ABSTRACT Background: Prior exposure to traumatic events significantly increases the risk of developing substance use disorders (SUD), while having SUD, in turn, elevates the likelihood of encountering additional traumatic events. Despite this relationship, the consequences of trauma frequently go undetected and untreated in this population. The trauma-focused intervention eye movement desensitisation and reprocessing (EMDR), a first-line treatment for post-traumatic stress disorder (PTSD), has shown promising therapeutic potential in SUD patients. However, its underlying neurobiological mechanisms remain unclear. This study aims to investigate the efficacy of EMDR in SUD patients with comorbid psychological trauma. Additionally, potential mechanisms of action of the intervention will be explored. The primary hypothesis is that integrating EMDR into standard SUD treatment will enhance substance use prognosis. Methods: Sixty-four patients with SUD and trauma symptomatology will be randomised into two groups. One group will receive EMDR trauma-focused intervention in 6-8 sessions alongside treatment as usual (TAU) (n = 32), while the control group will receive TAU only (n = 32). The primary outcome will be the time to relapse, assessed at baseline, immediately after treatment, and at 1- and 3-months follow-up. Additional measures include post-traumatic, anxiety, depressive symptoms and biological markers (hair/salivary cortisol levels, eye blink conditioning, and resting-state fMRI). Survival analysis and linear mixed models will be used to assess treatment effects. The trial is registered on ClinicalTrials.gov (NCT05488691). Discussion: This study addresses a critical gap in scientific literature and clinical practice by evaluating the efficacy of EMDR, in patients with SUD and comorbid trauma symptoms, through a combination of clinical and biological markers. The findings could lead to integration of personalised, trauma-focused interventions into public health services for patients with SUD.

Interspecies variations in eyeblink conditioning.

Cerebral Palsy (CP) is the most common motor disability in children and affects 1.5 - 2.5 out of every 1,000 live births. Adults with CP have poorer general cardiovascular health and higher total cholesterol levels than those without CP. These modifiable lifestyle factors could contribute to an increased risk for Alzheimer's disease (AD). Recent evidence confirms that people with CP are at a higher risk for developing AD and related dementias as they age. However, the nature of this link is still unclear: do early life stressors/environmental insults, like those associated with CP, cause changes in the brain that may lead to the development of disease later in life, or does the presence of modifiable lifestyle factors increase risk? To address this question, we combined the high-cholesterol-fed rabbit model of AD combined with the prenatal hypoxia-ischemia (HI) model of CP. At 70-80% gestation, blood flow was restricted to the uterus of a pregnant dam for 40 minutes causing global hypoxia in the rabbit kits in utero. Rabbit kits were born naturally at term. At weaning, rabbits were placed on a normal diet or a high cholesterol diet with copper in the drinking water. After 6 months on the diet, we performed trace eyeblink conditioning, object location memory, and novel object recognition to assess cognitive function. We also measured changes in protein expression to examine histopathological changes consistent with AD. We found a significant positive correlation between serum cholesterol levels and the length of time it took the rabbits to reach the threshold for learning in trace eyeblink conditioning. In novel object recognition, there is a main effect of HI. HI rabbits have a decreased preference for the object in a new location. When rabbits are fed a high cholesterol diet, HI leads to an increase in APP expression in the frontal cortex, but there are no differences between groups in GFAP expression. More work needs to be done to clarify the relationship between prenatal brain injury and susceptibility to AD, but our results support a role of early life brain injuries contributing to risk of AD.

BackgroundSystemic estrogen loss (menopause) is currently under investigation as a contributing factor in the observed increase in women's risk of Alzheimer's disease (AD). Despite the proven cognitive benefits and neuroprotective properties of endogenous estrogen, attempts to reduce AD risk by exogenous estrogen therapy have yielded controversial results. In this study, we investigated whether genistein, a phytoestrogen, can replace estrogen, rescue the effects of systemic estrogen loss on cognition, and potentially reduce AD risk.MethodWe induced menopause via gonadectomy (GDX) in mature female rabbits and administered genistein through a special diet. Rabbits were assigned to one of three experimental groups: GDX with genistein diet (GDX+GN), GDX with control diet (GDX+CON), and sham GDX with control diet (sGDX+CON). All rabbits received their respective diets for eight weeks before undergoing cognitive assessment with trace eyeblink conditioning, a test of associative learning and memory.ResultThe results show that dietary genistein improved learning and memory in gonadectomized rabbits across all cognitive indices, including the percentage of conditioned responses, onset latency, delayed memory recall, and response amplitude.ConclusionGenistein may provide cognitive benefits in estrogen‐deficient conditions and may be considered in place of estrogen therapy to rescue cognitive impairment and reduce AD risk in post‐menopausal women.

The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system and mutations in Kv1.2 leads to neurological disorders such as epilepsies and ataxias. In the cerebellum regulation of Kv1.2 is coupled to learning and memory. We have previously shown that blocking Kv1.2 by infusing its specific inhibitor Tityustoxin-kα (TsTX) into the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are not fully understood. We have previously shown that infusion of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the faciliatory effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression involves channel tyrosine phosphorylation and its endocytocytic removal from the cell surface. In this study we report that an excitatory chemical stimulus (50mM K+-100µM glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation of mGluR1 with DHPG reduced the amount of Kv1.2 detected by cell surface biotinylation in cerebellar slices. To determine the molecular pathways involved we used an unbiased mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with PKC-γ, CaMKII and Gq/G11, each of which had been shown in other studies to co-immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the interaction between Kv1.2 and PKC-γ since in HEK cells and hippocampal neurons Kv1.2 endocytosis is elicited by PKC activation. Here we show that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface Kv1.2 levels elicited by DHPG, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC.

SummaryNeural circuits must integrate sensory cues and their outcomes to guide learning, yet how distinct inhibitory interneurons contribute to this process remains a central question. This study investigates the specific roles of somatostatin (SST) and parvalbumin (PV) interneurons in the primary somatosensory cortex during trace eyeblink conditioning. We found that SST interneuron activity dynamically tracked the development of learned, anticipatory blinks, declining as learning progressed. Conversely, PV interneuron activity was coupled to locomotion, a proxy for arousal, following the aversive stimulus but was absent during the predictive cue window. Nicotinic cholinergic signaling selectively modulated SST responses. These results demonstrate a functional dissociation: SST interneurons refine predictive motor responses, while PV interneurons process affective arousal. This reveals a fundamental division of labor within cortical microcircuits for segregating and integrating distinct dimensions of learning.

Background: Hyperphosphorylated tau accumulation and neurofibrillary tangles (NFTs) are hallmarks of tauopathies, including Alzheimer’s disease (AD), and are strongly associated with cognitive decline. The rTg4510 mouse model, which expresses mutant human tau (P301L), develops progressive tauopathy in the absence of amyloid-β pathology, providing a valuable tool for investigating tau-driven neurodegeneration. Previous studies have demonstrated spatial and object-recognition memory deficits at six months of age, which can be prevented by doxycycline (DOX)-mediated suppression of tau expression. However, it remained unclear whether non-spatial hippocampal learning, particularly temporal associative learning, would be similarly affected. Methods: We evaluated six-month-old rTg4510 mice with or without DOX treatment. To control for potential motor confounds, we first assessed spontaneous home cage activity. We then tested hippocampus-dependent non-spatial learning using two paradigms: trace eyeblink conditioning (500-ms trace interval) and contextual fear conditioning. Results: General motor function remained intact; however, rTg4510 mice without DOX treatment exhibited increased rearing behavior. These mice demonstrated significant deficits in trace eyeblink conditioning acquisition, with particularly clear impairment on the final day of training. Contextual fear conditioning showed milder deficits. Analysis of response peak latency revealed subtle temporal processing abnormalities during early learning. Two months of DOX treatment initiated at four months of age prevented these learning deficits, confirming their association with tau overexpression. Conclusions: Our findings demonstrate that rTg4510 mice exhibit deficits in non-spatial temporal associative learning alongside previously reported spatial and object-recognition impairments. Trace eyeblink conditioning serves as a sensitive behavioral assay for detecting tau-related hippocampal dysfunction, and the prevention of learning deficits by DOX treatment highlights its potential utility as a translational biomarker for evaluating tau-targeted interventions.

Background: Metabolic syndrome is a constellation of medical conditions including central obesity, high blood sugar, and high triglycerides known to increase the risk of heart disease, stroke, and type 2 diabetes, with significant sex differences in the syndrome’s incidence and prevalence. These clinical symptoms may be accompanied by cognitive impairment. Methods: In the present experiment, we fed rabbits a diet high in fat and sugar (HFSD), assessed symptoms, and measured changes in cognition using trace eyeblink conditioning. Results: We show that a range of symptoms of metabolic syndrome resulted from HFSD in male and female rabbits and obesity, high blood sugar, and glucose intolerance were higher in male than female rabbits. Specifically, HFSD male rabbits gained more weight and had a higher body-mass index, more body fat, higher fasting glucose levels, and greater glucose intolerance. Importantly, using trace and delay eyeblink conditioning, we show that there was cognitive impairment because of the high-fat and high-sugar diet in both male and female rabbits, but this was greater in HFSD male rabbits than HFSD female rabbits. Conclusions: Metabolic syndrome modeled in rabbits fed a diet high in fat and sugar reflects trends in the adult population including central obesity, high blood sugar, and high triglycerides and cognitive impairment and provides an important model and test bed for assessing interventions.

IntroductionThere is a profound lack of electrophysiological data from the cerebellum in humans, as compared to animals, because it is difficult to record cerebellar activity non-invasively using magnetoencephalography (MEG) or electroencephalography (EEG). Recent developments in wearable MEG sensors hold potential to overcome this limitation, as they allow the placement of sensors closer to the cerebellum.MethodsWe leveraged the development of wearable optically pumped magnetometers to record on-scalp MEG (OP-MEG) during an established cerebellar learning paradigm—eyeblink conditioning. In four healthy human adults, we first validated that OP-MEG can reliably detect cerebellar responses by examining responses to an air puff stimulus.ResultsSignificant responses were observed in sensors positioned over the cerebellar region in all four adults in response to the air puff. We then indirectly tested the hypothesis that these responses reflect the population-level spiking activity of Purkinje cells. The air-puff–evoked responses diminished during the acquisition of conditioned responses, corresponding with previously observed changes in Purkinje cell activity in animals. Additionally, in three out of four participants, we observed a cerebellar evoked response just prior to the peak of the conditioned blink, resembling learning-associated shifts in Purkinje cell response latencies.DiscussionThis study demonstrates that OP-MEG is a viable method for recording cerebellar activity in humans. By bridging invasive animal recordings with non-invasive human neuroimaging, these findings provide further evidence of the cerebellum’s role in human learning.

The cerebellum has been considered to perform error-based supervised learning via long-term depression (LTD) at synapses between parallel fibers and Purkinje cells (PCs). Since the discovery of multiple synaptic plasticity other than LTD, recent studies have suggested that synergistic plasticity mechanisms could enhance the learning capability of the cerebellum. Indeed, we have proposed a concept of cerebellar learning as a reinforcement learning (RL) machine. However, there is still a gap between the conceptual algorithm and its detailed implementation. To close this gap, in this research, we implemented a cerebellar spiking network as an RL model in continuous time and space, based on known anatomical properties of the cerebellum. We confirmed that our model successfully learned a state value and solved the mountain car task, a simple RL benchmark. Furthermore, our model demonstrated the ability to solve the delay eyeblink conditioning task using biologically plausible internal dynamics. Our research provides a solid foundation for cerebellar RL theory that challenges the classical view of the cerebellum as primarily a supervised learning machine.

Eyeblink conditioning is mediated by similar cerebellar pathways in humans and animals and is typically investigated using delay or trace protocols. These studies show that humans can easily acquire eyeblink conditioning within a single day of training whereas animals usually require around 3-10 days of acquisition training before they consistently exhibit conditioned responses. We aimed to study how a multiple-day acquisition training, across 3 non-consecutive days of a month, with 100 trials per day affected learning in young human adults. We employed an embedded protocol in which the US is embedded within the CS duration without co-termination. Our findings show, for the first time in humans using this protocol, that learning improves substantially on days 2 and 3. Our findings encourage research into how human cerebellum mediates consolidation across several days of eyeblink conditioning as well as into the neurocognitive mechanisms of the relatively underexplored embedded eyeblink conditioning protocol.

Cerebellum (CB) interactions with forebrain systems contribute to learning cognitive and motor tasks, but the nature of these interactions is unknown. Trace eyeblink conditioning (EBC) is an excellent associative learning paradigm for examining interactions between forebrain systems and CB. The anterior cingulate cortex (ACC), central amygdala (AM), and CB are essential for trace EBC, and we previously recorded changes in neuronal activity in these areas during learning with paired presentations of the conditional stimulus (CS) and unconditional stimulus (US). We attributed changes in activity to learning CS-US contingency but did not report contingency manipulations. Here, we analyzed data from the same rats of both sexes during sessions with transitions from CS-US (paired) trials to CS-alone (extinction) trials and from CS-alone trials to CS-US trials. All three areas showed changes in activity with changes in contingency, during both stimuli and the intertrial interval (ITI). Subsets of ACC, AM, and CB neurons showed higher activity during paired trials, while others showed higher activity during extinction trials both during trial events and ITI. Within- and between-area neuronal synchrony and machine learning predictions of behavior from neuronal activity decreased during extinction trials relative to paired trials, indicating widespread effects of contingency change on the ACC-AM-CB network. The findings suggest that the ACC-AM-CB network encodes both within-trial stimulus contingency and between-trial task context. The transition from paired to extinction trials may decrease mossy fiber input consistency to CB from ACC and AM and decrease the likelihood of CRs.

ObjectivesEye movement desensitization and reprocessing (EMDR) is an effective evidence‐based treatment for post‐traumatic stress disorder. However, the therapeutic mechanisms underlying eye movements (EM) remain unclear. This study aimed to investigate the effect of horizontal EM on fear extinction learning in healthy individuals, using an Eye Blink Conditioning (EBC) task. This experimental paradigm has been widely used to explore associative fear learning and memory as a form of classical conditioning.MethodsHealthy participants were included to the study protocol and divided randomly into two groups. The EM group (n = 20) were asked to follow horizontally the experimenter's moving finger at the beginning of the extinction phase and the control group (n = 19) did not engage in any specific task. Sociodemographic and clinical information was collected. Percentage of conditioned response (CR) occurrence, time of onset and intensity between and within groups over longitudinal time were analysed using generalized multilevel mixed effects for repeated measures.ResultsResults showed accelerated extinction learning in the EM group, with an 18.2% probability of CR occurrence in the first block of extinction, compared to the control group (40.9%) (p‐value = .007).ConclusionsThe findings indicate that horizontal EM accelerates the extinction process in the EBC task. Therefore, this paradigm, used for studying associative learning and memory, could serve as an objective measure to investigate the mechanisms of action involved in desensitizing traumatic experiences during EMDR treatment.

Mossy fiber inputs are transformed into cerebellar Purkinje cell (PC) outputs by granule cell (GC)-dependent processing. Cerebellar dysfunction leads to motor, learning, emotional, and social deficits that are usually attributed to altered PC firing arising from impaired processing of mossy fiber inputs, even though PCs also fire independently of GCs. To isolate their contributions to cerebellum-dependent behaviors, we either disrupt GC signaling while leaving PC firing intact, or disrupt PC signaling to eliminate the influence of PCs. Experiments were performed in mice of both sexes. We find that both GC and PC signaling are essential for eyeblink conditioning and vestibulo-ocular reflex (VOR) learning. Remarkably, disrupting PC signaling impairs VOR, anxiety, and social behavior, but abolishing GC signaling does not. This establishes that while GC signaling is critical for motor learning, it does not influence many behaviors including those associated with autism-spectrum disorder. It suggests that GC-independent behaviors can potentially be rescued by restoring altered firing in downstream regions.

Nitric oxide (NO) is a versatile signaling molecule with significant roles in various physiological processes, including synaptic plasticity and memory formation. In the cerebellum, NO is produced by neural NO synthase and diffuses to influence synaptic changes, particularly at parallel fiber-Purkinje cell synapses. This study aims to investigate NO's role in cerebellar learning mechanisms using a biologically realistic simulation-based approach. We developed the NO Diffusion Simulator (NODS), a Python module designed to model NO production and diffusion within a cerebellar spiking neural network framework. Our simulations focus on the eye-blink classical conditioning protocol to assess the impact of NO modulation on long-term potentiation and depression at parallel fiber-Purkinje cell synapses. The results demonstrate that NO diffusion significantly affects synaptic plasticity, dynamically adjusting learning rates based on synaptic activity patterns. This metaplasticity mechanism enhances the cerebellum's capacity to prioritize relevant inputs and mitigate learning interference, selectively modulating synaptic efficacy. Our findings align with theoretical models, suggesting that NO serves as a contextual indicator, optimizing learning rates for effective motor control and adaptation to new tasks. The NODS implementation provides an efficient tool for large-scale simulations, facilitating future studies on NO dynamics in various brain regions and neurovascular coupling scenarios. By bridging the gap between molecular processes and network-level learning, this work underscores the critical role of NO in cerebellar function and offers a robust framework for exploring NO-dependent plasticity in computational neuroscience.

One is seldom aware of the anticipatory and preemptive feats that the eyeblink system achieves in daily life but it frequently protects the eye from projectiles gone awry and insects on apparent collision courses. This poor awareness is why predictive eyeblinks are considered a form of implicit learning. In motor neuroscience, implicit learning is considered to be slow and, eyeblink conditioning, in particular, is believed to be a rigid and inflexible cerebellar-dependent behavior. In cognitive neuroscience, however, implicit and automatic processes are thought to be rapidly acquired. Here we show that the eyeblink system is, in fact, capable of remarkable cognitive flexibility and can learn on more rapid timescales than previously expected. In a task where we yoked contextual learning of predictive eyeblinks and manual responses in humans, well-timed eyeblink responses flexibly adjusted to external context on each trial. The temporal precision of the predictive eyeblinks exceeded that of manual response times. Learning of the well-timed eyeblink responses was also more rapid than that for the manual response times. This pattern persevered with the use of a cognitive strategy, which seemed to accelerate both types of learning. These results suggest that behaviors associated with the cerebellar cortex that were previously believed to be inflexible and largely implicit, can demonstrate rapid and precise context-dependent temporal control.

Conditioned eye blink reflex in rabbits

Hippocampus alters visual representation to encode new memory.

Alcohol impairs learning and timing of conditioned eyeblink responses.

Dynamics of hippocampal reactivation for temporal association memory in mice.