Conditioned Eyeblink Response Research Articles

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.

Hypothermia increases cold-inducible protein expression and improves cerebellar-dependent learning after hypoxia ischemia in the neonatal rat.

Hypoxic ischemic encephalopathy remains a significant cause of developmental disability.1,2 The standard of care for term infants is hypothermia, which has multifactorial effects.3-5 Therapeutic hypothermia upregulates the cold-inducible protein RNA binding motif 3 (RBM3) that is highly expressed in developing and proliferative regions of the brain.6,7 The neuroprotective effects of RBM3 in adults are mediated by its ability to promote the translation of mRNAs such as reticulon 3 (RTN3).8 METHODS: Hypoxia ischemia or control procedure was conducted in Sprague Dawley rat pups on postnatal day 10 (PND10). Pups were immediately assigned to normothermia or hypothermia at the end of the hypoxia. In adulthood, cerebellum-dependent learning was tested using the conditioned eyeblink reflex. The volume of the cerebellum and the magnitude of cerebral injury were measured. A second study quantified RBM3 and RTN3 protein levels in the cerebellum and hippocampus collected during hypothermia. Hypothermia reduced cerebral tissue loss and protected cerebellar volume. Hypothermia also improved learning of the conditioned eyeblink response. RBM3 and RTN3 protein expression were increased in the cerebellum and hippocampus of rat pups subjected to hypothermia on PND10. Hypothermia was neuroprotective in male and female pups and reversed subtle changes in the cerebellum after hypoxic ischemic. Hypoxic ischemic produced tissue loss and a learning deficit in the cerebellum. Hypothermia reversed both the tissue loss and learning deficit. Hypothermia increased cold-responsive protein expression in the cerebellum and hippocampus. Our results confirm cerebellar volume loss contralateral to the carotid artery ligation and injured cerebral hemisphere, suggesting crossed-cerebellar diaschisis in this model. Understanding the endogenous response to hypothermia might improve adjuvant interventions and expand the clinical utility of this intervention.

Relationship of Impairments in Associative Learning With Intellectual Disability and Cerebellar Hypoplasia in Autistic Children.

The severity of autism spectrum disorder (ASD) varies widely and is associated with intellectual disability (ID) and brain dysmorphology. We tested the hypothesis that the heterogeneity of ASD can be accounted for, in part, by altered associative learning measured by eye-blink conditioning (EBC) paradigms, used to test for forebrain and cerebellar dysfunction across the full range of ASD severity and intellectual ability. Children in this cohort study were diagnosed with ASD or typical development (TD); most children were recruited from a 10-year longitudinal study. Outcome measures were the percentage and timing of conditioned eye-blink responses (CRs) acquired to a tone, recorded photometrically and related to measures of ASD severity, IQ, and age 2 brain morphometry by MRI. A sequence of trace and delay EBC was used. Analysis of variance, t test, and logistic regression (LR) were used. Sixty-two children were studied at school age. Nine children with ASD with ID since age 2 (ASD + ID; IQ = 49 ± 6; 11.9 ± 0.2 years old [±SD]) learned more slowly than 30 children with TD (IQ = 120 ± 16; 10.5 ± 1.5 years old [±SD]) during trace EBC and showed atypically early-onset CRs (1.4 SD pre-TD) related to hypoplasia of the cerebellum at age 2 but not of the amygdala, hippocampus, or cerebral cortex. Conversely, 16 children with ASD with robust intellectual development since age 2 (IQ = 100 ± 3; 12.0 ± 0.4 years old [±SD]) learned typically but showed early-onset CRs only during long-delay EBC (0.8 SD pre-TD) unrelated to hypoplasia of any measured brain area. Using 16 EBC measures, binary LR classified ASD and TD with 80% accuracy (95% CI = 72-88%), 81% sensitivity (95% CI = 69-92%), and 79% specificity (95% CI = 68-91%); multinomial LR more accurately classified children based on ID (94% accuracy, 95% CI = 89-100%) than ASD severity (85% accuracy, 95% CI = 77-93%). Separate analyses of 39 children with MRI (2.1 ± 0.3 years old [±SD]) indicated that cerebellar hypoplasia did not predict ASD + ID over ages 2-4 (Cohen d = 0.3) compared with early-onset CRs during age 11 trace EBC (Cohen d = -1.3). Trace EBC reveals the relationship between cerebellar hypoplasia and ASD + ID likely by engaging cerebrocerebellar circuits involved in intellectual ability and implicit timing. Follow-up prospective studies using associative learning can determine whether ID can be predicted in children with early ASD diagnoses.

Cerebellum-dependent associative learning is not impaired in a mouse model of neurofibromatosis type 1

Individuals with Neurofibromatosis type 1 (NF1) experience a high degree of motor problems. The cerebellum plays a pivotal role in motor functioning and the NF1 gene is highly expressed in cerebellar Purkinje cells. However, it is not well understood to what extent NF1 affects cerebellar functioning and how this relates to NF1 motor functioning. Therefore, we subjected global Nf1+/− mice to a cerebellum-dependent associative learning task, called Pavlovian eyeblink conditioning. Additionally, we assessed general motor function and muscle strength in Nf1+/− mice. To our surprise, we found that Nf1+/− mice showed a moderately increased learning rate of conditioned eyeblink responses, as well as improved accuracy in the adaptive timing of the eyeblink responses. Locomotion, balance, general motor function, and muscle strength were not affected in Nf1+/− mice. Together, our results support the view that cerebellar function in Nf1+/− mice is unimpaired.

Sustained Activity of Hippocampal Parvalbumin-Expressing Interneurons Supports Trace Eyeblink Conditioning in Mice.

Although recent studies have revealed an involvement of hippocampal interneurons in learning the association among time-separated events, its underlying cellular mechanisms remained not fully clarified. Here, we combined multichannel recording and optogenetics to elucidate how the hippocampal parvalbumin-expressing interneurons (PV-INs) support associative learning. To address this issue, we trained the mice (both sexes) to learn hippocampus-dependent trace eyeblink conditioning (tEBC) in which they associated a light flash conditioned stimulus (CS) with a corneal air puff unconditioned stimuli (US) separated by a 250 ms time interval. We found that the hippocampal PV-INs exhibited learning-associated sustained activity at the early stage of tEBC acquisition. Moreover, the PV-IN sustained activity was positively correlated with the occurrence of conditioned eyeblink responses at the early learning stage. Suppression of the PV-IN sustained activity impaired the acquisition of tEBC, whereas the PV-IN activity suppression had no effect on the acquisition of delay eyeblink conditioning, a hippocampus-independent learning task. Learning-associated augmentation in the excitatory pyramidal cell-to-PVIN drive may contribute to the formation of PV-IN sustained activity. Suppression of the PV-IN sustained activity disrupted hippocampal gamma but not theta band oscillation during the CS-US interval period. Gamma frequency (40 Hz) activation of the PV-INs during the CS-US interval period facilitated the acquisition of tEBC. Our current findings highlight the involvement of hippocampal PV-INs in tEBC acquisition and reveal insights into the PV-IN activity kinetics which are of key importance for the hippocampal involvement in associative learning.SIGNIFICANCE STATEMENT The cellular mechanisms underlying associative learning have not been fully clarified. Previous studies focused on the involvement of hippocampal pyramidal cells in associative learning, whereas the activity and function of hippocampal interneurons were largely neglected. We herein demonstrated the hippocampal PV-INs exhibited learning-associated sustained activity, which was required for the acquisition of tEBC. Furthermore, we showed evidence that the PV-IN sustained activity might have arisen from the learning-associated augmentation in excitatory pyramidal cell-to-PVIN drive and contributed to learning-associated augmentation in gamma band oscillation during tEBC acquisition. Our findings provide more mechanistic understanding of the cellular mechanisms underlying the hippocampal involvement in associative learning.

Cerebellar transcranial direct current stimulation modulates timing but not acquisition of conditioned eyeblink responses in SCA3 patients

BackgroundDelay eyeblink conditioning is an extensively studied motor learning paradigm that critically depends on the integrity of the cerebellum. In healthy individuals, modulation of cerebellar excitability using transcranial direct current stimulation (tDCS) has been reported to alter the acquisition and/or timing of conditioned eyeblink responses (CRs). It remains unknown whether such effects can also be elicited in patients with cerebellar disorders. ObjectiveTo investigate if repeated sessions of cerebellar tDCS modify acquisition and/or timing of CRs in patients with spinocerebellar ataxia type 3 (SCA3) and to evaluate possible associations between disease severity measures and eyeblink conditioning parameters. MethodsDelay eyeblink conditioning was examined in 20 mildly to moderately affected individuals with SCA3 and 31 healthy controls. After the baseline assessment, patients were randomly assigned to receive ten sessions of cerebellar anodal tDCS or sham tDCS (i.e., five days per week for two consecutive weeks). Patients and investigators were blinded to treatment allocation. The same eyeblink conditioning protocol was administered directly after the last tDCS session. The Scale for the Assessment and Rating of Ataxia (SARA), cerebellar cognitive affective syndrome scale (CCAS-S), and disease duration were used as clinical measures of disease severity. ResultsAt baseline, SCA3 patients exhibited significantly fewer CRs than healthy controls. Acquisition was inversely associated with the number of failed CCAS-S test items but not with SARA score. Onset and peak latencies of CRs were longer in SCA3 patients and correlated with disease duration. Repeated sessions of cerebellar anodal tDCS did not affect CR acquisition, but had a significant treatment effect on both timing parameters. While a shift of CRs toward the conditioned stimulus was observed in the sham group (i.e., timing became more similar to that of healthy controls, presumably reflecting the effect of a second eyeblink conditioning session), anodal tDCS induced a shift of CRs in the opposite direction (i.e., toward the unconditioned stimulus). ConclusionOur findings provide evidence that cerebellar tDCS is capable of modifying cerebellar function in SCA3 patients. Future studies should assess whether this intervention similarly modulates temporal processing in other degenerative ataxias.

Ventromedial Thalamus-Projecting DCN Neurons Modulate Associative Sensorimotor Responses in Mice.

The deep cerebellar nuclei (DCN) integrate various inputs to the cerebellum and form the final cerebellar outputs critical for associative sensorimotor learning. However, the functional relevance of distinct neuronal subpopulations within the DCN remains poorly understood. Here, we examined a subpopulation of mouse DCN neurons whose axons specifically project to the ventromedial (Vm) thalamus (DCNVm neurons), and found that these neurons represent a specific subset of DCN units whose activity varies with trace eyeblink conditioning (tEBC), a classical associative sensorimotor learning task. Upon conditioning, the activity of DCNVm neurons signaled the performance of conditioned eyeblink responses (CRs). Optogenetic activation and inhibition of the DCNVm neurons in well-trained mice amplified and diminished the CRs, respectively. Chemogenetic manipulation of the DCNVm neurons had no effects on non-associative motor coordination. Furthermore, optogenetic activation of the DCNVm neurons caused rapid elevated firing activity in the cingulate cortex, a brain area critical for bridging the time gap between sensory stimuli and motor execution during tEBC. Together, our data highlights DCNVm neurons' function and delineates their kinematic parameters that modulate the strength of associative sensorimotor responses.

Intracerebellar infusion of an mGluR1/5 agonist enhances eyeblink conditioning.

Cerebellar metabotropic glutamate receptor 1 (mGluR1) expressed by Purkinje cells may play an important role in learning-related cerebellar plasticity. Eyeblink conditioning (EBC) is a well-studied form of Pavlovian learning that engages discrete areas of cerebellar cortex and deep cerebellar nuclei. EBC is impaired in mGluR1 knockout mice. Here, we show that infusion of the mGluR1/5 agonist DHPG into the lobulus simplex region of cerebellar cortex facilitates EBC in rats. Infusion was made prior to Sessions 1 and 2 of EBC but the facilitatory effects persisted throughout subsequent, noninfusion sessions. The facilitatory effects were confined to frequency of eyeblink conditioned responses (CRs); there were no effects on amplitude or latency of CRs. There were also no effects on reflexive responding to the tone conditioned stimulus or eyelid stimulation unconditioned stimulus. The current results provide further evidence that cerebellar mGluR1 plays a role in cerebellar-dependent associative learning and complement previous studies using mGluR1 knockout mice. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

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.

Pavlovian eyeblink conditioning is severely impaired in tottering mice.

Cacna1a encodes the pore-forming α1A subunit of CaV2.1 voltage-dependent calcium channels, which regulate neuronal excitability and synaptic transmission. Purkinje cells in the cortex of cerebellum abundantly express these CaV2.1 channels. Here, we show that homozygous tottering (tg) mice, which carry a loss-of-function Cacna1a mutation, exhibit severely impaired learning in Pavlovian eyeblink conditioning, which is a cerebellar-dependent learning task. Performance of reflexive eyeblinks is unaffected in tg mice. Transient seizure activity in tg mice further corrupted the amplitude of eyeblink conditioned responses. Our results indicate that normal calcium homeostasis is imperative for cerebellar learning and that the oscillatory state of the brain can affect the expression thereof.NEW & NOTEWORTHY In this study, we confirm the importance of normal calcium homeostasis in neurons for learning and memory formation. In a mouse model with a mutation in an essential calcium channel that is abundantly expressed in the cerebellum, we found severely impaired learning in eyeblink conditioning. Eyeblink conditioning is a cerebellar-dependent learning task. During brief periods of brain-wide oscillatory activity, as a result of the mutation, the expression of conditioned eyeblinks was even further disrupted.

Enhanced Acquisition and Retention of Conditioned Eyeblink Responses in Veterans Expressing PTSD Symptoms: Modulation by Lifetime History of Mild Traumatic Brain Injury.

Enhanced acquisition of eyeblink conditioning is observed in active duty military and veterans expressing PTSD symptoms (PTSD+) and those expressing temperamental vulnerabilities to develop PTSD after traumatic experiences, such as behaviorally inhibited temperament. There is a growing literature showing persistent cerebellar abnormalities in those experiencing mild traumatic brain injury (mTBI+) as well as linkages between mTBI and PTSD. With the dependency of eyeblink conditioning on cerebellar processes, the impact of mTBI on eyeblink conditioning in veterans expressing PTSD is unknown. The present study assessed eyeblink conditioning in veterans during two sessions separated by 1 week. With a focus on the accelerated learning of veterans expressing PTSD, training utilized a protocol which degrades learning through interspersing conditioned stimulus (CS) exposures amongst delay-type trials of CS and unconditional stimulus (US) co-terminating trials. Faster acquisition of the eyeblink conditioned responses (CR) was observed in PTSD during Week 1. The Week 2 assessment revealed an interaction of mTBI and PTSD, such that asymptotic performance of PTSD+ was greater than PTSD− among mTBI− veterans, whereas these groups did not differ in mTBI+ veterans. To further examine the relationship between enhanced sensitivity to acquire eyeblink conditioning and PTSD, cluster analysis was performed based on performance across training sessions. Those with enhanced sensitivity to acquire eyeblink conditioned responses expressed more PTSD symptoms, which were specific to Cluster C symptoms of avoidance, in addition to greater behavioral inhibition. These results support the continued investigation of the conditioned eyeblink response as a behavioral indicator of stress-related psychopathology.

Long-term effects of cerebellar anodal transcranial direct current stimulation (tDCS) on the acquisition and extinction of conditioned eyeblink responses

Cerebellar transcranial direct current stimulation (tDCS) has been reported to enhance the acquisition of conditioned eyeblink responses (CR), a form of associative motor learning. The aim of the present study was to determine possible long-term effects of cerebellar tDCS on the acquisition and extinction of CRs. Delay eyeblink conditioning was performed in 40 young and healthy human participants. On day 1, 100 paired CS (conditioned stimulus)–US (unconditioned stimulus) trials were applied. During the first 50 paired CS–US trials, 20 participants received anodal cerebellar tDCS, and 20 participants received sham stimulation. On days 2, 8 and 29, 50 paired CS–US trials were applied, followed by 30 CS-only extinction trials on day 29. CR acquisition was not significantly different between anodal and sham groups. During extinction, CR incidences were significantly reduced in the anodal group compared to sham, indicating reduced retention. In the anodal group, learning related increase of CR magnitude tended to be reduced, and timing of CRs tended to be delayed. The present data do not confirm previous findings of enhanced acquisition of CRs induced by anodal cerebellar tDCS. Rather, the present findings suggest a detrimental effect of anodal cerebellar tDCS on CR retention and possibly CR performance.

Interaction of Fear Conditioning with Eyeblink Conditioning Supports the Sensory Gating Hypothesis of the Amygdala in Men.

Inhibition of the amygdala slows down acquisition of conditioned eyeblink responses (CRs). Based on the two-stage or two-factor theory of aversive conditioning, amygdala-dependent conditioned fear is a necessary prerequisite to acquire eyeblink CRs but is no longer needed after eyeblink CRs are attained. According to the sensory gating hypothesis of the amygdala, on the other hand, the amygdala modulates the salience of unconditioned stimuli (USs) and conditioned stimuli (CSs) in eyeblink conditioning. We tested these two opposing assumptions in five groups of 20 young and healthy men. On day 1, three groups underwent fear acquisition training followed by acquisition of eyeblink CRs. On the next day (day 2), extinction was tested. In group 1, fear and eyeblink extinction trials overlapped; in group 2, fear and eyeblink extinction trials alternated; and in group 3, fear extinction trials were followed by eyeblink extinction trials. Groups 4 and 5 were control conditions testing fear and eyeblink conditioning only. Preceding fear acquisition training facilitated acquisition of conditioned eyeblinks. Concomitant fear extinction impeded extinction of eyeblink CRs, which was accompanied by increased autonomic responses. Fear extinction, however, was not significantly altered by concomitant eyeblink extinction. Recall of fear CRs on day 2 was facilitated in group 1, suggesting additive response summation. Findings are difficult to explain with the two-stage theory of aversive conditioning, which predicts the suppression of conditioned fear once conditioned eyeblinks are acquired. Facilitated acquisition and impeded extinction of eyeblink CRs, however, are in accordance with the sensory-gating hypothesis of the amygdala.

Prefrontal but not cerebellar tDCS attenuates renewal of extinguished conditioned eyeblink responses.

An extended neural network is known to underlie extinction learning. As yet, comparatively little is known about the possible contribution of the cerebellum and the dorsolateral prefrontal cortex (dlPFC). In the present study, transcranial direct current stimulation (tDCS) was used to provide further evidence that the dlPFC and the cerebellum are involved in extinction-related processes. A total of 100 young and healthy human participants were randomly assigned to one of five stimulation groups: (1) anodal tDCS of the cerebellum, (2) cathodal tDCS of the cerebellum, (3) anodal tDCS of the dlPFC, (4) cathodal tDCS of the dlPFC, and (5) sham stimulation. Participants underwent delay eyeblink conditioning using an A-B-A/B renewal paradigm. Two different colors of background light (orange and blue) were used as contexts. On day 1, acquisition of conditioned eyeblink responses was performed in context A, followed by extinction in context B. tDCS was applied during extinction. On day 2, extinction recall was tested in contexts A and B with higher incidence of conditioned responses in acquisition context A compared to extinction context B indicating renewal effects. All groups showed significant effects of acquisition of conditioned eyeblink responses and significant effects of extinction. There was no significant difference in extinction between stimulation groups. During extinction recall, renewal effects were present in all groups, except the group which had received anodal tDCS of the dlPFC during extinction. In the present study, no direct effects of dlPFC or cerebellar tDCS were demonstrated on extinction. Anodal tDCS of the dlPFC, but not the cerebellum, resulted in delayed effects on context-related processes of extinction, possibly explained by shifting attention away from the context and towards the conditioned stimulus during extinction learning. Anodal tDCS of the dlPFC attenuated context-related recall of learned aversive responses. Effects of tDCS, however, were weak and need to be confirmed in future studies. Lack of cerebellar tDCS effects do not exclude a possible role of the cerebellum in extinction-related processes, and are likely explained by methodological limitations of cerebellar tDCS.

Inactivation of the interpositus nucleus during unpaired extinction does not prevent extinction of conditioned eyeblink responses or conditioning-specific reflex modification.

For almost 75 years, classical eyeblink conditioning has been an invaluable tool for assessing associative learning processes across many species, thanks to its high translatability and well-defined neural circuitry. Our laboratory has adapted the paradigm to extensively detail associative changes in the rabbit reflexive eyeblink response (unconditioned response, UR), characterized by postconditioning increases in the frequency, size, and latency of the UR when the periorbital shock unconditioned stimulus (US) is presented alone, termed conditioning-specific reflex modification (CRM). Because the shape and timing of CRM closely resembles the conditioned eyeblink response (CR) to the tone conditioned stimulus (CS), we previously tested whether CRs and CRM share a common neural substrate, the interpositus nucleus of the cerebellum (IP), and found that IP inactivation during conditioning blocked the development of both CRs and the timing aspect of CRM. The goal of the current study was to examine whether extinction of CRs and CRM timing, accomplished simultaneously with unpaired CS/US extinction, also involves the IP. Results showed that muscimol inactivation of the IP during extinction blocked CR expression but not extinction of CRs or CRM timing, contrasting with the literature showing IP inactivation prevents CR extinction during CS-alone presentations. The continued presence of the US throughout the unpaired extinction procedure may have been sufficient to overcome IP blockade, promoting plasticity in the cerebellar cortex and/or extracerebellar components of the eyeblink conditioning pathway that can modulate extinction of CRs and CRM timing. Results therefore add support to the distributed plasticity view of cerebellar learning. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Reduced avoidance coping in male, but not in female rats, after mild traumatic brain injury: Implications for depression

Although there is evidence that traumatic brain injury (mTBI) induces emotional sequelae in rats, it is unclear whether the phenotype is reminiscent of major depressive disorder (MDD) or posttraumatic stress disorder (PTSD). Three behavioral protocols with oppositional indicators for MDD or PTSD were assessed: acoustic startle responses (ASRs), eyeblink conditioning, and instrumental escape/avoidance (E/A) learning. Female and male rats were exposed to lateral fluid percussion injury (LFPi) consistent with mild TBI (mTBI) or sham (SHAM) surgery. Experiment 1 suggested that the acquisition of the classically conditioned eyeblink responses was unaffected by mTBI infemale and male rats. In Experiment 2, male and female mTBI rats acquired instrumental escape responses similar to their SHAM counterparts. Avoidance expression of female mTBI rats did not differ appreciably from female SHAM rats. However, male mTBI rats expressed avoidance at a lower rate than male SHAM rats over training. Poor coping in male rats emerged with repeated exposure to stress, suggesting that depressive behaviors in mTBI develop over time and with continued demand from stress. Severely attenuated ASRs were evident in female and male mTBI rats compared to respective SHAM rats throughout testing across the two experiments. Overall, signs among the three bidirectional assessments during the subacute period after mTBI were more indicative of MDD-like, than PTSD-like sequelae.

Role of human prefrontal cortex in the modulation of conditioned eyeblink responses

Classical conditioning of the eyeblink reflex (EBC) is a simple form of associative motor learning. EBC is heavily dependent on cerebellar function, but experimental studies also suggest that the prefrontal cortex (PFC) orchestrates a neuronal network which interacts with the cerebellum to mediate the conditioned eyeblink responses (CR).To further investigate the role of PFC for EBC in humans, we aimed in this study at assessing whether acquisition of CR can be modulated by focal repetitive transcranial magnetic stimulation (rTMS) given as theta burst stimulation (TBS) over the dorsolateral PFC (DLPFC).A standard delay conditioning paradigm with a 540 ms tone as conditioned stimulus (CS) coterminating with a 100 ms air puff as unconditioned stimulus (US) was used in a total of 60 healthy subjects (35 female, 25 male, mean age 28.4 ± 2.4 years). One hundred paired CS-US trials and 30 extinction CS alone trials were given. TBS was applied over the DLPFC ipsilaterally to the US during the acquisition phase. Subjects were randomly assigned to three groups (n = 20) using excitatory intermittent TBS (iTBS), inhibitory continuous TBS (cTBS) or sham stimulation.CR acquisition was significantly enhanced by iTBS (mean total CR incidence 63.1 ± 6.5%) and significantly reduced by cTBS (13 ± 2%) compared to sham stimulation (25.1 ± 6.7%).We provide thus physiological evidence that the acquisition of this type of associative learning is critically modulated by PFC activity in humans.

Spontaneous recovery of conditioned eyeblink responses is associated with transiently decreased cerebellar theta activity in guinea pigs

Behavioral studies have demonstrated that extinguished conditioned eyeblink responses (CR) can spontaneously recover after extinction. However, the neural mechanisms underlying this process are still unclear. We have shown that spontaneous cerebellar theta activity was predictive of subsequent CR extinction. Here, we sought to further evaluate the association between spontaneous recovery and cerebellar theta activity in behaving guinea pigs. It was found that trace conditioning training significantly diminished the degree of spontaneous recovery during extinction sessions as compared to delay training. Moreover, by recording local field potential in the cerebellum of guinea pigs undergoing an eyeblink conditioning extinction task, we found that spontaneous recovery of delay-paradigm CRs was associated with transiently decreased CS-evoked theta activity in the cerebellum. These findings suggest that decreased CS-evoked cerebellar theta activity may contribute to the neural process that is important for the spontaneous recovery of extinguished motor memory. Future studies are needed to clarify the neural mechanism underlying changed cerebellar theta activity during altered behavioral contingencies.

Abnormal eyeblink conditioning is an early marker of cerebellar dysfunction in preclinical SCA3 mutation carriers

BackgroundSpinocerebellar ataxias (SCAs) are a group of autosomal dominantly inherited degenerative diseases. As the pathological process probably commences years before the first appearance of clinical symptoms, preclinical carriers of a SCA mutation offer the opportunity to study the earliest stages of cerebellar dysfunction and degeneration. Eyeblink classical conditioning (EBCC) is a motor learning paradigm, crucially dependent on the integrity of the olivocerebellar circuit, and has been shown to be able to detect subtle alterations of cerebellar function, which might already be present in preclinical carriers.MethodsIn order to acquire conditioned responses, we performed EBCC, delay paradigm, in 18 preclinical carriers of a SCA3 mutation and 16 healthy, age-matched controls by presenting repeated pairings of an auditory tone with a supraorbital nerve stimulus with a delay interval of 400 ms.ResultsPreclinical carriers acquired significantly less conditioned eyeblink responses than controls and learning rates were significantly reduced. This motor learning defect was, however, not associated with the predicted time to onset.ConclusionsEBCC is impaired in preclinical carriers of a SCA3 mutation, as a result of impaired motor learning capacities of the cerebellum and is thus suggestive of cerebellar dysfunction. EBCC can be used to detect but probably not monitor preclinical cerebellar dysfunction in genetic ataxias, such as SCA3.

Extinction and Renewal of Conditioned Eyeblink Responses in Focal Cerebellar Disease.

Extinction of conditioned aversive responses (CR) has been shown to be context-dependent. The hippocampus and prefrontal cortex are of particular importance. The cerebellum may contribute to context-related processes because of its known connections with the hippocampus and prefrontal cortex. Context dependency of extinction can be demonstrated by the renewal effect. When CR acquisition takes place in context A and is extinguished in context B, renewal refers to the recovery of the CR in context A (A-B-A paradigm). In the present study acquisition, extinction and renewal of classically conditioned eyeblink responses were tested in 18 patients with subacute focal cerebellar lesions and 18 age- and sex-matched healthy controls. Standard delay eyeblink conditioning was performed using an A-B-A paradigm. All cerebellar patients underwent a high-resolution T1-weighted brain MRI scan to perform lesion-symptom mapping. CR acquisition was not significantly different between cerebellar and control participants allowing to draw conclusions on extinction. CR extinction was significantly less in cerebellar patients. Reduction of CR extinction tended to be more likely in patients with lesions in the lateral parts of lobule VI and Crus I. A significant renewal effect was present in controls only. The present data provide further evidence that the cerebellum contributes to extinction of conditioned eyeblink responses. Because acquisition was preserved and extinction took place in another context than acquisition, more lateral parts of the cerebellar hemisphere may contribute to context-related processes. Furthermore, lack of renewal in cerebellar patients suggest a contribution of the cerebellum to context-related processes.