Elsevier

Methods

Volume 33, Issue 4, August 2004, Pages 312-321
Methods

Detection of transmitter release with carbon fiber electrodes

https://doi.org/10.1016/j.ymeth.2004.01.004Get rights and content

Abstract

The use of constant voltage amperometry is described as an analytical technique for the detection of biogenic amines. This technique can be used to measure the amount of transmitter released from individual organelles and to determine the kinetic properties of transmitter discharge from the organelle providing unique insight into properties of signal transmission from secretory cells.

Introduction

The quantal theory of chemical transmission between neurons proposes that neurotransmitter is discharged from synaptic vesicles at specialized sites in the nerve terminal, the active zones [13]. This hypothesis has formed the foundation of our current understanding of interneuronal communication, but the biophysical properties of transmitter release and the molecular steps involved in promoting the exocytotic step are not fully known. One way to study the process of release is by electrochemical detection which is the focus of this article. Electrochemical methods have been recently refined by the development of small-sized carbon fibers that can be used as amperometric sensors for the detection of transmitter release from single cells. Amperometry monitors the oxidation of transmitter molecules at the surface of the carbon fiber. This technique enables a highly sensitive measurement of secretion and can detect release at high temporal resolution when the fiber is placed in close proximity to the releasing cell surface. Exocytosis from individual vesicles is then monitored as spike-like oxidation currents. Currently amperometry allows the detection of exocytosis only in a limited number of cells, because not many secreted products are readily oxidizable. The most intensively studied substances are catecholamines (e.g., epinephrine, norepinephrine or dopamine) and indolamines such as serotonin. On the other hand, amperometry is so exquisitely sensitive that minute amounts of transmitter released from the smallest secretory organelle [24], the small synaptic vesicle, are still detectable [6], [8].

Beside studying release of certain neurotransmitters, amperometry has also been exploited to investigate the secretion of neuropeptides [34]. Still, due to the small amount of neuropeptide molecules and the limited number of oxidizable residues the detection of neuropeptide release is more difficult. Nevertheless, the secretion of a small number of peptides (and proteins) has been studied, including insulin [1], α-melanocyte stimulating hormone [34], and gonadotrophin-releasing hormone [23]. Further, related analytical electrochemical techniques (including voltammetry) are widely used to study mixtures of peptides, often after complexation with copper [47]. However, given that peptides and oxidizable transmitters, such as the catecholamines, can be found in dense-core granules in the same cell type [45], studies on secretion of catecholamines may be used to infer mechanisms that govern neuropeptide release.

Amperometry belongs to a group of loosely related voltammetric techniques (comprising fast cyclic voltammetry, differential pulse voltammetry, and others) which offer the experimentator distinct advantages. For instance, cyclic voltammetry allows the identification of substances released from the cell [31], but, has the inherent disadvantage that the temporal resolution of the measurement is limited by the duration of the periodic voltage pattern (triangle wave) applied to the electrode. In contrast, constant voltage amperometry does not provide information about the nature of the oxidized substance, but is far superior when kinetics of secretion are analyzed.

A number of excellent reviews have appeared in the last few years, which cover the benefits and limitations of different electrochemical techniques [10], [27], [42], [43].

The field of electrochemical measurements of secretion from single cells was pioneered by the laboratories of Julian Millar and Mark Wightman [17], [31]. Subsequently, the benefits of amperometric measurements have been exploited by monitoring in detail the temporal profile of transmitter discharge from various vesicle types [4], [6], [8], [9], [11], [25], [29], [33], [38], [40], [44], [48], [50], [51]. In non-neuronal cells, amperometrically recorded events are sometimes preceded by a small “foot”-signal [11] which reflects the diffusion of transmitter through a slowly dilating fusion pore formed at the onset of exocytosis [4]. Similar signals were observed upon exocytosis of large dense-core vesicles in serotonergic neurons [6], suggesting that the initial formation of a fusion pore is a universal step in promoting exocytosis of secretory organelles.

An elegant combination of amperometry together with capacitance tracking, also referred to as ‘patch amperometry,’ makes use of a carbon fiber that is inserted inside a patch pipette which is then sealed onto the surface of a cell. Exocytosis of granules within the small membrane area circumscribed by the pipette tip is monitored by changes in membrane capacitance. In parallel, amperometry allows for detection of transmitter release providing direct insight into the mechanism how the exocytotic fusion pore modulates content release from the same organelle. This technique has been used to investigate the transmitter-conducting state of initial fusion pores as well as the regulation of ‘kiss-and-run’ exocytosis by calcium [2], [3]. Recently, it has also been applied to cell-free membrane patches since some vesicles remain attached to the membrane after excision [14].

Wightman and co-workers found the amount and rate of secretion of granular content from both mast cell vesicles and chromaffin granules to be affected by changes in the ionic environment, suggesting that an intravesicular, proteinaceous matrix and its decondensation kinetics may modulate transmitter discharge from these types of organelles [36], [40]. Thus, amperometry makes it possible to unravel various parameters of secretion and enables comparative analyses of transmitter release from different types of organelles.

As an example of the practical use of amperometry, we describe its application to cultured Retzius cells of the leech (Hirudo medicinalis) as a model system to study serotonin release. Retzius cells form a rapidly acting serotonergic connection when they are co-cultured with a chemoreceptive “follower cell” [5], [7], [16], [19]. Furthermore, these neurons maintain their capability to synthesize, store, and release serotonin in isolation [22]. Due to the large size of these cells (diameter of about 80 μm), the releasing cell surface can be directly approached with the detector, a feature of crucial importance for studying release at high time resolution.

In this chapter, we describe recording techniques that are routinely used in our laboratory to monitor exocytosis of single vesicle from cultured Retzius cells.

Section snippets

Preparation of Retzius cells from the leech (H. medicinalis)

Details regarding the techniques for identifying Retzius cells and removing (‘aspiration method’) them from leech ganglia were described elsewhere [19]. Here, only a brief description on the preparation and further handling of the Retzius cells is given. Leibowitz L-15 medium (GIBCO, Gaithersburg, MD) supplemented with 6% fetal bovine serum (GIBCO), glucose, 6 mg/ml, and gentamycin, 0.1 mg/ml (Sigma, St. Louis, MO) is used as culture medium. Desheathed ganglia are incubated for 1 h in culture

Principles of amperometric measurements

In amperometric measurements, the carbon fiber electrode is held at a constant potential (e.g., >650 or 800 mV for serotonin and catecholamines, respectively) exceeding the redox potential of the substance of interest. When molecules such as epinephrine or serotonin hit the carbon surface, electrons are transferred and a current can be measured. This oxidation reaction occurs faster than the diffusion-based transport of molecules to the electrode. Consequently, electrolysis generates a

Fabrication of carbon fiber electrodes

Various techniques for fabricating carbon fiber electrodes have been reported differing mainly in how the carbon fiber is mounted and insulated [12], [21], [26], [28], [30], [37], [41], [49]. We describe here two methods. The first, developed by Robert Chow, Ludolf von Rüden, and Erwin Neher, involves a polyethylene insulation [10], [11]. This method is inexpensive and has been used successfully to measure secretion from various cell types [6], [11], [50], [51]; (Zhou et al., 1996). We found

Instrumentation

The instrumentation for amperometric measurements is the same as that used for conventional patch-clamp recordings. The widespread use of the patch-clamp method has been paralleled by an increased availability of equipment. The choice of an amplifier depends on kinetics and amplitude of the signal to be studied. The current gain, bandwidth, and noise of the amplifier should be considered. We use an EPC-7 amplifier which is well suited for amperometric measurements. We feel that a detailed

Refinement of recording techniques

Recordings of transmitter release from small synaptic vesicles (SSV) and large dense-core vesicles (LDCV) in Retzius cells are best obtained from the very tip of the cell’s axon stump (Fig. 4). This is the preferential site of synapse formation if Retzius cells are co-cultured with chemoreceptive neurons [32]. Furthermore, at these sites, Retzius cells have spherical clusters of SSV (surrounded by LDCV) directly opposing the plasma membrane, as has been demonstrated by electron microscopy [6],

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