Exo-endocytosis at mossy fiber terminals: Toward capacitance measurements in cells with arbitrary geometry Christopher Kushmerick and Henrique von Gersdorff* The Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 xocytosis and endocytosis are real time as a decrease in membrane ments have been made on secretory ubiquitous cellular phenomena capacitance back to baseline resting lev- cells for which the compact isopotential necessary for diverse functions els (6–9). approximation seems, prima facie,tobe such as secretion, internal sig- Most measurements obtained to date justified, including adrenal chromaffin Enaling, protein traffic, and motility. have relied on one of two general tech- cells (10), mast cells (11), and neuroen- Many different techniques have been niques to relate membrane current to docrine cells (12), which secrete via developed to assay exocytosis and endo- capacitance (6, 9). Time-domain meth- large dense-core vesicles. In addition, cytosis, but to date only electrical mea- ods use the amplitude and time course small clear-core synaptic vesicle fusion surements of plasma membrane capaci- of membrane current relaxations after and membrane retrieval have been mea- tance have had the time resolution step changes in electrical potential to sured from retinal bipolar cell terminals necessary to capture both the fusion and determine cell membrane parameters. (2, 3, 13), hair cells (4, 5), and photore- reuptake of small clear-core vesicle ceptors (14). However, these sensory membrane during fast neurotransmis- neurons contain nonconventional rib- sion. In this issue of PNAS, Hallermann bon-type active zones (3, 13, 15). et al. (1) present capacitance measure- These are the first Recently, attempts have been made to ments from hippocampal mossy fiber measure exocytosis in cells with complex nerve terminals during stimulated exocy- membrane capacitance geometry and multiple electrical com- tosis. These are the first time-resolved partments by using capacitance mea- membrane capacitance measurements measurements from surements. Hsu and Jackson (16) first from bona fide (albeit relatively large) bona fide bouton-type showed that capacitance measurements bouton-type synaptic terminals, with di- are possible in pituitary slices from ameters of Ϸ3 ␮m and a resting capaci- synaptic terminals. nerve varicosities (with attached axons) tance of Ϸ1 pF. The results obtained that secrete both via dense- and clear- not only provide important data on ba- core vesicles (17). Mennerick et al. (18) sic synaptic properties of this nerve ter- used the time-domain method to analyze Frequency-domain methods use the minal, in particular on the vesicle pool the electrotonic structure of the intact phase shift between an applied sinusoi- size and the question of multivesicular goldfish retinal bipolar cell, consisting of dal membrane potential and the induced release, but also provide an example of a soma and terminal connected by a current to calculate the complex imped- how to extend capacitance measure- short axon. They showed that a two- ance of the circuit. In both techniques ments from cells with simple and com- compartment model adequately de- voltage-dependent conductances are pact geometry to more general classes scribed their recordings and, under the avoided (pharmacologically and by care- of neurons and nerve terminals. assumption of high membrane resis- ful choice of membrane potential) such The accessibility of exocytosis and tance, allowed for unique determination that only passive (capacitive and leak) endocytosis to voltage-clamp measure- of the resistance of the axon and the currents remain. ments stems from the fact that these capacitance of the soma and terminal. A Regardless of the choice of technique, processes, by their nature, change the third example, the calyx of Held, is a interpretation of the data depends on surface area of the plasma membrane. giant nerve terminal in the brainstem the use of an a priori electrical model of Because the plasma membrane acts as ascending auditory pathway. This syn- the preparation. The simplest model, an electrical capacitor, changes in sur- apse, unlike the bipolar cell synapse, appropriate for a compact isopotential face area can be detected as changes in contains conventional active zones. De- cell, consists of the membrane capaci- its total capacitance. Thus by electrical pending on the length of the attached tance in parallel with its conductance measurements, net changes in plasma axon, the calyx appears as one or more membrane (i.e., exocytosis minus endo- (6). The rest of the circuit (recording electrotonic compartments (19). Sun cytosis) can be measured. Coupled with patch pipette, cytoplasm, and extracellu- and Wu (20) used frequency-domain voltage protocols that open voltage- lar path) is combined into the series re- capacitance measurements and the com- gated calcium channels, this technique sistance term for a total of three passive pact isopotential cell approximation to can be used to follow membrane parameters (Cm, Gm, and Rs; see Fig. 1). measure exocytosis in calyces that be- 2ϩ changes during Ca -dependent secre- In this case, theoretical time-domain haved as a single electrical compartment tion of neurotransmitters. If exocytosis responses consist of single exponential (i.e., calyces that appeared to have a and endocytosis are temporally distinct relaxations from which the three cell single exponential current relaxation in phenomena, the rate of exocytosis can parameters can be uniquely determined. response to a step hyperpolarization). be determined by measuring capacitance Likewise, for a sinusoidal excitation, the Using the same measurement technique, jumps triggered by step depolarizations components of current in phase and 90° of different durations (see refs. 2–5). In out of phase with the voltage stimulus addition, synaptic vesicle membrane re- can be analyzed by using standard cir- See companion article on page 8975. trieval (or reinternalization via endocy- cuit theory to determine Cm, Gm, and *To whom correspondence should be addressed. E-mail: tosis) after fusion can be monitored in Rs. To date, most capacitance measure- [email protected]. 8618–8620 ͉ PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633427100 Downloaded by guest on October 2, 2021 COMMENTARY the mossy fiber terminal, during which Ca2ϩ influx occurred. They found two components of capacitance increase in response to varying length depolariza- tions: a fast component with a time con- stant near 1 ms and a second, slower component with a time constant near 20 ms. They interpreted these components as distinct pools of synaptic vesicles with differential release kinetics. Recovery of membrane capacitance (i.e., putative endocytosis) after a maximally effective stimulation that depleted the releasable pool of vesicles occurred on a time scale of several seconds. Perhaps the most provocative aspect of the article concerns the quantitative estimate of the rate of vesicle release per active zone and implications for multivesicular release (i.e., release of more than one vesicle per active zone per action potential) (22). The argu- ments stem from the observation that during a prolonged depolarizing voltage step, the calculated release corre- Fig. 1. Model circuits for different cell geometries. (A) Spherical isopotential cells. These cells can be sponded, on average, to more than one described by three passive circuit parameters: membrane capacitance (Cm), membrane conductance (Gm), vesicle per active zone per millisecond. and series resistance (Rs, a composite parameter usually dominated by the resistance of the attached If release were uniform in time during whole-cell mode recording patch pipette but also including the resistance of the cytoplasmic and the depolarization, this would imply that extracellular paths). (B) Two-compartment model proposed by Mennerick et al. (18) to describe goldfish lateral inhibition of release of vesicles retinal bipolar neurons. This four-component model assumes negligibly low membrane conductance and from the same active zone, if present, axon capacitance. R2 is the axon resistance, C1 and C2 are the synaptic terminal and cell soma capacitance, Ͻ and R1 is series resistance of a patch pipette recording from the synaptic terminal. (C) General n- was very short-lived ( 1 ms, compare compartment model described by 3n parameters. with action potential duration half-width of Ϸ0.6 ms). In fact, during the fast component of release, the rate per ac- Taschenberger et al. (21) demonstrated The authors then used their model tive zone may have been much higher. empirically that capacitance jumps fol- cell to test several time- and frequency- Assuming that all the increase in capaci- lowing exocytosis were similar across domain capacitance measurements tech- tance signal is due to exocytosis of syn- calyces, independent of whether the niques to determine which most faith- aptic vesicles that are docked at active structure consisted of one or two elec- fully reported simulated changes in the zones, this measurement implies that trical compartments. Thus it seems that electrical parameters of the bouton. multivesicular release occurs at this syn- capacitance measurements in the calyx Notwithstanding the complex structure, apse. The list of synapses that experi- of Held are not greatly affected by the they found that time- or frequency-do- ence multivesicular release has been attached