International Laboratory of Plant Neurobiology
After the first successful Symposium on Plant Neurobiology, held in Florence in 2005, and thank to the funding from Ente Cassa di Risparmio di Firenze, the LINV started its activity as a par
t of the University of Florence. Under the leadership of Prof. Stefano Mancuso (University of Florence) and Prof. František Baluška (University of Bonn), a high number of graduated students, PhD and post-docs have been active in several aspects of plant neurobiology, a new view and concept of plants that combines physiology, ecology, and molecular biology.
The expertise of LINV on electrophysiology is mostly related to the fields of stress physiology, plant tropisms and plant rhythms. In particular, the group members have raised and acquired a deep knowledge of a wide range of advanced electrophysiological methods such as: transmembrane potential; spectroscopy impedance; vibrating probe technique; MEA technique etc.
The molecular biology research in the LINV is mainly focused on determining the function of specific proteins and identifying how in plant cells environment these specific proteins can impact cell function, development and cell-to-cell interactions and communications. A wide variety of molecular, biochemical and proteomic techniques are here available in order to analyze the function of the proteins and the interactions that occurs between them. Advanced techniques of confocal microscopy for monitoring several proteins in normal and over-expression conditions are applied to better understand the 1) structural and functional analysis of proteins and their complexes, 2) characterization of protein-protein interactions, 3) the in vivo dynamics of specific proteins.
The LINV owns advanced microscopy equipment: a Leica TCS SP5 confocal microscopy, a Zeiss fluorescence Stereo Discovery V12 and a Zeiss fluorescence inverted Axio observer Z1, which is equipped with the ApoTome system, and combined with a micromanipulation and microinjection system.
Multi-Elecrode Array (MEA)
ELECTRICAL NETWORK ACTIVITY OF THE ROOT APEX
The MEA set-up is used as a noninvasive method for monitoring spontaneous and evoked activity (spikes) as a change in the electrical potential of the intercellular space surrounding the cell. Spikes are a particular class of electrophysiological signals that depend on the excitably characteristics of cells and on their nature to generate Action Potentials (APs); APs base on the depolarization and repolarization of cell membranes due to ions flux (Ca+, K+ and Cl-). Every treatments that alter the ability of membrane to exchange ions reflect on the electrical activity of the cells, thus on the signals recordable. A typical setup for MEA recording is based on metal microelectrodes fabricated on a planar chip, discrete-element preamplifiers located close to the MEA device and a multi-wire cable that conducts the pre-amplified analog signals to a data acquisition card. We have already reported (Masi et al. 2009, PNAS 106:4048-4053) time recordings of single-unit spike activity with MEAs in acute slice of Zea mays L. root apex. Extracellular electrical activity (spikes) can be recorded simultaneously from 60 electrodes (30 μm diameter) with high spatial and temporal resolution. The nature of spike shapes has been studied on each MEA electrodes (200 μm interelectrode spacing). This new technique allows us to map functionally discrete regions of the root and to observe the space-time relationships and the spontaneous electrical activity of the root apex both in normal condition and in response to stress (drug treatment, gravistimulation, heat, electrical stimulation etc). Synchronous events and the propagation of signals can also be investigated.
The Vibrating Probe Technique
The Vibrating Probe Technique is a non-invasive method for measurements of net ion fluxes (or gas molecules) from plant cells and leaving tissues thanks to the use of selective microelectrodes (custom made or commercial probes).
The advantage of this approach is basically the direct measurement of ion fluxes in a simple, quick, continuous and non-invasive manner. Two features lie at the core of the high resolution ion-selective probes: the properties of the commercially available liquid exchange membranes and the self-referencing noise and drift reduction.
No other available technique meets the exacting requirements of the non-invasive ion-selective probe, which permits measuring steady ion fluxes across the plasma membrane of single cells with a high temporal and spatial resolution. Moreover, this approach permits repeated observations on the same tissue over hours or days, due to the fast response time.
In this sense the “vibrating probe technique” complements the other methods available to observe the movements of ions across membranes or within cells.
In our lab, this technique can be used to measure K+, Ca2+, Cl-, H+, O2, NO and H2O2 in plant tissues and it is a very effective method to assess plant physiological status and plant stress.
Electrical Impedance Spectroscopy
A repeatable and non-destructive method to study the behavior and the properties of cell membranes is Electrical Impedance Spectroscopy (EIS). It is a diagnostic method based on the study of the passive electrical properties (determined by the observation of the tissue electrical response to the injection of external electrical energy) of a material (Azzarello et al., 2006). When an AC is applied to a cell, its membranes act as capacitors due to their capacitive reactive elements. Cell membranes have high impedance at low frequencies, while the impedance decreases with increasing frequency. Accordingly, the current flows in the apoplastic space, where ions are the main current carriers, which determine the total impedance. The symplastic space becomes conductive and at high frequencies the symplastic and apoplastic resistance form a parallel circuitry. The impedance and phase angles of materials can be measured by a multiple frequency impedance analyser (impedance meter) that is able to scan each sample at different frequencies. The data obtained from EIS are commonly analyzed by fitting them to an equivalent circuit model, composed by resistors and capacitors. The applications of electrical impedance measurement in plant tissues are numerous. In fact, this technique can be used to determine physiological conditions of plant tissues, particularly in relation to cold acclimation, freezing injury, dormancy induction, nutritional deficiency and heat injury. Electrical impedance measurements provided a means of non-destructively analyzing variation in intra- and extracellular resistances and in the condition of the membranes.