Yoon H. Cho

Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells

We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.

Kainate receptors act as conditional amplifiers of spike transmission at hippocampal mossy fiber synapses.

Hippocampal mossy fiber (Mf) synapses are viewed as conditional detonators, assisting CA3 cells in complex network functions. By analyzing mice deficient for GluK2 (GluR6), GluK3 (GluR7) and GluK5 (KA2) genes we show that kainate receptors (KARs) play a crucial role in the control of synaptic integration and spike transmission efficacy at Mf synapses. We dissected out the role of the different KAR functions at Mf synapses and we show that presynaptic and postsynaptic KARs concur to amplify unitary Mf synaptic inputs to trigger spike discharge within a wide range of frequencies (from 1 to 50 Hz). Moreover, KARs strongly favor spike transmission in response to patterns of presynaptic activity mimicking in vivo dentate granule cell activity. By amplifying spike transmission, KARs also facilitate the induction of associative long-term potentiation in CA3. Hence the actions of KARs as amplifiers of spike transmission contribute largely to the “conditional detonator” function of Mf synapses and are likely important for spatial information processing.

Spatial Location Learning in Mice with Ibotenate Lesions of Entorhinal Cortex or Subiculum

This study examined the effects of ibotenate lesions of either the entorhinal cortex (EC) or the subiculum (SUB) on the ability of mice to memorize a single spatial location (initial discrimination), and on their capacity to switch to a new location (transfer) following the initial learning in an eight-arm radial maze. Results indicated that mice with ibotenate lesions of the EC or SUB were impaired in postoperative acquisition of the spatial discrimination task, making more reference, but not working memory, errors and displaying fewer first correct response trials than sham-operated control mice. Furthermore, additional damage to the ventral hippocampus exacerbated the impairment of performance induced by lesions of the SUB alone. In addition, all mice, except for the combined lesion group, exhibited similar performance levels when they were trained to choose another arm of the maze that had not previously been baited (transfer). These findings suggest that both the EC and the SUB play important roles in spatial information processing in mice.

Changes in striatal procedural memory coding correlate with learning deficits in a mouse model of Huntington disease.

In hereditary neurodegenerative Huntington disease (HD), early cognitive impairments before motor deficits have been hypothesized to result from dysfunction in the striatum and cortex before degeneration. To test this hypothesis, we examined the firing properties of single cells and local field activity in the striatum and cortex of pre–motor-symptomatic R6/1 transgenic mice while they were engaged in a procedural learning task, the performance on which typically depends on the integrity of striatum and basal ganglia. Here, we report that a dramatically diminished recruitment of the vulnerable striatal projection cells, but not local interneurons, of R6/1 mice in coding for the task, compared with WT littermates, is associated with severe deficits in procedural learning. In addition, both the striatum and cortex in these mice showed a unique oscillation at high γ-frequency. These data provide crucial information on the in vivo cellular processes in the corticostriatal pathway through which the HD mutation exerts its effects on cognitive abilities in early HD.

Sex-Dependent Changes in Social Behaviors in Motor Pre-Symptomatic R6/1 Mice

Background The R6/1 mouse line is one of the most widely employed models of Huntington Disease (HD), a complex syndrome characterized by motor and non-motor deficits. Surprisingly, its behavioral phenotype during the early phases of the pathology when the motor impairments are not manifest yet has been poorly investigated. It is also not clear whether the expression of HD-like symptoms at the pre-motor stage in this mouse model differs between the two sexes. Methods Male and female 12 weeks-old R6/1 mice and their wild-type littermates were tested on a battery of tests modeling some of the major neuropsychiatric non-motor symptoms of HD: alterations in social interest, social interaction and communication, as well as disturbances in prepulse inhibition of the acoustic startle response (PPI) and circadian patterns of activity. The lack of motor symptoms was confirmed during the entire experimental period by means of the tail test for clasping. Results R6/1 mice displayed marked alterations in all social behaviors which were mainly observed in males. Male R6/1 animals were also the only ones showing reduced body weight. Both male and female transgenic mice displayed mild alterations in the circadian activity patterns, but no deficits in PPI. Conclusions These results demonstrate the validity of the R6/1 mouse in mimicking selected neuropsychiatric symptoms of HD, the social deficits being the clearest markers of the pre-motor phase of the pathology. Furthermore, our data suggest that male R6/1 mice are more suitable for future studies on the early stages of HD.

Design of a twin tetrode microdrive and headstage for hippocampal single unit recordings in behaving mice.

A new, easy to construct electrode, microdrive and headstage for electrophysiological recording system which is specifically adapted for freely behaving mice is described. The system uses printed circuit boards and light, flexible cables to enable the animals free movement for behavioral testing. A clip attachment system permits rapid and secure connection of the headstage and cables to the microdrive assembly on the animals head. The current system provides eight recording channels, but the design can be modified to accommodate additional channels.

Differential effects of ibotenate lesions of the CA1 subfield of the hippocampus on a delayed-nonmatching-to-place task as a function of preoperative training in mice

This study examined the effects of ibotenate lesions of the hippocampal CA1 subfield on spatial working memory (delayed-nonmatching-to-place [DNMTP] task) as a function of the existence of preoperative training in mice. The task was varied in its level of difficulty according to either the number of interpolated arm visits (occupied delays) or the length of free time delays imposed between place sample presentation and subsequent recognition. Results indicate that when the tasks were first well learned and the CA1 subfield was subsequently lesioned, only slight impairments were observed during both reacquisition of the DNMTP rule and the problem containing five interpolated visits. When CA1 pyramidal cells were lesioned prior to any training, CA1-lesioned subjects showed general performance deficits regardless of the delay studied (occupied or free). A shift from isolated rule task to the same task tested simultaneously with more difficult variants produced marked performance deficits even for the previously mastered DNMTP rule task. These results suggest that preoperative training reduces subsequent lesion-induced memory deficits and modifies the pattern of postoperative working-memory performance; lesions of the hippocampal CA1 field contribute to the impairments of not only mnemonic capability per se but also reference memory components of the DNMTP task underlying the procedural and cognitive demands required for correct performance of the task.

Differential involvement of prefrontal cortex, striatum, and hippocampus in DRL performance in mice

Behavioral effects of neurotoxic lesions of the hippocampus, medial prefrontal (prelimbic, infralimbic and anterior cingulate) cortex or dorsal striatum were assessed using a DRL-10s schedule in mice. Post-operative acquisition data indicate that mice with hippocampal, but not prefrontal or striatal lesions received fewer reinforcements during daily 30-min sessions, and were less efficient in the timing of their responses. Additional analysis of inter-response-time (IRT) distributions revealed that the responses of hippocampal-lesioned mice exhibited undistinguishable responses for short IRTs (up to 9s). In addition, prefrontal-lesioned mice demonstrated a degradation of performance with further testing, and a flattened IRT distribution at late test phase, while striatal-lesioned mice behaved similarly to sham-lesioned mice. These results are interpreted in terms of known functions of the hippocampus in behavioral inhibition, and of the prefrontal cortex in executive control/decision making (and time production).

The Entorhinal Cortex and a Delayed Non-matching-to-place Task in Mice: Emphasis on Preoperative Training and Presentation Procedure

This study examined the effects of ibotenate lesions of the entorhinal cortex (EC) on performance of a spatial recognition memory task, the delayed non‐matching‐to‐place task (DNMTP), varying in level of difficulty according to the number of interpolated arm visits between sample‐place presentation and subsequent recognition in mice. Results of experiment 1, designed to test the rate of acquisition of the task, showed that experimental animals were impaired in the basic non‐matching task. However, with further training they were able to learn the task. Impairments were also observed when the amount of interpolating information inserted was gradually increased, and then all problem difficulties were pseudorandomly tested. There was no measurable recovery of function over time when the animals were retested on the task ∼1 month later. Experiment 2 showed that the animals that received extensive training on the task prior to lesions of the EC were only transiently impaired in the DNMTP task. These data suggest that the EC plays an important role in acquisition rather than retention of the spatial recognition memory task.

Sleep Physiology Alterations Precede Plethoric Phenotypic Changes in R6/1 Huntington's Disease Mice

In hereditary neurodegenerative Huntington’s disease (HD), there exists a growing consideration that sleep and circadian dysregulations may be important symptoms. It is not known, however, whether sleep abnormalities contribute to other behavioral deficits in HD patients and mouse models. To determine the precise chronology for sleep physiology alterations and other sensory, motor, psychiatric and cognitive symptoms of HD, the same R6/1 HD transgenics and their wild-type littermates were recorded monthly for sleep electroencephalogram (EEG) together with a wide range of behavioral tests according to a longitudinal plan. We found an early and progressive deterioration of both sleep architecture and EEG brain rhythms in R6/1 mice, which are correlated timely with their spatial working memory impairments. Sleep fragmentation and memory impairments were accompanied by the loss of delta (1-4Hz) power in the transgenic mice, the magnitude of which increased with age and disease progression. These precocious sleep and cognitive impairments were followed by deficits in social behavior, sensory and motor abilities. Our data confirm the existence and importance of sleep physiology alterations in the widely used R6/1 mouse line and highlight their precedence over other plethoric phenotypic changes. The brainwave abnormalities, may represent a novel biomarker and point to innovative therapeutic interventions against HD.