Classical Eyeblink Conditioning Research Articles

Mesoscale simulations predict the role of synergistic cerebellar plasticity during classical eyeblink conditioning.

According to the motor learning theory by Albus and Ito, synaptic depression at the parallel fibre to Purkinje cells synapse (pf-PC) is the main substrate responsible for learning sensorimotor contingencies under climbing fibre control. However, recent experimental evidence challenges this relatively monopolistic view of cerebellar learning. Bidirectional plasticity appears crucial for learning, in which different microzones can undergo opposite changes of synaptic strength (e.g. downbound microzones-more likely depression, upbound microzones-more likely potentiation), and multiple forms of plasticity have been identified, distributed over different cerebellar circuit synapses. Here, we have simulated classical eyeblink conditioning (CEBC) using an advanced spiking cerebellar model embedding downbound and upbound modules that are subject to multiple plasticity rules. Simulations indicate that synaptic plasticity regulates the cascade of precise spiking patterns spreading throughout the cerebellar cortex and cerebellar nuclei. CEBC was supported by plasticity at the pf-PC synapses as well as at the synapses of the molecular layer interneurons (MLIs), but only the combined switch-off of both sites of plasticity compromised learning significantly. By differentially engaging climbing fibre information and related forms of synaptic plasticity, both microzones contributed to generate a well-timed conditioned response, but it was the downbound module that played the major role in this process. The outcomes of our simulations closely align with the behavioural and electrophysiological phenotypes of mutant mice suffering from cell-specific mutations that affect processing of their PC and/or MLI synapses. Our data highlight that a synergy of bidirectional plasticity rules distributed across the cerebellum can facilitate finetuning of adaptive associative behaviours at a high spatiotemporal resolution.

Cerebellar interpositus nucleus exhibits time-dependent errors and predictive responses

Learning is a functional state of the brain that should be understood as a continuous process, rather than being restricted to the very moment of its acquisition, storage, or retrieval. The cerebellum operates by comparing predicted states with actual states, learning from errors, and updating its internal representation to minimize errors. In this regard, we studied cerebellar interpositus nucleus (IPn) functional capabilities by recording its unitary activity in behaving rabbits during an associative learning task: the classical conditioning of eyelid responses. We recorded IPn neurons in rabbits during classical eyeblink conditioning using a delay paradigm. We found that IPn neurons reduce error signals across conditioning sessions, simultaneously increasing and transmitting spikes before the onset of the unconditioned stimulus. Thus, IPn neurons generate predictions that optimize in time and shape the conditioned eyeblink response. Our results are consistent with the idea that the cerebellum works under Bayesian rules updating the weights using the previous history.

A mini-review of the pathophysiology of task-specific tremor: insights from electrophysiological and neuroimaging findings

Task-specific tremor (TST) is a specific type of tremor that occurs when performing or attempting to perform a specific task, such as writing or playing a musical instrument. The clinical entity of TST remains heterogeneous. Some TSTs can only be induced by conducting a specific task, while others can be elicited when adopting a particular position simulating a task. The pathophysiology of TST is controversial. Whether TST is an isolated tremor syndrome, a spectrum of dystonic tremor syndrome (DTS), or essential tremor (ET) is not yet clear. Evidence from electrophysiological studies suggests that TST patients have normal reciprocal inhibition responses but abnormal motor cortical excitability, especially relating to the maladaptive long-interval intracortical inhibitory circuitry. The blink recovery study and eyeblink classical conditioning studies demonstrated possible hyperexcitability of the brainstem circuits and cerebellar dysfunction in patients with TST. Functional MRI studies have further shown that patients with TST have reduced functional connectivity in the cerebellum, similar to patients with DTS and ET. Due to variable methodologies and the sparsity of functional MRI studies in TST, it remains uncertain if patients with TST share the connectivity abnormalities between the cortical or subcortical areas that have been demonstrated in patients with DTS. Comprehensive electrophysiological and functional neuroimaging studies may help to elucidate the pathophysiology of TST.

Electrophysiological Activity from the Eye Muscles, Cerebellum and Cerebrum During Reflexive (Classical Pavlovian) Versus Voluntary (Ivanov-Smolensky) Eye-Blink Conditioning.

We report an experiment to investigate the role of the cerebellum and cerebrum in motor learning of timed movements. Eleven healthy human subjects were recruited to perform two experiments, the first was a classical eye-blink conditioning procedure with an auditory tone as conditional stimulus (CS) and vestibular unconditional stimulus (US) in the form of a double head-tap. In the second experiment, subjects were asked to blink voluntarily in synchrony with the double head-tap US preceded by a CS, a form of Ivanov-Smolensky conditioning in which a command or instruction is associated with the US. Electrophysiological recordings were made of extra-ocular EMG and EOG at infra-ocular sites (IO1/2), EEG from over the frontal eye fields (C3'/C4') and from over the posterior fossa over the cerebellum for the electrocerebellogram (ECeG). The behavioural outcomes of the experiments showed weak reflexive conditioning for the first experiment despite the double tap but robust, well-synchronised voluntary conditioning for the second. Voluntary conditioned blinks were larger than the reflex ones. For the voluntary conditioning experiment, a contingent negative variation (CNV) was also present in the EEG leads prior to movement, and modulation of the high-frequency EEG occurred during movement. US-related cerebellar activity was prominent in the high-frequency ECeG for both experiments, while conditioned response-related cerebellar activity was additionally present in the voluntary conditioning experiment. These results demonstrate a role for the cerebellum in voluntary (Ivanov-Smolensky) as well as in reflexive (classical Pavlovian) conditioning.

A Cerebellum-Inspired Spiking Neural Model With Adapting Rate Neurons

The cerebellum plays an important role in smooth and coordinated motor control. Precise control requires the cerebellum to regulate movements in both space and time domains. Although many cerebellar models have been proposed, most of them focus on motor coordination, motor learning, or timing distinctly. Therefore, it is necessary to develop a cerebellar model which contributes to the proper execution of movements via motor learning that displays temporal specificity. In this paper, we proposed a novel spiking neural network model to realize cerebellar processing with strong biomimicry, which is based on adapting rate neurons and has a cerebellum-inspired structure as well as biologically plausible cerebellar divergence/convergence ratios. The model was tested with the eyeblink classical conditioning (EBCC) experimental task. The simulation results verify that our implementation has improvements in both signal encoding and learning speed than previous studies. Furthermore, compared with neurophysiological data, our model shows a similar learning trend and can represent the fluctuation of the learning curves. It is demonstrated that our proposed cerebellar model experimentally reproduces the key features of the EBCC task and provides a new way to understand the timing and learning control neural mechanisms of the cerebellum.

Synaptic Mechanisms of Delay Eyeblink Classical Conditioning: AMPAR Trafficking and Gene Regulation in an In Vitro Model.

An in vitro model of delay eyeblink classical conditioning was developed to investigate synaptic plasticity mechanisms underlying acquisition of associative learning. This was achieved by replacing real stimuli, such as an airpuff and tone, with patterned stimulation of the cranial nerves using an isolated brainstem preparation from turtle. Here, our primary findings regarding cellular and molecular mechanisms for learning acquisition using this unique approach are reviewed. The neural correlate of the in vitro eyeblink response is a replica of the actual behavior, and features of conditioned responses (CRs) resemble those observed in behavioral studies. Importantly, it was shown that acquisition of CRs did not require the intact cerebellum, but the appropriate timing did. Studies of synaptic mechanisms indicate that conditioning involves two stages of AMPA receptor (AMPAR) trafficking. Initially, GluA1-containing AMPARs are targeted to synapses followed later by replacement by GluA4 subunits that support CR expression. This two-stage process is regulated by specific signal transduction cascades involving PKA and PKC and is guided by distinct protein chaperones. The expression of the brain-derived neurotrophic factor (BDNF) protein is central to AMPAR trafficking and conditioning. BDNF gene expression is regulated by coordinated epigenetic mechanisms involving DNA methylation/demethylation and chromatin modifications that control access of promoters to transcription factors. Finally, a hypothesis is proposed that learning genes like BDNF are poised by dual chromatin features that allow rapid activation or repression in response to environmental stimuli. These in vitro studies have advanced our understanding of the cellular and molecular mechanisms that underlie associative learning.

Behavioral and Transcriptome Profiling of Heterozygous Rab10 Knock-Out Mice.

A central question in the field of aging research is to identify the cellular and molecular basis of neuroresilience. One potential candidate is the small GTPase, Rab10. Here, we used Rab10+/- mice to investigate the molecular mechanisms underlying Rab10-mediated neuroresilience. Brain expression analysis of 880 genes involved in neurodegeneration showed that Rab10+/- mice have increased activation of pathways associated with neuronal metabolism, structural integrity, neurotransmission, and neuroplasticity compared with their Rab10+/+ littermates. Lower activation was observed for pathways involved in neuroinflammation and aging. We identified and validated several differentially expressed genes (DEGs), including Stx2, Stx1b, Vegfa, and Lrrc25 (downregulated) and Prkaa2, Syt4, and Grin2d (upregulated). Behavioral testing showed that Rab10+/- mice perform better in a hippocampal-dependent spatial task (object in place test), while their performance in a classical conditioning task (trace eyeblink classical conditioning, TECC) was significantly impaired. Therefore, our findings indicate that Rab10 differentially controls the brain circuitry of hippocampal-dependent spatial memory and higher-order behavior that requires intact cortex-hippocampal circuitry. Transcriptome and biochemical characterization of these mice suggest that glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D or GluN2D) is affected by Rab10 signaling. Further work is needed to evaluate whether GRIN2D mediates the behavioral phenotypes of the Rab10+/- mice. We conclude that Rab10+/- mice described here can be a valuable tool to study the mechanisms of resilience in Alzheimer's disease (AD) model mice and to identify novel therapeutical targets to prevent cognitive decline associated with normal and pathologic aging.

The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome

BackgroundFragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment.MethodsWe performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4.ResultsWe found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by β adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, β adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice.LimitationsWe cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells.ConclusionsOur study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.

Electrophysiological activity from over the cerebellum and cerebrum during eye blink conditioning in human subjects.

We report the results of an experiment in which electrophysiological activity was recorded from the human cerebellum and cerebrum in a sample of 14 healthy subjects before, during and after a classical eye blink conditioning procedure with an auditory tone as conditional stimulus and a maxillary nerve unconditional stimulus. The primary aim was to show changes in the cerebellum and cerebrum correlated with behavioral ocular responses. Electrodes recorded EMG and EOG at peri-ocular sites, EEG from over the frontal eye-fields and the electrocerebellogram (ECeG) from over the posterior fossa. Of the 14 subjects half strongly conditioned while the other half were resistant. We confirmed that conditionability was linked under our conditions to the personality dimension of extraversion-introversion. Inhibition of cerebellar activity was shown prior to the conditioned response, as predicted by Albus (1971). However, pausing in high frequency ECeG and the appearance of a contingent negative variation (CNV) in both central leads occurred in all subjects. These led us to conclude that while conditioned cerebellar pausing may be necessary, it is not sufficient alone to produce overt behavioral conditioning, implying the existence of another central mechanism. The outcomes of this experiment indicate the potential value of the noninvasive electrophysiology of the cerebellum.

Role of cerebellar cortex in associative learning and memory in guinea pigs.

Time-related cognitive function refers to the capacity of the brain to store, extract, and process specific information. Previous studies demonstrated that the cerebellar cortex participates in advanced cognitive functions, but the role of the cerebellar cortex in cognitive functions is unclear. We established a behavioral model using classical eyeblink conditioning to study the role of the cerebellar cortex in associative learning and memory and the underlying mechanisms. We performed an investigation to determine whether eyeblink conditioning could be established by placing the stimulating electrode in the middle cerebellar peduncle. Behavior training was performed using a microcurrent pulse as a conditioned stimulus to stimulate the middle cerebellar peduncle and corneal blow as an unconditioned stimulus. After 10 consecutive days of training, a conditioned response was successfully achieved in the Delay, Trace-200-ms, and Trace-300-ms groups of guinea pigs, with acquisition rates of >60%, but the Trace-400-ms and control groups did not achieve a conditioned stimulus-related blink conditioned response. It could be a good model for studying the function of the cerebellum during the establishment of eyeblink conditioning.

Cerebellum involvement in dystonia: insights from a spiking neural network model during associative learning.

Dystonia is a neurological movement disorder characterized by twisting and repetitive movements or abnormal fixed postures. This complex brain disease has usually been associated with damages to the Basal Ganglia. However, recent studies point out the potential role of the cerebellum. Indeed, motor learning is impaired in dystonic patients, e.g. during eyeblink classical conditioning, a typical cerebellum-driven associative learning protocol, and rodents with local cerebellar damages exhibit dystonic movements. Alterations in the olivocerebellar circuit connectivity have been identified as a potential neural substrate of dystonia. Here, we investigated this hypothesis through simulations of eyeblink conditioning driven by a realistic spiking model of the cerebellum. The pathological model was generated by decreasing the signal transmission from the Inferior Olive to cerebellar cortex, as observed in animal experiments. The model was able to reproduce a reduced acquisition of eyeblink motor responses, with also an unproper timing. Indeed, this pathway is fundamental to drive cerebellar cortical plasticity, which is the basis of cerebellum-driven motor learning. Exploring different levels of damage, the model predicted the possible amount of underlying impairment associated with the misbehavior observed in patients. Simulations of other debated lesions reported in mouse models of dystonia will be run to investigate the cerebellar involvement in different types of dystonia. Indeed, the eyeblink conditioning phenotype could be used to discriminate between them, identifying specific deficits in the generation of motor responses. Future studies will also include simulations of pharmacological or deep brain stimulation treatments targeting the cerebellum, to predict their impact in improving symptoms.

Cerebellum Involvement in Dystonia During Associative Motor Learning: Insights From a Data-Driven Spiking Network Model.

Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements, postures, or both. Although dystonia is traditionally associated with basal ganglia dysfunction, recent evidence has been pointing to a role of the cerebellum, a brain area involved in motor control and learning. Cerebellar abnormalities have been correlated with dystonia but their potential causative role remains elusive. Here, we simulated the cerebellar input-output relationship with high-resolution computational modeling. We used a data-driven cerebellar Spiking Neural Network and simulated a cerebellum-driven associative learning task, Eye-Blink Classical Conditioning (EBCC), which is characteristically altered in relation to cerebellar lesions in several pathologies. In control simulations, input stimuli entrained characteristic network dynamics and induced synaptic plasticity along task repetitions, causing a progressive spike suppression in Purkinje cells with consequent facilitation of deep cerebellar nuclei cells. These neuronal processes caused a progressive acquisition of eyelid Conditioned Responses (CRs). Then, we modified structural or functional local neural features in the network reproducing alterations reported in dystonic mice. Either reduced olivocerebellar input or aberrant Purkinje cell burst-firing resulted in abnormal learning curves imitating the dysfunctional EBCC motor responses (in terms of CR amount and timing) of dystonic mice. These behavioral deficits might be due to altered temporal processing of sensorimotor information and uncoordinated control of muscle contractions. Conversely, an imbalance of excitatory and inhibitory synaptic densities on Purkinje cells did not reflect into significant EBCC deficit. The present work suggests that only certain types of alterations, including reduced olivocerebellar input and aberrant PC burst-firing, are compatible with the EBCC changes observed in dystonia, indicating that some cerebellar lesions can have a causative role in the pathogenesis of symptoms.

117 The role of the cerebellum in cortical myoclonus: a neurophysiological study

ObjectiveTo investigate whether primary writing tremor (PWT) and dystonia share a common pathophysiology.BackgroundPWT is probably the most common form of task-specific tremor. Its pathophysiology is still debated and there is controversy about it being a type of focal dystonia, a variant of essential tremor or a separate entity.MethodsWe recruited 7 patients with PWT, who underwent the following neurophysiological tests: blink recovery cycle (BRC), eye-blink classical conditioning (EBCC), short and long-intracortical inhibition (SICI-LICI). Moreover, motor-evoked potentials (MEP), SICI and LICI were measured pre and post paired associative stimulation (PAS) plasticity paradigm. Data were compared with those obtained in 7 dystonic patients (DYT).ResultsData analysis for BRC showed a similar inhibition between DYT and PWT. There was no difference between DYT and PWT in the number of conditioned response (CR) during. Baseline SICI and LICI were similar in the two patients’ group, but whereas they were unchanged in PWT after PAS, they were reduced in DYT.ConclusionsThe present results suggest that PWT and DYT have similar brainstem excitability and associative learning. Cortical plasticity induced by PAS had a different effect on SICI and LICI in the two groups. Overall this data suggests that PWT and DYT share some pathophysiological mechanisms.a.latorre@ucl.ac.uk

250 Is primary writing tremor a forme fruste of dystonia?

ObjectiveTo investigate whether primary writing tremor (PWT) and dystonia share a common pathophysiology.BackgroundPWT is probably the most common form of task-specific tremor. Its pathophysiology is still debated and there is controversy about it being a type of focal dystonia, a variant of essential tremor or a separate entity.MethodsWe recruited 7 patients with PWT, who underwent the following neurophysiological tests: blink recovery cycle (BRC), eye-blink classical conditioning (EBCC), short and long-intracortical inhibition (SICI-LICI). Moreover, motor-evoked potentials (MEP), SICI and LICI were measured pre and post paired associative stimulation (PAS) plasticity paradigm. Data were compared with those obtained in 7 dystonic patients (DYT).ResultsData analysis for BRC showed a similar inhibition between DYT and PWT. There was no differ- ence between DYT and PWT in the number of conditioned response (CR) during. Baseline SICI and LICI were similar in the two patients’ group, but whereas they were unchanged in PWT after PAS, they were reduced in DYT.ConclusionsThe present results suggest that PWT and DYT have similar brainstem excitability and associa- tive learning. Cortical plasticity induced by PAS had a different effect on SICI and LICI in the two groups. Overall this data suggests that PWT and DYT share some pathophysiological mechanisms.a.latorre@ucl.ac.uk

Emotional Face Expressions Influence the Delay Eye-blink Classical Conditioning

Recent evidence raised the importance of the cerebellum in emotional processes, with specific regard to negative emotions. However, its role in the processing of face emotional expressions is still unknown. This study was aimed at assessing whether face emotional expressions influence the cerebellar learning processes, using the delay eyeblink classical conditioning (EBCC) as a model. Visual stimuli composed of faces expressing happy, sad and neutral emotions were used as conditioning stimulus in forty healthy subjects to modulate the cerebellum-brainstem pathway underlying the EBCC. The same stimuli were used to explore their effects on the blink reflex (BR) and its recovery cycle (BRRC) and on the cerebellar-brain inhibition (CBI). Data analysis revealed that the learning component of the EBCC was significantly reduced following the passive view of sad faces, while the extinction phase was modulated by both sad and happy faces. By contrast, BR, BRRC and CBI were not significantly affected by the view of emotional face expressions. The present study provides first evidence that the passive viewing of faces displaying emotional expressions, are processed by the cerebellum, with no apparent involvement of the brainstem and the cerebello-cortical connection. In particular, the view of sad faces, reduces the excitability of the cerebellar circuit underlying the learning phase of the EBCC. Differently, the extinction phase was shortened by both happy and sad faces, suggesting that different neural bases underlie learning and extinction of emotions expressed by faces.

A biochemical mechanism for time-encoding memory formation within individual synapses of Purkinje cells.

Within the classical eye-blink conditioning, Purkinje cells within the cerebellum are known to suppress their tonic firing rates for a well defined time period in response to the conditional stimulus after training. The temporal profile of the drop in tonic firing rate, i.e., the onset and the duration, depend upon the time interval between the onsets of the conditional and unconditional training stimuli. Direct stimulation of parallel fibers and climbing fiber by electrodes was found to be sufficient to reproduce the same characteristic drop in the firing rate of the Purkinje cell. In addition, the specific metabotropic glutamate-based receptor type 7 (mGluR7) was found responsible for the initiation of the response, suggesting an intrinsic mechanism within the Purkinje cell for the temporal learning. In an attempt to look for a mechanism for time-encoding memory formation within individual Purkinje cells, we propose a biochemical mechanism based on recent experimental findings. The proposed mechanism tries to answer key aspects of the "Coding problem" of Neuroscience by focusing on the Purkinje cell's ability to encode time intervals through training. According to the proposed mechanism, the time memory is encoded within the dynamics of a set of proteins-mGluR7, G-protein, G-protein coupled Inward Rectifier Potassium ion channel, Protein Kinase A, Protein Phosphatase 1 and other associated biomolecules-which self-organize themselves into a protein complex. The intrinsic dynamics of these protein complexes can differ and thus can encode different time durations. Based on their amount and their collective dynamics within individual synapses, the Purkinje cell is able to suppress its own tonic firing rate for a specific time interval. The time memory is encoded within the effective dynamics of the biochemical reactions and altering these dynamics means storing a different time memory. The proposed mechanism is verified by both a minimal and a more comprehensive mathematical model of the conditional response behavior of the Purkinje cell and corresponding dynamical simulations of the involved biomolecules, yielding testable experimental predictions.

Behavior and Regional Cortical BOLD Signal Fluctuations Are Altered in Adult Rabbits After Neonatal Volatile Anesthetic Exposure.

Neonatal and infant exposure to volatile anesthetics has been associated with long-term learning, memory, and behavioral deficits. Although early anesthesia exposure has been linked to a number of underlying structural abnormalities, functional changes associated with these impairments remain poorly understood. To investigate the relationship between functional alteration in neuronal circuits and learning deficiency, resting state functional MRI (rsfMRI) connectivity was examined in adolescent rabbits exposed to general anesthesia as neonates (1 MAC isoflurane for 2 h on postnatal days P8, P11, and P14) and unanesthetized controls before and after training with a trace eyeblink classical conditioning (ECC) paradigm. Long-range connectivity was measured between several key regions of interest (ROIs), including primary and secondary somatosensory cortices, thalamus, hippocampus, and cingulate. In addition, metrics of regional BOLD fluctuation amplitudes and coherence, amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF), and regional homogeneity (ReHo) were calculated. Our results showed that the trace ECC learning rate was significantly lower in the anesthesia-exposed group. No anesthesia-related changes in long-range connectivity, fALFF, or ReHo were found between any ROIs. However, ALFF was significantly higher in anesthesia-exposed rabbits in the primary and secondary somatosensory cortices, and ALFF in those areas was a significant predictor of the learning performance for trace ECC. The absence of anesthesia-related changes in long-range thalamocortical connectivity indicates that functional thalamocortical input is not affected. Higher ALFF in the somatosensory cortex may indicate the developmental disruption of cortical neuronal circuits after neonatal anesthesia exposure, including excessive neuronal synchronization that may underlie the observed cognitive deficits.

Learning-Dependent Transcriptional Regulation of BDNF by its Truncated Protein Isoform in Turtle.

The vertebrate brain-derived neurotrophic factor (BDNF) gene produces a number of alternatively spliced transcripts only some of which generate the BDNF protein required for synaptic plasticity and learning. Many of the transcripts are uncharacterized and are of unknown biological significance. Previously, we described alternative splicing within the protein-coding sequence of the BDNF gene in the pond turtle (tBDNF) that generates a functionally distinct truncated protein isoform (trcBDNF) that is regulated during a neural correlate of eyeblink classical conditioning in ex vivo brainstem preparations. We hypothesized that trcBDNF has a dominant negative function because of its anticorrelated expression pattern compared to its full-length BDNF counterpart. The data presented here suggests that trcBDNF functions as a transcriptional repressor of a conditioning-inducible downstream tBDNF promoter that controls expression of full-length BDNF required for learning. First, expression of full-length transcripts is negatively correlated with trcBDNF; transcripts are inhibited when endogenous trcBDNF is ectopically induced and expressed when trcBDNF is inhibited. Second, ChIP-qPCR assays of a recombinant trcBDNF protein, RtrcBDNF, show strong binding to the downstream tBDNF exon III promoter that corresponds with inhibition of conditioning. Third, deletions of the C-terminus of RtrcBDNF result in inhibition of promoter binding and conditioning acquisition when a tropomyosin receptor kinase B (TrkB) binding site is accounted for. Finally, microinjection of RtrcBDNF directly into brainstem preparations inhibits conditioning. These data reveal a new mechanism of activity-dependent BDNF transcriptional regulation and suggest that BDNF is an autoregulatory gene. How generalizable this mechanism is across plasticity genes remains to be elucidated.

The Claustrum is Involved in Cognitive Processes Related to the Classical Conditioning of Eyelid Responses in Behaving Rabbits.

It is assumed that the claustrum (CL) is involved in sensorimotor integration and cognitive processes. We recorded the firing activity of identified CL neurons during classical eyeblink conditioning in rabbits, using a delay paradigm in which a tone was presented as conditioned stimulus (CS), followed by a corneal air puff as unconditioned stimulus (US). Neurons were identified by their activation from motor (MC), cingulate (CC), and medial prefrontal (mPFC) cortices. CL neurons were rarely activated by single stimuli of any modality. In contrast, their firing was significantly modulated during the first sessions of paired CS/US presentations, but not in well-trained animals. Neuron firing rates did not correlate with the kinematics of conditioned responses (CRs). CL local field potentials (LFPs) changed their spectral power across learning and presented well-differentiated CL–mPFC/CL–MC network dynamics, as shown by crossfrequency spectral measurements. CL electrical stimulation did not evoke eyelid responses, even in trained animals. Silencing of synaptic transmission of CL neurons by the vINSIST method delayed the acquisition of CRs but did not affect their presentation rate. The CL plays an important role in the acquisition of associative learning, mostly in relation to the novelty of CS/US association, but not in the expression of CRs.

A New 3-Day Standardized Eyeblink Conditioning Protocol to Assess Extinction Learning From Infancy to Adulthood.

Associative learning can be observed from the neonatal period onward, providing opportunities to examine changes in basic learning and memory abilities. One method that is suitable to study associative learning is classical eyeblink conditioning (EBC) which is dependent on the cerebellum. Extinction learning can be systematically investigated in this paradigm by varying the context during learning and extinction. Because of methodological difficulties and ethical challenges, no studies have compared extinction learning using EBC across human development. Our goal was to test feasibility of a 3-day delay EBC paradigm that can be used from infancy to adulthood. Acceptance/safety was tested especially for infancy by investigating attrition rates and parental report on infant wellbeing. On a paradigm side, we tested if the paradigm leads to successful acquisition and extinction. An air puff served as unconditional stimulus (US) and a tone as conditional stimulus (CS). On day 1 during acquisition, participants received 36 US–CS pairings in context A. On day 2, participants received 12 acquisition trials in context A to consolidate association learning, followed by 48 extinction trials (tone alone presentations) in context B. Renewal was assessed on day 3 and incorporated 12 CS alone trials presented in both the acquisition context and the extinction context. Eyeblink responses were videotaped and coded offline. The protocol was tested with 12–36-months-old infants (N = 72), adolescents (N = 8), and adults (N = 8). Concerning the acceptance/safety side, attrition ranged from 21 to 58% in infant samples due to the complex preparation of the children for the paradigm. However, attrition is equal to or lower than other infant learning paradigms. Parents of infant samples were very interested in the paradigm and reported low levels of infant stress, exhaustion, and negative feelings during the sessions. Data quality was very high, and no participant had to be excluded because of insufficient data. Concerning the paradigm side, participants showed successful acquisition and extinction as a group. The procedure is ethically sound, feasible, tolerated by many infants, and acceptable among parents. The data show successful acquisition and extinction rates, making the paradigm a valuable tool for investigating developmental changes in extinction learning over the lifespan.