Interpositus Research Articles

Sensorimotor content of multi-unit activity recorded in the paramedian lobule of the cerebellum using carbon fiber microelectrode arrays.

The cerebellum takes in a great deal of sensory information from the periphery and descending signals from the cerebral cortices. It has been debated whether the paramedian lobule (PML) in the rat and its paravermal regions that project to the interpositus nucleus (IPN) are primarily involved in motor execution or motor planning. Studies that have relied on single spike recordings in behaving animals have led to conflicting conclusions regarding this issue. In this study, we tried a different approach and investigated the correlation of field potentials and multi-unit signals recorded with multi-electrode arrays from the PML cortex along with the forelimb electromyography (EMG) signals in rats during behavior. Linear regression was performed to predict the EMG signal envelopes using the PML activity for various time shifts (±25, ±50, ±100, and ± 400 ms) between the two signals to determine a causal relation. The highest correlations (~0.5 on average) between the neural and EMG envelopes were observed for zero and small (±25 ms) time shifts and decreased with larger time shifts in both directions, suggesting that paravermal PML is involved both in processing of sensory signals and motor execution in the context of forelimb reaching behavior. EMG envelopes were predicted with higher success rates when neural signals from multiple phases of the behavior were utilized for regression. The forelimb extension phase was the most difficult to predict while the releasing of the bar phase prediction was the most successful. The high frequency (>300 Hz) components of the neural signal, reflecting multi-unit activity, had a higher contribution to the EMG prediction than did the lower frequency components, corresponding to local field potentials. The results of this study suggest that the paravermal PML in the rat cerebellum is primarily involved in the execution of forelimb movements rather than the planning aspect and that the PML is more active at the initiation and termination of the behavior, rather than the progression.

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.

Topographic organization in the cerebellar nuclei and inferior olive in relation to cerebellar hemispheric lobules in the mouse: Distinction between crus I and neighboring lobules.

The parallel closed-loop topographic connections between subareas of the inferior olive (IO), cerebellar cortex, and cerebellar nuclei (CN) define the fundamental modular organization of the cerebellum. The cortical modules or zones are organized into longitudinal zebrin stripes which are extended across transverse cerebellar lobules. However, how cerebellar lobules, which are related to the cerebellar functional localization, are incorporated into the olivo-cortico-nuclear topographic organization has not been fully clarified. In the present study, we analyzed the lobular topography in the CN and IO by making 57 small bidirectional tracer injections in the lateral zebrin-positive stripes equivalent with C2, D1, and D2 zones in every hemispheric lobule in zebrin stripe-visualized mice. C2, D1, and D2 zones were connected to the lateral part of the posterior interpositus nucleus (lPIN), and caudal and rostral parts of the lateral nucleus (cLN, rLN), respectively, and from the rostral part of the medial accessory olive (rMAO), and ventral and dorsal lamellas of the PO (vPO, dPO), respectively, as reported. Within these areas, crus I was specifically connected to the ventral parts of the lPIN, cLN, and rLN, and from the rostrolateral part of the rMAO and the lateral parts of the vPO and dPO. The results indicated that the cerebellar modules have lobule-related subdivisions and that crus I is topographically distinct from other lobules. We speculate that crus I and crus I-connected subdivisions in the CN and IO are involved more in nonmotor functions than other neighboring areas in the mouse.

Inhibiting cholinergic signalling in the cerebellar interpositus nucleus impairs motor behaviour.

The role of neuromodulators in the cerebellum is not well understood. In particular, the behavioural significance of the cholinergic system in the cerebellum is unknown. To investigate the importance of cerebellar cholinergic signalling in behaviour, we infused acetylcholine receptor antagonists, scopolamine and mecamylamine, bilaterally into the rat cerebellum (centred on interpositus nucleus) and observed the motor effects through a battery of behavioural tests. These tests included unrewarded behaviour during open field exploration and a horizontal ladder walking task and reward-based beam walking and pellet reaching tasks. Infusion of a mix of the antagonists did not impair motor learning in the horizontal ladder walking or the reaching task but reduced spontaneous movement during open field exploration, impaired coordination during beam walking and ladder walking, led to fewer reaches in the pellet reaching task, slowed goal-directed reaching behaviour and reduced reward pellet consumption in a free access to food task. Infusion of the muscarinic antagonist scopolamine on its own resulted in deficits in motor performance and a reduction in the number of reward pellets consumed in the free access to food task. By contrast, infusion of the nicotinic antagonist mecamylamine on its own had no significant effect on any task, except beam walking traversal time, which was reduced. Together, these data suggest that acetylcholine in the cerebellar interpositus nucleus is important for the execution and coordination of voluntary movements mainly via muscarinic receptor signalling, especially in relation to reward-related behaviour.

Cerebellar contribution to the regulation of defensive states.

Despite fine tuning voluntary movement as the most prominently studied function of the cerebellum, early human studies suggested cerebellar involvement emotion regulation. Since, the cerebellum has been associated with various mood and anxiety-related conditions. Research in animals provided evidence for cerebellar contributions to fear memory formation and extinction. Fear and anxiety can broadly be referred to as defensive states triggered by threat and characterized by multimodal adaptations such as behavioral and cardiac responses integrated into an intricately orchestrated defense reaction. This is mediated by an evolutionary conserved, highly interconnected network of defense-related structures with functional connections to the cerebellum. Projections from the deep cerebellar nucleus interpositus to the central amygdala interfere with retention of fear memory. Several studies uncovered tight functional connections between cerebellar deep nuclei and pyramis and the midbrain periaqueductal grey. Specifically, the fastigial nucleus sends direct projections to the ventrolateral PAG to mediate fear-evoked innate and learned freezing behavior. The cerebellum also regulates cardiovascular responses such as blood pressure and heart rate-effects dependent on connections with medullary cardiac regulatory structures. Because of the integrated, multimodal nature of defensive states, their adaptive regulation has to be highly dynamic to enable responding to a moving threatening stimulus. In this, predicting threat occurrence are crucial functions of calculating adequate responses. Based on its role in prediction error generation, its connectivity to limbic regions, and previous results on a role in fear learning, this review presents the cerebellum as a regulator of integrated cardio-behavioral defensive states.

Degradation of Perineuronal Nets in the Cerebellar Interpositus Nucleus Ameliorated Social Deficits in Shank3-deficient Mice

Perineuronal nets (PNNs) are structures that contain extracellular matrix chondroitin sulfate proteoglycan and surround the soma and dendrites of various neuronal cell types. They are involved in synaptic plasticity and undertake important physiological functions. Altered expression of PNNs has been demonstrated in the brains of autism-related animal models. However, the underlying mechanism is still unknown. In this study, we demonstrated that the PNNs in the cerebellum are involved in modulating social and repetitive/inflexible behaviors in Shank3B–/– mice, an established animal model of autism spectrum disorder. First, we performed wisteria floribunda agglutinin staining of the whole brain of Shank3B–/– mice, and found wisteria floribunda agglutinin-positive PNNs are significantly increased in the cerebellar interpositus nucleus (IntP) in Shank3B–/– mice compared to control littermates. After degradation of PNNs in the IntP by chondroitinase ABC, the repetitive behaviors of Shank3B–/– mice were decreased, while their social behaviors were ameliorated. These results suggested that PNNs homeostasis is involved in the regulation of social behavior, revealing a potential therapeutic strategy targeting PNNs in the IntP for the treatment of autism spectrum disorder.

Functional properties of eyelid conditioned responses and involved brain centers.

For almost a century the classical conditioning of nictitating membrane/eyelid responses has been used as an excellent and feasible experimental model to study how the brain organizes the acquisition, storage, and retrieval of new motor abilities in alert behaving mammals, including humans. Lesional, pharmacological, and electrophysiological approaches, and more recently, genetically manipulated animals have shown the involvement of numerous brain areas in this apparently simple example of associative learning. In this regard, the cerebellum (both cortex and nuclei) has received particular attention as a putative site for the acquisition and storage of eyelid conditioned responses, a proposal not fully accepted by all researchers. Indeed, the acquisition of this type of learning implies the activation of many neural processes dealing with the sensorimotor integration and the kinematics of the acquired ability, as well as with the attentional and cognitive aspects also involved in this process. Here, we address specifically the functional roles of three brain structures (red nucleus, cerebellar interpositus nucleus, and motor cortex) mainly involved in the acquisition and performance of eyelid conditioned responses and three other brain structures (hippocampus, medial prefrontal cortex, and claustrum) related to non-motor aspects of the acquisition process. The main conclusion is that the acquisition of this motor ability results from the contribution of many cortical and subcortical brain structures each one involved in specific (motor and cognitive) aspects of the learning process.

Resting State Functional Connectivity Signatures of MRgFUS Vim Thalamotomy in Parkinson's Disease: A Preliminary Study

Magnetic Resonance-guided high-intensity Focused Ultrasound (MRgFUS) of the thalamic ventral intermediate nucleus (Vim) for tremor has increasingly gained interest as a new non-invasive alternative to standard neurosurgery. Resting state functional connectivity (rs-FC) correlates of MRgFUS have not been extensively investigated yet. A region of interest (ROI)-to-ROI rs-FC MRI “connectomic” analysis focusing on brain regions relevant for tremor was conducted on 15 tremor-dominant patients with Parkinson's disease who underwent MRgFUS. We tested whether rs-FC between tremor-related areas was modulated by MRgFUS at 1 and 3 months post-operatively, and whether such changes correlated with individual clinical outcomes assessed by the MDS-UPDRS-III sub items for tremor. Significant increase in FC was detected within bilateral primary motor (M1) cortices, as well as between bilateral M1 and crossed primary somatosensory cortices, and also between pallidum and the dentate nucleus of the untreated hemisphere. Correlation between disease duration and FC increase at 3 months was found between the putamen of both cerebral hemispheres and the Lobe VI of both cerebellar hemispheres, as well as between the Lobe VI of untreated cerebellar hemisphere with bilateral supplementary motor area (SMA). Drop-points value of MDS-UPDRS at 3 months correlated with post-treatment decrease in FC, between the anterior cingulate cortex and bilateral SMA, as well as between the Lobe VI of treated cerebellar hemisphere and the interpositus nucleus of untreated cerebellum. Tremor improvement at 3 months, expressed as percentage of intra-subject MDS-UPDRS changes, correlated with FC decrease between bilateral occipital fusiform gyrus and crossed Lobe VI and Vermis VI. Good responders (≥50% of baseline tremor improvement) showed reduced FC between bilateral SMA, between the interpositus nucleus of untreated cerebellum and the Lobe VI of treated cerebellum, as well as between the untreated SMA and the contralateral putamen. Good responders were characterized at baseline by crossed hypoconnectivity between bilateral putamen and M1, as well as between the putamen of the treated hemisphere and the contralateral SMA. We conclude that MRgFUS can effectively modulate brain FC within the tremor network. Such changes are associated with clinical outcome. The shifting mode of integration among the constituents of this network is, therefore, susceptible to external redirection despite the chronic nature of PD.

Three-Dimensional Heterogeneity of Cerebellar Interposed Nucleus-Recipient Zones in the Thalamic Nuclei

The cerebellum is conceptualized as a processor of complex movements and is also endowed with roles in cognitive and emotional behaviors. Although the axons of deep cerebellar nuclei are known to project to primary thalamic nuclei, macroscopic investigation of the characteristics of these projections, such as the spatial distribution of recipient zones, is lacking. Here, we studied the output of the cerebellar interposed nucleus (IpN) to the ventrolateral (VL) and centrolateral (CL) thalamic nuclei using electrophysiological recording in vivo and trans-synaptic viral tracing. We found that IpN stimulation induced mono-synaptic evoked potentials (EPs) in the VL but not the CL region. Furthermore, both the EPs induced by the IpN and the innervation of IpN projections displayed substantial heterogeneity across the VL region in three-dimensional space. These findings indicate that the recipient zones of IpN inputs vary between and within thalamic nuclei and may differentially control thalamo-cortical networks.

Action-based organization of a cerebellar module specialized for predictive control of multiple body parts

The role of the cerebellum in predictive motor control and coordination has been thoroughly studied during movements of a single body part. In the real world, however, actions are often more complex. Here, we show that a small area in the rostral anterior interpositus nucleus (rAIN) of the mouse cerebellum is responsible for generating a predictive motor synergy that serves to protect the eye by precisely coordinating muscles of the eyelid, neck, and forelimb. Within the rAIN region, we discovered a new functional category of neurons with unique properties specialized for control of motor synergies. These neurons integrated inhibitory cutaneous inputs from multiple parts of the body, and their activity was correlated with the vigor of the defensive motor synergy on a trial-by-trial basis. We propose that some regions of the cerebellum are organized in poly-somatotopic"action maps" to reduce dimensionality and simplify motor control during ethologically relevant behaviors.

Mapping of long-term cognitive and motor deficits in pediatric cerebellar brain tumor survivors into a cerebellar white matter atlas

PurposeDiaschisis of cerebrocerebellar loops contributes to cognitive and motor deficits in pediatric cerebellar brain tumor survivors. We used a cerebellar white matter atlas and hypothesized that lesion symptom mapping may reveal the critical lesions of cerebellar tracts.MethodsWe examined 31 long-term survivors of pediatric posterior fossa tumors (13 pilocytic astrocytoma, 18 medulloblastoma). Patients underwent neuronal imaging, examination for ataxia, fine motor and cognitive function, planning abilities, and executive function. Individual consolidated cerebellar lesions were drawn manually onto patients’ individual MRI and normalized into Montreal Neurologic Institute (MNI) space for further analysis with voxel-based lesion symptom mapping.ResultsLesion symptom mapping linked deficits of motor function to the superior cerebellar peduncle (SCP), deep cerebellar nuclei (interposed nucleus (IN), fastigial nucleus (FN), ventromedial dentate nucleus (DN)), and inferior vermis (VIIIa, VIIIb, IX, X). Statistical maps of deficits of intelligence and executive function mapped with minor variations to the same cerebellar structures.ConclusionWe identified lesions to the SCP next to deep cerebellar nuclei as critical for limiting both motor and cognitive function in pediatric cerebellar tumor survivors. Future strategies safeguarding motor and cognitive function will have to identify patients preoperatively at risk for damage to these critical structures and adapt multimodal therapeutic options accordingly.

Developmental timetables and gradients of neurogenesis in cerebellar Purkinje cells and deep glutamatergic neurons: A comparative study between the mouse and the rat.

The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([3 H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [3 H]TdR on embryonic days (E) 11-12, E12-13, E13-14, E14-15, E15-16, and E16-17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.

Fiber Microdissection Technique for Demonstrating the Deep Cerebellar Nuclei and Cerebellar Peduncles.

The cerebellum is one of the most primitive and complex parts of the human brain. The fiber microdissection technique can be extremely useful for neurosurgeons to understand the topographical organization of the cerebellum's important contents, such as the deep cerebellar nuclei and the cerebellar peduncles, and their relationship with the brain stem. To dissect the deep cerebellar nuclei and the cerebellar peduncles using the fiber microdissection technique. Under the operating microscope, 5 previously frozen, formalin-fixed human cerebellums and brain stems were dissected from the superior surface, and 5 were dissected from the inferior surface. Each stage of the process is described. The primary dissection tools were handmade, thin, wooden spatulas with tips of various sizes, toothpicks, and a fine regulated suction. In 15 simplified dissection steps (6 for the superior surface and 9 for the inferior surface), the deep cerebellar nuclei (dentate, interpositus, and fastigial) and the cerebellar peduncles (inferior, middle, and superior) are delineated. Their anatomical relationships with each other and other neighboring structures are demonstrated. The anatomy of the deep cerebellar nuclei and the cerebellar peduncles are clearly defined and understood through the use of the fiber microdissection technique. These stepwise dissections will guide the neurosurgeon in acquiring a topographical understanding of these complex and deep structures of the cerebellum. This knowledge, along with radiological information, can help in planning the most appropriate surgical strategy for various lesions of the cerebellum.

Long-Term Aberrations To Cerebellar Endocannabinoids Induced By Early-Life Stress

Emerging evidence points to the role of the endocannabinoid system in long-term stress-induced neural remodeling with studies on stress-induced endocannabinoid dysregulation focusing on cerebral changes that are temporally proximal to stressors. Little is known about temporally distal and sex-specific effects, especially in cerebellum, which is vulnerable to early developmental stress and is dense with cannabinoid receptors. Following limited bedding at postnatal days 2–9, adult (postnatal day 70) cerebellar and hippocampal endocannabinoids, related lipids, and mRNA were assessed, and behavioral performance evaluated. Regional and sex-specific effects were present at baseline and following early-life stress. Limited bedding impaired peripherally-measured basal corticosterone in adult males only. In the CNS, early-life stress (1) decreased 2-arachidonoyl glycerol and arachidonic acid in the cerebellar interpositus nucleus in males only; (2) decreased 2-arachidonoyl glycerol in females only in cerebellar Crus I; and (3) increased dorsal hippocampus prostaglandins in males only. Cerebellar interpositus transcriptomics revealed substantial sex effects, with minimal stress effects. Stress did impair novel object recognition in both sexes and social preference in females. Accordingly, the cerebellar endocannabinoid system exhibits robust sex-specific differences, malleable through early-life stress, suggesting the role of endocannabinoids and stress to sexual differentiation of the brain and cerebellar-related dysfunctions.

New Cerebello-Cortical Pathway Involved in Higher-Order Oculomotor Control.

The cerebellum and the basal ganglia play an important role in the control of voluntary eye movement associated with complex behavior, but little is known about how cerebellar projections project to cortical eye movement areas. Here we used retrograde transneuronal transport of rabies virus to identify neurons in the cerebellar nuclei that project via the thalamus to supplementary eye field (SEF) of the frontal cortex of macaques. After rabies injections into the SEF, many neurons in the restricted region, the ventral aspects of the dentate nucleus (DN), the caudal pole of the DN, and the posterior interpositus nucleus (PIN) were labeled disynaptically via the thalamus, whereas no neuron labeling was found in the anterior interpositus nucleus (AIN). The distribution of the labeled neurons was dorsoventrally different from that of DN and PIN neurons labeled from the motor cortex. In the basal ganglia, a large number of labeled neurons were confined to the dorsomedial portion of the internal segment of the globus pallidus (GPi) as more neurons were labeled in the inner portion of the GPi (GPii) than in the outer portion of the GPi (GPio). This is the first evidence of a projection between cerebellum/basal ganglia and the SEF that could enable the cerebellum to modulate the cognitive control of voluntary eye movement.

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).

Quantitative organization of the excitatory synapses of the primate cerebellar nuclei: further evidence for a specialized architecture underlying the primate cerebellum

The cerebellar intrinsic connectivity is of remarkable regularity with a similar build repeated many times over. However, several modifications of this basic circuitry occur that can provide important clues to evolutionary adaptations. We have observed differences in the wiring of the cerebellar output structures (the deep cerebellar nuclei, DCN) with higher dendritic length density in the phylogenetically newer DCN. In rats, we showed that an increase in wiring is associated with an increase in the presynaptic vesicular glutamate transporter 1 (vGluT1). In this study, we have extended our analysis to the rhesus monkey and can show similarities and differences between the two species. The similarities confirm a higher density in vGluT1+ boutons in the lateral (LN/dentate) and posterior interpositus nucleus compared to the phylogenetically older DCN. In general, we also observe a lower density of vGluT1 and 2+ boutons in the monkey, which however, yields a similar number of excitatory boutons per neuron in both species. The only exception is the vGlut1+ boutons in the macaque LN/dentate, which showed a significantly lower number of vGluT1+ boutons per neuron. We also detected a higher percentage of co-labelled vGluT1 and 2 boutons in the macaque than we found in the rat. In summary, these results confirm that the hyposcalled dendrites of the monkey LN/dentate also show a lower number of vGluT1+ boutons per neuron. These results provide further support of our model relating the dendritic morphology of the LN/dentate neurons to the morphology of the specially enlarged LN/dentate nucleus in primates.

Offline consolidation of spatial memory: Do the cerebellar output circuits play a role? A study utilizing a Morris water maze protocol in male Wistar rats

To address whether the cerebellum takes part to spatial memory consolidation related to navigation, male Wistar rats were trained daily (4 days), in a Morris water maze to found a submerged escape platform by use of distal cues (place training test). Retention of the allocentric map was evaluated in the probe test (without platform), before the place test. Bilateral shutdown of deep cerebellar nuclei was carried by infusion of the GABA-A agonist muscimol (0.25 µl at 1 µg/µl) immediately after each place test. Histology revealed a dorsal dentate nucleus (DDN) group, with muscimol diffusion confined to dentate nuclei, and a ventromedial/dentate nuclei (VMDN) group, with muscimol additionally involving fastigial, interpositus and vestibular nuclei. In the place test, Vehicle, DDN and VMDN groups reduced latency and distance to the platform over the 4 days and within the single day, indicative of efficient acquisition and working memory; navigational trajectories however differed in that, while Vehicle and DDN groups evolved to use direct paths, VMDN group indulged to navigate in proximity to the platform, suggesting an impairment in refining the spatial map. In the probe test VMDN, unlike Vehicle and DDN animals, failed to develop a preference for the quadrant where the platform was previously located, indicating a consolidation deficit.In conclusion, ventromedial cerebellar related structures may contribute to the process of consolidation of an allocentric spatial memory: their inactivation may have impaired the offline integration of idiothetic information with allothetic signals within the navigational network, leading to a coarse resolution map.

3004 Effects of Early Life Stress on Adult Behavioral and Neural Outcomes in Rats

OBJECTIVES/SPECIFIC AIMS: Early life stress is known to greatly impact neurodevelopment during critical periods, conferring risk for various psychopathologies, including the onset and exacerbation of schizophrenia and anxiety disorders. The endocannabinoid system is highly integrated into the stress response and may be one means by which early life stress produces such deleterious effects. Using a naturalistic, ecologically valid animal model, this study explored interactions between the stress response and endocannabinoid systems within the cerebellum, a region dense with the CB1 endocannabinoid receptors and shown to be susceptible to stress. METHODS/STUDY POPULATION: This study explored behavioral and neural impacts of early life stress in Long-Evans rats reared with or without limited access to bedding material during postnatal day (PND) 2-9. Corticosterone (CORT) levels were measured at PND8 and 70. During PND50-70, rats were assessed on Novel Object Recognition to test memory, Rotarod to evaluate cerebellar integrity, Elevated Plus Maze to assay anxiety, Social Preference, and Eyeblink Conditioning, a cerebellar-dependent and endocannabinoid-mediated task. Lipid analysis was performed on PND70 tissue samples of cerebellar interpositus (IP) nucleus via high-performance liquid chromatography and tandem mass spectrometry. RESULTS/ANTICIPATED RESULTS: Both male and female rats experiencing early life stress exhibited significantly impaired recognition memory (N = 16-20/group). Female rats having undergone stress exhibited decreased social preference compared to normally reared females (N = 11/group). Stressed males showed facilitated eyblink conditioning compared to normally reared males (N = 7-9/group). There were no group differences in rotarod or elevated plus maze performance or CORT levels at PND8 or 70 across rearing groups. At PND70, male rats experiencing early life stress exhibited a significant decrease in 2-arachidonoyl glycerol (2-AG) and arachidonic acid levels in the IP nucleus compared to normally reared males (N = 8-9/group). Compared to normally reared females, those experiencing early life stress exhibited a significant increase in prostaglandin E2 levels in the IP nucleus (N = 6-7/group). DISCUSSION/SIGNIFICANCE OF IMPACT: Early life stress, induced by limited bedding, resulted in sex-specific behavioral and lipid impairments. Results suggest that stress causes long-term alterations in endocannabinoid dynamics in males in the cerebellar IP nucleus and sex-related lipids in female cerebellum. These changes may contribute to observed long-term behavioral aberrations. Moreover, findings suggest these behavioral changes may be the result of negative-feedback dysfunction (as evidenced by decreased endocannabinoids in males) or increased neural inflammation or proliferation (as evidenced by increased prostaglandins in females). Future analysis will quantify mRNA and protein for cannabinoid receptors to better characterize aberrations to this system. Moreover, other neural regions dense with cannabinoid receptors (i.e., PFC, hippocampus) will be investigated. This work provides a basis for understanding stress impacts on the development of cognitive deficits observed in psychotic and anxiety disorders. Specifically, facilitation of eyblink conditioning complements research in humans with anxiety disorders. Broadly, understanding stress-related endocannabinoid dysregulation may provide insights into risks for, and the development of, psychopathology and uncover novel therapeutic targets with high translational power.

Muscarinic Receptor Modulation of the Cerebellar Interpositus Nucleus In Vitro

How the cerebellum carries out its functions is not clear, even for its established roles in motor control. In particular, little is known about how the cerebellar nuclei (CN) integrate their synaptic and neuromodulatory inputs to generate cerebellar output. CN neurons receive inhibitory inputs from Purkinje cells, excitatory inputs from mossy fibre and climbing fibre collaterals, as well as a variety of neuromodulatory inputs, including cholinergic inputs. In this study we tested how activation of acetylcholine receptors modulated firing rate, intrinsic properties and synaptic transmission in the CN. Using in vitro whole-cell patch clamp recordings from neurons in the interpositus nucleus, the acetylcholine receptor agonist carbachol was shown to induce a short-term increase in firing rate, increase holding current and decrease input resistance of interpositus CN neurons. Carbachol also induced long-term depression of evoked inhibitory postsynaptic currents and a short-term depression of evoked excitatory postsynaptic currents. All effects were shown to be dependent upon muscarinic acetylcholine receptor activation. Overall, the present study has identified muscarinic receptor activation as a modulator of CN activity.