Response of single spinal motoneurones to transcranial magnetic simulation in healthy subjects and patients with upper motor neurone disorders

The problem addressed by this study was: How does the human corticospinal tract influence the discharge of spinal motoneurones and what are the effects of neurological disease? The method employed was to study the firing probability of 78 tonically active single motor units of the upper limb following transcranial magnetic stimulation. This was performed in healthy subjects and in a group of patients with different upper motor neurone (UMN) disorders. The inducing current flowed in an anticlockwise direction through a circular coil which was positioned tangentially at the vertex. Two peaks were produced in the peri-stimulus time histogram. The primary peak (PP) had an onset latency in healthy subjects ranging from 13 ms (deltoid and biceps) to 31 ms (first dorsal interosseous muscle) (FDI) and had a short duration of 4.6 ±1.7 ms (mean ± SD). PP frequently consisted of 1-3 sub-peaks, with a mean intermodal interval of 1.4 ms for FDI and 2.9 ms for forearm and upper arm muscles. This interval probably reflects the maximal rise time of one in a sequence of excitatory postsynaptic potentials (EPSPs) at the motoneurone. An increase either in the interval between the stimulus and the preceding voluntary discharge, or in the intensity of stimulation, raised the probability of discharges occurring within PP and influenced their latency. The secondary peak (SP) had an onset latency in FDI ranging from 56-90 ms and a long duration of 20.9 ±12.0 ms. Evidence suggests that SP was caused by the rising phase of a late EPSP mediated via a pathway which included a peripheral afferent component. When compared with healthy subjects, PP in UMN patients was found to be either normal, absent, delayed and dispersed (by up to 28 ms and 21 ms, respectively) or found to consist of sub-peaks with abnormally long inter- modal intervals. These findings suggest specific mechanisms including cortical inexcitability, variable degrees of slowing in the velocity of propagation in descending fibres, frequency dependent conduction block, delay between EPSPs caused by the operation of more than one pathway and ineffective spatial or temporal summation at the spinal motoneurone

Sound Encoding in the Mouse Cochlea: Molecular Physiology and Optogenetic Stimulation

Afferent synapses between inner hair cells (IHCs) and spiral ganglion neurons in the cochlea translate sound information into a discrete spike code, providing us the opportunity to directly observe the output of the cochlea. The availability of mutant strains with genetic hearing impairment makes the mouse a valuable species to investigate the molecular mechanisms of cochlear function. In this thesis, mouse was used as a model species to study cochlear sound encoding by recording single unit activities from auditory nerve fibers (ANFs) in vivo. First, developmental changes of ANF responses before and after hearing onset were characterized as an introduction on how normal ANF responses mature during the early postnatal age. Spontaneous bursting activity from ANFs/cochlear nucleus neurons was observed before hearing onset. After hearing onset, the average spontaneous and evoked spike rates of single ANFs increased, while tuning threshold and frequency selectivity improved between p14-15 to p20-21. To gain insight into the role of synaptic organization in cochlear and ANF function, mice carrying targeted mutations of presynaptic scaffold protein Bassoon were analyzed. IHCs of mice that are deficient of the central portion of the presynaptic scaffold protein Bassoon (BSNΔEx4/5) were previously shown to mostly lack synaptic ribbons and to have a smaller readily releasable pool of synaptic vesicles and reduced exocytosis, resulting in lower firing rates of ANFs. To distinguish better between the effects of the Bassoon mutation and those of the loss of the synaptic ribbon, the BSNΔEx4/5 phenotype was compared with that of a newly generated gene trap mutant (BSNgt), which has an intermediate phenotype in terms of the fraction of ribbon occupied active zones, presumably due to leaky expression of a small amount of Bassoon protein. The mean distance between the remaining ribbons and the active zone was greater in BSNgt than in wildtype and the synaptic calcium channel clusters had reduced immunostaining reactivity. The BSNgt IHCs showed a slightly less severe reduction of peak Ca2+ currents and sustained exocytosis compared to BSNΔEx4/5. However, IHC fast exocytosis and single unit responses of ANFs showed almost identical response properties between the two mutants. These data suggest that it is not the physical presence or absence of a synaptic ribbon but rather the disruption of presynaptic ultrastructure (e.g. abnormal calcium channel clustering, looser ribbon anchorage) that mainly determines the synaptic phenotype of Bassoon mutants.  Next, the ANF responses of Black Swiss mice (BLSW) were characterized. BLSW mice have inherited early onset sensorineural hearing loss and susceptibility to audiogenic seizures due to a mutation in the Gipc3 gene. BLSW ANFs showed higher tuning thresholds and broader frequency selectivity, which is consistent with a previous report of OHC dysfunction. Interestingly, BLSW ANFs had elevated spontaneous discharge activity, indicating that Gipc3 is a key molecular player not only for normal OHC but also for IHC function.  Upon hearing loss due to HC dysfunction, the remaining ANFs can be electrically stimulated to restore the sense of hearing. The number of useful frequency channels using electrical stimulation is limited by the spread of current. Focused optical stimulation may allow for more selective activation of ANFs compared to electrical stimulation. In the last part of the thesis, spiking activity was measured in response to laser light stimulation in ANFs and cochlear nucleus neurons of mice with constitutive expression of the light-gated ion channel Channelrhodopsin-2 and virus-mediated expression of the faster ChR2 variant CatCh.