Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

Use of magnetic stimulation to elicit motor evoked potentials, somatosensory evoked potentials, and H-reflexes in non-sedated rodents.

Assessment of locomotor function of rodents may be supplemented using electrophysiological tests which monitor the integrity of ascending and descending tracts as well as the focal circuitry of the spinal cord in non-sedated rodents. Magnetically induced SSEPs (M-SSEPs) were elicited in rats by activating the hindpaw using magnetic stimulation (MS). M-SSEP response latencies were slightly longer than those elicited by electrical stimulation. M-SSEPs were eliminated following selective dorsal column lacerations of the spinal cord, indicating that they were transmitted via this tract. Magnetically induced motor evoked potentials (M-MEPs) were elicited in mice following transcranial MS and recorded from the gastrocnemius muscles. M-MEPs performed on myelin deficient mice demonstrated longer onset latencies and smaller amplitudes than in wild-type mice. Magnetically induced H-reflexes (MH-reflexes) which assess local circuitry in the lumbosacral area of the spinal cord were performed in rats. This response disappeared following an L3 contusion spinal cord injury, however, kainic acid (KA) injection at L3, known to selectively destroy interneurons, caused a shorter latency and an increase in the amplitude of the MH-reflex. M-SSEPs and MH-reflexes in rats and M-MEPs in mice compliment locomotor evaluation in assessing the functional integrity of the spinal cord under normal and pathological conditions in the non-sedated animal.

Expression of Zfhep/deltaEF1 protein in palate, neural progenitors, and differentiated neurons.

Zfhep/deltaEF1 is essential for embryonic development. We have investigated the expression pattern of Zfhep protein during mouse embryogenesis. We show expression of Zfhep in the mesenchyme of the palatal shelves, establishing concordance of expression with the reported cleft palate of the deltaEF1-null mice. Zfhep protein is strongly expressed in proliferating progenitors of the nervous system. In most regions of the brain, post-mitotic cells stop expressing Zfhep when they migrate out of the ventricular zone (VZ) and differentiate. However, in the hindbrain, Zfhep protein is also highly expressed in post-mitotic migratory neuronal cells of the precerebellar extramural stream that arise from the neuroepithelium adjacent to the lower rhombic lip. Also, Zfhep is expressed as cells migrate from a narrow region of the pons VZ towards the trigeminal nucleus. Co-expression with Islet1 shows that Zfhep is expressed in motor neurons of the trigeminal nucleus of the pons, but not in the inferior olive motor neurons at E12.5. Therefore, Zfhep is strongly expressed in a tightly regulated pattern in proliferating neural stem cells and a subset of neurons. Zfhep protein is also strongly expressed in trigeminal ganglia, and is moderately expressed in other cranial ganglia. In vitro studies have implicated Zfhep as a repressor of myogenesis, however, we find that Zfhep protein expression increases during muscle differentiation.