Extending the realm of membrane capacitance measurements to nerve terminals with complex morphologies

Almost thirty years ago, Neher & Marty (1982) showed that time-resolved membrane capacitance (Cm) measurements have the sensitivity to monitor single vesicle fusion (exocytosis) events and subsequent membrane retrieval (endocytosis) in living neuroendocrine cells. Almers & Neher (1987) then showed that while Cm measurements are exquisitely sensitive to exocytosis, they are not a very specific measure of secretion, since non-exocytotic processes like membrane pinocytosis and/or gating-charge movements of transmembrane proteins can produce Cm changes, and moreover, these changes may be insidiously Ca2+ dependent. Since those pioneering papers, Cm measurements have been extended to several different types of excitable and non-excitable secretory cells, and also to sensory neurons, like photoreceptors (Rieke & Schwartz, 1994; Bartoletti et al. 2010), and to epithelial cells, like hair cells (Parsons et al. 1994). Most of this work has employed quasi-spherical, isopotential and one-compartment secretory cells, which are ideally suited for this measurement technique. Conventional neurons, however, have nerve terminals with complex geometries and multiple bouton-type endings. The first paper to extend Cm measurements to nerve terminals in brain slices was by Hsu & Jackson (1996) using pituitary nerve varicosities on an unmyelinated axon (beads-on-a-string morphology). Cm measurements were then extended by Mennerick et al. (1997) to a more complex secretory cell: the intact goldfish retinal bipolar neuron, which contains multiple ribbon-type active zones in its large synaptic terminal. A two-compartmental RC circuit model was developed for bipolar cells with soma and terminal compartments connected by a thin cylindrical axonal resistance. It was shown that Cm measurements were possible with the patch pipette located in either the soma or terminal, provided one followed certain precautions to avoid potential artifacts. Capacitance measurements are also feasible in another giant and specialized nerve terminal with a large readily releasable pool of vesicles, the calyx of Held (Sun & Wu, 2001; Taschenberger et al. 2002). More recently, Hallermann et al. (2003) showed how to successfully perform Cm measurements at hippocampal mossy fibre bouton-type terminals (∼3 μm diameter), which contain conventional active zones. This seminal paper showed that a careful reconstruction of nerve terminal and axonal morphology, followed by a morphology-based equivalent electrical model of the nerve terminal and axon, was necessary to implement reliable Cm measurements in more conventional CNS nerve terminals. Now a paper in The Journal of Physiology by Oltedal & Hartveit (2010) advances this steady progress in extending the realm of Cm measurements by showing that it is possible to record Cm changes directly from small mammalian rod bipolar cell synaptic terminals. The authors use rat rod bipolar cells that have ∼45-μm-long axons and two to three axon terminal boutons (a grapes-on-a-vine morphology; the size of the terminal bouton attached to the recording pipette is ∼2–4 μm). Their computer simulations suggest that in order to detect a Cm increase at the axon terminal with high reliability, one should either use soma-end recordings with low lock-in sine wave frequency (∼100 Hz), or the more technically challenging direct terminal-end recordings with high lock-in sine wave frequency (∼2 kHz). An effective way to minimize noise in Cm measurements is to increase the amplitude of the lock-in voltage-clamp sine wave (Lindau & Neher, 1988). But the higher the amplitude of the sine wave, the more likely it is to activate voltage-gated currents, and the resulting non-linearities may produce Cm artifacts. Interestingly, the authors find that a high-frequency sine wave (∼2 kHz) at a Vhold of −65 mV with an amplitude of ±30 mV (peak to peak) produces very little activation of voltage-gated Ca2+ channels. This type of lock-in stimulus may thus be used for reliable Cm measurements in retinal bipolar cells. Using this approach the authors find a high rate of exocytosis following the activation of Ca2+ current and strong short-term synaptic depression mediated by the rapid depletion of a relatively small vesicle pool. Glutamate release thus has a highly transient nature at this synapse following a strong stimulus. These results are in remarkable quantitative agreement with previous paired recordings between rat rod bipolar cells and amacrine cells (Singer & Diamond, 2003). Nevertheless, rather contradictory results were also reported from acutely dissociated mouse rod bipolar cells (Zhou et al. 2006), which showed no statistical difference of Cm changes in soma-end and terminal-end recordings using a 1 kHz lock-in sine wave. In order to understand the apparent contradiction, the authors also built a model for the mouse rod bipolar cell based on its morphology (a relatively short and thick axon: 32 μm length, 1 μm diameter) and showed the feasibility to Cm measurements from soma-end recordings. Taken together, geometric considerations and a judicious choice of lock-in sine wave parameters should become necessary prerequisites for future Cm measurements in more complex nerve terminals. Remarkably, in the same issue of The Journal of Physiology, Palmer (2010) also reports Cm measurements from bipolar cells, but couples these with paired recordings between bipolar cell terminals and ganglion cell layer neurons in goldfish retina. These tour-de-force and elegant experiments reveal that a presynaptic depolarizing step to the bipolar cell terminal evokes both AMPA/kainate receptor- and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in connected ganglion cell layer neurons. A linear relationship between bipolar cell terminal exocytosis (Cm jumps) and total EPSC charge transfer indicates that glutamate receptor saturation and desensitization are not present in this ribbon-type synapse. Finally, the quantal content of the evoked EPSCs was estimated by the analysis of miniature EPSCs in ganglion cell layer neurons. The readily releasable pool of vesicles was composed of about 23 vesicles, which corresponds to the number of vesicles docked at the bottom row of the synaptic ribbon. The ganglion cell layer neuron is thus apparently able of sense the full complement of fusing synaptic vesicles in the dyad synapse formed by a single synaptic ribbon and two postsynaptic neurons. In summary, the careful and thorough analysis of Oltedal & Hartveit (2010) provides us now with a detailed roadmap for future Cm measurements of nerve terminals with more complex morphologies, while the paired recordings of Palmer (2010) provide an independent confirmation that these Cm measurements are indeed bona fide proxies of transmitter secretion. So twenty years after a lecture at Stockholm where Erwin Neher challenged us to extend Cm measurements to nerve terminals by stating: ‘Unfortunately, nerve terminals are usually not accessible to the kind of biophysical investigations described here (i.e. Cm measurements)’, we are finally beginning to meet that challenge.