Development and Optimization of a Multilayer Rat Purkinje Neuron Culture.

Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.

Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration.

OBJECTIVE: Identify the subcellular location and potential binding partners of two cerebellar degeneration-related proteins, CDR2L and CDR2, associated with anti-Yo-mediated paraneoplastic cerebellar degeneration. METHODS: Cancer cells, rat Purkinje neuron cultures, and human cerebellar sections were exposed to cerebrospinal fluid and serum from patients with paraneoplastic cerebellar degeneration with Yo antibodies and with several antibodies against CDR2L and CDR2. We used mass spectrometry-based proteomics, super-resolution microscopy, proximity ligation assay, and co-immunoprecipitation to verify the antibodies and to identify potential binding partners. RESULTS: We confirmed the CDR2L specificity of Yo antibodies by mass spectrometry-based proteomics and found that CDR2L localized to the cytoplasm and CDR2 to the nucleus. CDR2L co-localized with the 40S ribosomal protein S6, while CDR2 co-localized with the nuclear speckle proteins SON, eukaryotic initiation factor 4A-III, and serine/arginine-rich splicing factor 2. INTERPRETATION: We showed that Yo antibodies specifically bind to CDR2L in Purkinje neurons of PCD patients where they potentially interfere with the function of the ribosomal machinery resulting in disrupted mRNA translation and/or protein synthesis. Our findings demonstrating that CDR2L interacts with ribosomal proteins and CDR2 with nuclear speckle proteins is an important step toward understanding PCD pathogenesis.

A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates.

The capacity of pluripotent stem cells both for self-renewal and to differentiate into any cell type have made them a powerful tool for studying human disease. Protocols for efficient differentiation towards cardiomyocytes using defined, serum-free culture medium combined with small molecules have been developed, but thus far, limited to larger formats. We adapted protocols for differentiating human pluripotent stem cells to functional human cardiomyocytes in a 96-well microplate format. The resulting cardiomyocytes expressed cardiac specific markers at the transcriptional and protein levels and had the electrophysiological properties that confirmed the presence of functional cardiomyocytes. We suggest that this protocol provides an incremental improvement and one that reduces the impact of heterogeneity by increasing inter-experimental replicates. We believe that this technique will improve the applicability of these cells for use in developmental biology and mechanistic studies of disease.

CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration.

The pathogenesis of Yo-mediated paraneoplastic cerebellar degeneration (PCD) is unclear. We applied cerebrospinal fluid and serum from PCD patients as well as CDR2 and CDR2L antibodies to neuronal tissue, cancer cell lines, and cells transfected with recombinant CDR2 and CDR2L to elucidate which is the major antigen of Yo antibodies. We found that Yo antibodies bound endogenous CDR2L, but not endogenous CDR2. However, Yo antibodies can bind the recombinant CDR2 protein used in routine clinical testing for these antibodies. Because Yo antibodies only bind endogenous CDR2L, we conclude that CDR2L is the major antigen of Yo antibodies in PCD. ANN NEUROL 2019;86:316-321.

Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis.

Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs. Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.

A novel, diffusely infiltrative xenograft model of human anaplastic oligodendroglioma with mutations in FUBP1, CIC, and IDH1.

Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.

Balancing Arc synthesis, mRNA decay, and proteasomal degradation: maximal protein expression triggered by rapid eye movement sleep-like bursts of muscarinic cholinergic receptor stimulation.

Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

The Arc of synaptic memory.

The immediate early gene Arc is emerging as a versatile, finely tuned system capable of coupling changes in neuronal activity patterns to synaptic plasticity, thereby optimizing information storage in the nervous system. Here, we attempt to overview the Arc system spanning from transcriptional regulation of the Arc gene, to dendritic transport, metabolism, and translation of Arc mRNA, to post-translational modification, localization, and degradation of Arc protein. Within this framework we discuss the function of Arc in regulation of actin cytoskeletal dynamics underlying consolidation of long-term potentiation (LTP) and regulation of AMPA-type glutamate receptor endocytosis underlying long-term depression (LTD) and homeostatic plasticity. Behaviorally, Arc has a key role in consolidation of explicit and implicit forms of memory, with recent work implicating Arc in adaptation to stress as well as maladaptive plasticity connected to drug addiction. Arc holds considerable promise as a "master regulator" of protein synthesis-dependent forms of synaptic plasticity, but the mechanisms that modulate and switch Arc function are only beginning to be elucidated.

SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway.

Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

Improved learning and memory in aged mice deficient in amyloid beta-degrading neutral endopeptidase.

BACKGROUND: Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-beta (Abeta) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. METHODOLOGY/PRINCIPAL FINDINGS: We found that while endogenous Abeta concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Abeta deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Abeta degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. CONCLUSIONS/SIGNIFICANCE: Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases.

Gender-dependent ATPA-induced changes in long-term potentiation in the rat lateral amygdala.

There is increasing evidence that kainate receptors contribute to both postsynaptic and presynaptic signaling not only in the hippocampus but also in the amygdala. The present study demonstrates that low concentrations of the specific kainate GLU(K5) receptor agonist, ATPA, depressed baseline activity in the lateral nucleus of the rat amygdala (LA), induced by stimulation of external capsule fibers or by intranuclear stimulation in horizontal brain slices. ATPA reduced high-frequency-induced long-term potentiation (LTP) in males while it enhanced LTP in females during certain phases of the estrus cycle. In untreated slices from females, LA-LTP differed depending on the phase of the estrus cycle. In addition, we show for the first time that the p38 mitogen-activated protein (MAP) kinase inhibitor, SKF 86002, reduced LA-LTP. In males, the effects of ATPA and SKF 86002 were not additive. To the contrary, in females, the exposure to ATPA in control plus SKF 86002 increases LTP relative to control plus SKF 86002 alone. Thus, we demonstrate that the effectiveness of GLU(K5) stimulation on plasticity changes in the amygdala is gender-dependent and that the MAP kinase pathway might be involved in males.

Activation of kainate GLU(K5) transmission rescues kindling-induced impairment of LTP in the rat lateral amygdala.

The amygdala is a component of the limbic system that plays a central role in emotional behavior and certain psychiatric diseases. Pathophysiological alterations of neuronal excitability in the amygdala are characteristic features of temporal lobe epilepsy and certain (epilepsy accompanying) psychiatric illnesses such as anxiety and depressive disorders. The role of kainate receptors in the activity of synaptic networks, in brain function, and diseases is still poorly understood. Various kainate receptor subtypes have been shown to contribute to synaptic transmission and modulate presynaptic release of glutamate and gamma-aminobutyric acid (GABA). Several lines of evidence point to the importance of GLU(K5) kainate receptors in epilepsy. In this study we investigated the role of specific GLU(K5) kainate receptor in the lateral nucleus of the amygdala (LA). The cellular mechanisms for emotional learning in the amygdala are believed to be the result of changes in synaptic transmission efficacy, similar to long-term potentiation (LTP). Here, we used both field potential and intracellular recordings in horizontal rat amygdala slices, and showed that LTP in the LA, induced by high-frequency stimulation of afferents running within LA, is impaired 48 h after the last induced seizure. This kindling-induced impairment was reversed by the specific kainate GLU(K5) agonist ATPA. Partial blockade of GABAergic transmission with the specific GABA(A) receptor antagonist SR95531 also significantly facilitated the induction of early LA-LTP, but only partially abolished the kindling-induced impairment of LA-LTP. This study shows that the stimulation of the GLU(K5) kainate receptor subtype rescues the kindling-induced impairment of LA-LTP at least within 48 h after the last seizure. Therefore, GLU(K5) kainate receptor subunits are involved in kindling-induced plasticity changes in the amygdala.

Input-specific long-term potentiation in the rat lateral amygdala of horizontal slices.

Long-term potentiation (LTP) at input synapses to the lateral nucleus of the amygdala (LA) is a candidate mechanism for memory storage during fear learning. Cellular mechanisms of LTP have been nearly exclusively investigated in coronal brain slices. In our experiments, we used a horizontal brain slice preparation of rats that preserved most of the connections to cortical areas and the hippocampus. The stimulation electrodes were located either within the external capsule (EC) or the LA. The aim of the present study was to investigate the mechanisms of LTP induced either by weak theta burst stimulation (TBS) or strong high frequency stimulation (HFS) using the two different stimulation sites. Whereas both TBS and HFS of afferences running through the LA induced stable LTP, TBS failed to induce LTP of EC-inputs to the LA. The present findings also show that LTP in the LA exhibits vulnerability at different time windows after induction. The time window was dependent on the kind of stimulated afferences. Later LTP becomes resistant to disruption by low frequency stimulation. We could show that both used inputs depended on NMDA receptors for LTP-induction. LTP induced by stimulation of fibers within the LA was not altered by nifedipine (10 microM). In contrast, EC-induced LTP was dependent on L-type voltage-gated calcium channels (VGCC). Finally, we found a higher magnitude of LTP in females using TBS, whereas HFS did not cause gender-specific differences. Our study supports the conclusion that the form of LA-LTP depend on which afferences are activated and what pattern of stimulation is used to induce LTP.

Kindling-induced changes in plasticity of the rat amygdala and hippocampus.

Temporal lobe epilepsy (TLE) is often accompanied by interictal behavioral abnormalities, such as fear and memory impairment. To identify possible underlying substrates, we analyzed long-term synaptic plasticity in two relevant brain regions, the lateral amygdala (LA) and the CA1 region of the hippocampus, in the kindling model of epilepsy. Wistar rats were kindled through daily administration of brief electrical stimulations to the left basolateral nucleus of the amygdala. Field potential recordings were performed in slices obtained from kindled rats 48 h after the last induced seizure, and in slices from sham-implanted and nonimplanted controls. Kindling resulted in a significant impairment of long-term potentiation (LTP) in both the LA and the CA1, the magnitude of which was dependent on the number of prior stage V seizures. Saturation of CA1-LTP, assessed through repeated spaced delivery of high-frequency stimulation, occurred at lower levels in kindled compared to sham-implanted animals, consistent with the hypothesis of reduced capacity of further synaptic strengthening. Furthermore, theta pulse stimulation elicited long-term depression in the amygdala in nonimplanted and sham-implanted controls, whereas the same stimulation protocol stimulation caused LTP in kindled rats. In conclusion, kindling differentially affects the magnitude, saturation, and polarity of LTP in the CA1 and LA, respectively, most likely indicating an activity-dependent mechanism in the context of synaptic metaplasticity.

Repeated ethanol exposure and withdrawal impairs human fear conditioning and depresses long-term potentiation in rat amygdala and hippocampus.

BACKGROUND: In rats, repeated episodes of alcohol consumption and withdrawal (RWD) impair fear conditioning to discrete cues. METHODS: Fear conditioning was measured in human binge drinkers as the increased startle response in the presence of a CS+ conditioned to aversive white noise. Secondly, the ability of tone CSs, paired with footshock, to induce c-fos expression, a marker of neuronal activity, in limbic structures subserving emotion was studied in rats. Additionally, consequences of RWD on subsequent induction of long term potentiation (LTP) in external capsule/lateral amygdala and Schaffer collateral/hippocampus CA1 pathways were studied in rat brain slices. RESULTS: Fear conditioning was impaired in young human binge drinkers. The ability of fear-conditioned CSs to increase c-fos expression in limbic brain areas was reduced following RWD, as was LTP induction. Rats conditioned prior to RWD, following RWD showed generalization of conditioned fear from the tone CS+ to a neutral control stimulus, and a novel tone. CONCLUSIONS: Binge-like drinking impairs fear conditioning, reduces LTP, and results in inappropriate generalization of learned fear responses. We propose a mechanism whereby RWD-induced synaptic plasticity reduces capacity for future learning, while allowing unconditioned stimuli access to neuronal pathways underlying conditioned fear.

Alcohol withdrawal and conditioning.

This review contains the proceedings from a symposium held at the RSA conference in 2003 on "Alcohol Withdrawal and Conditioning." The presentations covered a range of interactions between conditioning and alcohol withdrawal, in both animal behavior and the clinic. Dr. D.N. Stephens first described his studies exploring the consequences of alcohol dependence and repeated experience of withdrawal on the conditioning process. His data suggested that repeated withdrawal from moderate alcohol intake impairs amygdala-dependent mechanisms for learning about aversive events. Dr. H. Becker then detailed studies examining the consequences of repeated ethanol withdrawal experience on subsequent ethanol drinking behavior in mice, and conditions in which motivational properties of odor cues that are associated with different phases of ethanol withdrawal influence such relapse behavior. The data suggested that cues associated with acute withdrawal or "recovery" from withdrawal may serve as modulating factors in influencing subsequent ethanol drinking behavior, and that the timing of the cues determines their consequences. Dr. F. Weiss described recent findings from animal models of relapse that suggested the efficacy of alcohol-associated contextual stimuli in eliciting alcohol-seeking behavior resembles the endurance of conditioned cue reactivity and cue-induced cocaine craving in humans. The interactive effects of stress and ethanol-related environmental stimuli were found to be dependent on concurrent activation of endogenous opioid and corticotropin-releasing factor systems. Conditioning factors (i.e., exposure to drug-associated stimuli) and stress could therefore interact to augment vulnerability to relapse. Dr. C. Drummond then addressed the clinical aspects of conditioning during alcohol withdrawal and described studies showing exposure of alcoholics to alcohol-related cues elicited greater subjective and physiological responses than exposure to neutral cues. The former responsivity showed a relationship with a measure of motivation to drink alcohol. Finally, Dr. C. Cunningham provided a summary of the concepts involved in the presentations and discussed the conditioning processes that affect behavior during and after alcohol withdrawal.

Long-term depression in horizontal slices of the rat lateral amygdala.

Long-term depression (LTD) is an enduring decrease in synaptic efficacy and is thought to underlie memory. In contrast to investigations of plasticity mechanisms in the amygdala in rat coronal slices, this study was done in horizontal slices. Field excitatory postsynaptic potentials (fEPSPs) and EPSPs, respectively, were recorded extracellularly and intracellularly from the lateral nucleus of the amygdala (LA). We show that low-frequency stimulation (LFS) induces LTD in the LA, when stimulation electrodes were located in the LA. No significant differences were found between females and males. In dependence of strain variations, a reduction of GABAergic inhibition either reduced the magnitude of LTD or was a prerequisite for the induction of extracellularly recorded LA-LTD. Theta pulse stimulation (TPS) of afferents within the LA caused a weaker LTD than LFS. Theta burst stimulation (TBS) given 20 min after the end of LFS reversed LTD, whereas high-frequency stimulation (HFS) resulted in long-term potentiation (LTP) that was significantly stronger than that obtained in naive slices. Therefore, primed induction of LTD facilitates high-frequency-induced LTP in the rat lateral amygdala. NMDARs as well as group II mGluRs were involved in the mediation of LA-LTD. In contrast to data obtained by stimulation of afferents running within the LA, LFS of the external capsule fibers induced a weak LA-LTD, and TPS was not able to induce LTD. This study showed for the first time that LTD can be induced in the LA by standard LFS (900 pulses at 1 Hz) and that LTP stimuli reversed LTD. The results also provide further evidence for the broad sensitivity of synaptic plasticity mechanisms to the history of prior activity.