A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.
Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high-information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes.
In an attempt to integrate biological components with silicon-based devices and systems, artificial silane surfaces have been successfully used to grow motoneurons in a defined environment. In this study we characterized the morphology and electrophysiology of purified rat embryonic (E14) motoneurons grown on a self-assembled monolayer (SAM) of N-1[3-(trimethoxysilyl)propyl]diethylenetriamine (DETA) versus that on ornithine/laminin surfaces in serum-free media. On DETA motoneurons were flat and grew more processes, whereas on ornithine/laminin they tended to aggregate. The membrane time constant, a characteristic associated with electrotonic compactness, was significantly longer for motoneurons grown on DETA. Other electrophysiological parameters were similar for the motoneurons on the different surfaces. This is the first study where purified ventral horn motoneurons were cultured in a completely defined (nonbiological surface, serum-free) environment.
Specific neuronal subtypes, especially motoneurons (MNs), derived from human stem cells provide a significant therapeutic potential for spinal cord diseases such as ALS and spinal cord injury. So far in vitro, MNs have only been successfully induced from embryonic stem cells (hESC) and human fetal cortical progenitors. Although neural progenitors from spinal cord would be a likely source for generating MNs, there has been no study reporting successful in vitro differentiation of MNs from spinal cord progenitors. This study first characterized a polyclonal spinal cord stem cell line isolated from an eight-week fetus. Then a paradigm was introduced to successfully induce MNs from this cell line, which was demonstrated by the immuostaining using the MN markers HB9, Islet1 and ChAT. The combination of HB9 and ChAT (Choline Acetyl Transferase) immunostainings indicated that ~20% of the cells were MNs after this induction protocol. The presence of other cell types in the differentiated culture was also analyzed. Finally, the electrophysiological properties of these differentiated MNs were characterized to confirm their functional integrity. The majority of these MNs fired repetitive action potentials (APs), which is an indicator of functional maturation. The recordings of spontaneous Excitatory Postsynaptic Currents (EPSCs) confirmed the formation of synapses onto these MNs. This study reports the first successful differentiation of MNs from human spinal cord stem cells in vitro, providing a novel approach for obtaining functional MNs when designing the therapeutic strategy for spinal cord diseases or injuries.
Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber-granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13-16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes ( approximately 30 pA), 20-80% rise times (approximately 1.2 ms), decay time constants ( approximately 10 ms), and half-widths (approximately 8 ms). The mean failure rate was high (approximately 70%) in both groups, and in both groups activation of N-methyl-D-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber-granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber-granule cell synapses.
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