cerebellar learning. To investigate how these instructive signals are encoded, we recorded the activity of individual climbing fibers during cerebellar-dependent eyeblink conditioning in mice. Our findings show that climbing fibers signal both the unexpected delivery and the unexpected omission of the periocular airpuff that serves as the instructive signal for eyeblink conditioning. In addition, we report the surprising discovery that climbing fibers activated by periocular airpuffs also respond to stimuli from other sensory modalities, if those stimuli are novel or if they predict that the periocular airpuff is about to be presented. This pattern of climbing fiber activity is strikingly similar to the responses of dopamine neurons during reinforcement learning, which have been shown to encode a particular type of instructive signal known as a temporal difference prediction error.

Involvement of muscarinic acetylcholine receptors in conditional discrimination task in eyeblink conditioning in mice

Involvement of muscarinic acetylcholine receptors in conditional discrimination task in eyeblink conditioning in mice

Trace Eyeblink Conditioning in Mice Is Dependent upon the Dorsal Medial Prefrontal Cortex, Cerebellum, and Amygdala: Behavioral Characterization and Functional Circuitry

Trace eyeblink conditioning is useful for studying the interaction of multiple brain areas in learning and memory. The goal of the current work was to determine whether trace eyeblink conditioning could be established in a mouse model in the absence of elicited startle responses and the brain circuitry that supports this learning. We show here that mice can acquire trace conditioned responses (tCRs) devoid of startle while head-restrained and permitted to freely run on a wheel. Most mice (75%) could learn with a trace interval of 250 ms. Because tCRs were not contaminated with startle-associated components, we were able to document the development and timing of tCRs in mice, as well as their long-term retention (at 7 and 14 d) and flexible expression (extinction and reacquisition). To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented. Furthermore, inactivation of the cerebellum with muscimol completely abolished tCRs, demonstrating that learned responses were driven by the cerebellum. Finally, inactivation of the mPFC and amygdala in trained animals nearly abolished tCRs. Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons. The data provide the first report of trace eyeblink conditioning in mice in which tCRs were driven by the cerebellum and required a localized region of mPFC for acquisition. The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

Reevaluation of the role of parallel fiber synapses in delay eyeblink conditioning in mice using Cbln1 as a tool

The delay eyeblink conditioning (EBC) is a cerebellum-dependent type of associative motor learning. However, the exact roles played by the various cerebellar synapses, as well as the underlying molecular mechanisms, remain to be determined. It is also unclear whether long-term potentiation (LTP) or long-term depression (LTD) at parallel fiber (PF)–Purkinje cell (PC) synapses is involved in EBC. In this study, to clarify the role of PF synapses in the delay EBC, we used mice in which a gene encoding Cbln1 was disrupted (cbln1-/- mice), which display severe reduction of PF–PC synapses. We showed that delay EBC was impaired in cbln1-/- mice. Although PF-LTD was impaired, PF-LTP was normally induced in cbln1-/- mice. A single recombinant Cbln1 injection to the cerebellar cortex in vivo completely, though transiently, restored the morphology and function of PF–PC synapses and delay EBC in cbln1-/- mice. Interestingly, the cbln1-/- mice retained the memory for at least 30 days, after the Cbln1 injection’s effect on PF synapses had abated. Furthermore, delay EBC memory could be extinguished even after the Cbln1 injection’s effect were lost. These results indicate that intact PF–PC synapses and PF-LTD, not PF-LTP, are necessary to acquire delay EBC in mice. In contrast, extracerebellar structures or remaining PF–PC synapses in cbln1-/- mice may be sufficient for the expression, maintenance, and extinction of its memory trace.

Dimethoate accelerates the extinction of eyeblink conditioning in mice

In agriculture, organophosphates are frequently used as insecticides and pesticides. These compounds decrease acetylcholine esterase (AChE) activity, thereby provoking an accumulation of the neurotransmitter acetylcholine at synapses and resulting in the over-stimulation of acetylcholine receptors. Using trace paradigms, we investigated the effects of dimethoate, a widely used organophosphate insecticide, on the classical conditioning of eyelid responses, a hippocampal-dependent mouse model of associative learning. Mice were conditioned with a trace shock-SHOCK paradigm having first implanted stimulating electrodes in the supraorbitary nerve and recording electrodes in the ipsilateral orbicularis oculi muscle. When these mice were injected with dimethoate (5, 20, 50mg/kg/day) they were capable of acquiring associative learning, and the latency and amplitude of their unconditioned eyelid responses were unaffected by the administration of the pesticide. However, dimethoate administration led to the rapid extinction of conditioned responses, suggesting that this organophosphate accelerates the extinction of this form of associative learning. Analysis of the motor function of these mice using the rotarod performance test revealed that motor function and performance clearly deteriorated following dimethoate administration, with no improvements over the following 4 days. Together these findings indicate that dimethoate accelerates the extinction of acquired conditioned responses, affecting associative learning and memory, and it impairs motor function and performance in mice.

Amygdala, deep cerebellar nuclei and red nucleus contribute to delay eyeblink conditioning in C57BL /6 mice

That the cerebellum plays an essential role in delay eyeblink conditioning is well established in the rabbit, but not in the mouse. To elucidate the critical brain structures involved in delay eyeblink conditioning in mice, we examined the roles of the deep cerebellar nuclei (DCN), the amygdala and the red nucleus (RN) through the use of electrolytic lesions and reversible inactivation. All mice received eyeblink training of 50 trials during a daily session in the higher-intensity conditioned stimulus (CS) condition (10 kHz, 70 dB). DCN lesions caused severe ataxia; nonetheless, the mice acquired conditioned responses (CRs). Reversible inactivation of DCN, by muscimol (MSC) injection, led to a severe CR impairment in the early sessions of conditioning; however, in later sessions, the mice acquired CRs. Amygdala lesions impaired the acquisition of CRs, which did not reach the level of sham-operated mice, even after prolonged training sessions. MSC injections into the lateral amygdala severely impaired CRs, which began to recover after the removal of MSC. RN inactivation with MSC completely abolished CRs, and removal of MSC immediately restored CRs to the level of control mice. The results indicate that: (i) the DCN are important, but not essential, at least for the late acquisition in mouse eyeblink conditioning; (ii) the amygdala plays an important role in the acquisition and expression of CRs; and (iii) the RN is essential for the expression of CRs. Our findings reveal the various brain areas critically involved in mouse eyeblink conditioning, which include the cerebellum, amygdala and RN.

Cerebellar and extracerebellar involvement in mouse eyeblink conditioning: the ACDC model

Over the past decade the advent of mouse transgenics has generated new perspectives on the study of cerebellar molecular mechanisms that are essential for eyeblink conditioning. However, it also appears that results from eyeblink conditioning experiments done in mice differ in some aspects from results previously obtained in other mammals. In this review article we will, based on studies using (cell-specific) mouse mutants and region-specific lesions, re-examine the general eyeblink behavior in mice and the neuro-anatomical circuits that might contribute to the different peaks in the conditioned eyeblink trace. We conclude that the learning process in mice has at least two stages: An early stage, which includes short-latency responses that are at least partly controlled by extracerebellar structures such as the amygdala, and a later stage, which is represented by well-timed conditioned responses that are mainly controlled by the pontocerebellar and olivocerebellar systems. We refer to this overall concept as the Amygdala-Cerebellum-Dynamic-Conditioning Model (ACDC model).

Cerebellar mechanisms underlying encoding and consolodation of claccical eyeblink conditioning in mice

Trafficking of G protein-coupled receptors (GPCRs) to the plasma membrane is a pivotal process to fulfill their biological functions. Metabotropic glutamate receptors (mGluRs; mGluR1-8) are expressed throughout the CNS and are important for modulating synaptic transmission and plasticity. Group I mGluRs, including mGluR1 and mGluR5, have long intracellular C-terminal tails containing multiple protein binding domains and sites for phosphorylation and ER retention. We have now investigated some of the structural determinants for mGluR5 trafficking to the plasma membrane by studying a series of truncations and ligand binding mutants. We also take advantage of dimer formation between the extracellular domain (ECD) of mGluR5 and design an ECD based surface-binding assay to evaluate dimerization and surface expression of mGluR5 containing various truncations or point mutations. We found that the C terminus is not essential for mGluR5 surface expression. In contrast, the 7th transmembrane domain (TM7) plays a critical role in its surface expression in both heterologous cells and neurons. Furthermore, a ligand binding mutation within the ECD of mGluR5 (Y64A/T174A) that blocks ligand binding impairs both surface expression and dimerization of mGluR5 in neurons. The integrity of both the whole 7TM domain and the C- terminal tail of mGluR5 are also important for stabilizing dimerization with the ECD. Thus multiple domains regulate dimerization and trafficking of mGluR5.This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’.

Image analysis of the trace eyeblink conditioning in mice

Image analysis of the trace eyeblink conditioning in mice

Amygdala plays important roles in acquisition and retention of delay eyeblink conditioning in mice

Amygdala plays important roles in acquisition and retention of delay eyeblink conditioning in mice

Dependence on the cerebellum and the medial prefrontal cortex in mouse eyeblink conditioning

Dependence on the cerebellum and the medial prefrontal cortex in mouse eyeblink conditioning