HAL Id: hal-00331615
https://hal.archives-ouvertes.fr/hal-00331615
Submitted on 17 Oct 2008
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Tools for electrophysiology labs: a C coded dynamic-clamp on DSP board (aC/DC).
Sofiane Boussa, Jennifer Pasquier, Francois Leboulenger, Frank Le Foll, Alain Faure
To cite this version:
Sofiane Boussa, Jennifer Pasquier, Francois Leboulenger, Frank Le Foll, Alain Faure. Tools for electro- physiology labs: a C coded dynamic-clamp on DSP board (aC/DC).. Deuxième conférence française de Neurosciences Computationnelles, ”Neurocomp08”, Oct 2008, Marseille, France. �hal-00331615�
TOOLS FOR ELECTROPHYSIOLOGY LABS: A C CODED DYNAMIC- CLAMP ON DSP BOARD (aCDC)
Sofiane Boussa1-2, Jennifer Pasquier2, François Leboulenger2, Frank Le Foll2 and Alain Faure1
1Groupe de recherche en électrotechnique et automatique du Havre (GREAH)
2Laboratoire d’écotoxicologie - milieux aquatiques (LEMA) 25, rue Philippe Lebon, 76058, Le Havre, France
sofiane.boussa@univ-lehavre.fr
ABSTRACT
Dynamic-clamp represents an archetypal interdisciplinary approach in the field of computational neuroscience because it aims at establishing a dialog in real time between i) a numerical model and a spiking cell and ii) between neurobiologists and computer programmers. In this paper, we present a relative simple hardware solution for an efficient dynamic-clamp application. The prototype has been built around a DSP board (dsPIC 33F, Microchip INC). Our results demonstrate the high speed and flexibility of our setup with two examples using frog melanotrope cells as biological counterpart. First the fast inward sodium has been numerically-blocked in physiological recording solutions, and secondly, artificial autaptic GABAergic connections have been graft as recurrent neuroepithelial synapses on melanotrophs. Further experiments are in progress to examine the influences of the nature, amplitude and time-course of autaptic currents in cell spiking profile.
KEY WORDS
Dynamic-clamp, DSP, computational instrumentation, Melanotrophs, sodium current, autapses.
1. Introduction
In 1940s, George Marmont and Kenneth Cole have developed (independently) the voltage-clamp technique, allowing a precise control of membrane potential in excitable cells and, simultaneously, a recording of membrane currents. Shortly after, this technique has been used by A.L Hodgkin and A.F Huxley to describe the inward Na+ and outward K+ currents responsible for action potentials [1]. Thereafter, abundant significant electrophysiological studies based on the voltage-clamp technique were carried out to determine the voltage- dependence characteristics of members of the vast family of ion channels proteins. In the late 1970s, Erwin Neher and Bert Sakmann have developed the patch- clamp technique, which can be viewed as a great improvement of the voltage-clamp technique. By using the patch-clamp approach, it became possible to control not only a cell but also a piece of membrane (the patch) containing a panel of signaling proteins. Patch-clamp recordings were therefore extensively employed on numerous cell types. By varying patch-clamp configurations and with the help of both excellent
signal-to-noise ratio electronics and advanced analysis tools, the understanding of single-channel properties of ion conductances have been achieved at the molecular level [2, 3].
However, a step forward to a more integrative echelon requires identification of the role of each particular ion conductance in action potential shaping and, as a consequence, in spike firing patterns. This implies experiments consisting in adding or ablating dynamically ion conductances to spiking neurons or networks, in order to observe real-time modifications in the discharge behavior and integrative properties of nerve cells. A particular interest would concern the involvement of synaptic plasticity (potentiation, depression, use-dependent gain, synchrony and latency, shunting effect for example) and voltage-gated ion channel kinetic modulations in spike timing regulation.
In 1993, the dynamic-clamp method was introduced to inject or subtract artificial conductances in biological neurons [4, 5]. However, up to now, there is no consensus concerning the best technical solution to perform a versatile and reliable dynamic-clamp.
Moreover, there is no available commercial setup. Thus, electrophysiology labs have proposed distinctive solutions, reviewed by Prinz and Abbott, to achieve the goal of a genuine dynamic-clamp [6]. Among of them, a majority of systems are based either on analog devices/controllers or on friendly interfaces under Windows and Linux real time kernel [7, 8]. The major difficulty, shared by all implementations, remains the necessity of fast data sampling and fast numerical integration to solve differential equations [9]. Windows or Linux-operated platforms are preferred by neurobiologists because of their use convenience and their open possibilities, but are very dependent on the electrophysiological setup and introduce jitter and latency. On the other hand, hardware platforms provides better performances, but are penalized by their complexity and total cost, when components, compilers and drivers are included.
In the present work, we propose a low cost (US$300) hardware solution for dynamic-clamp, based on a DSP1, coded in C language. It can be used with any eletrophysiological recording setup (presently integrated on a patch-clamp amplifier and digitizer supplied by Axon Instruments). The performance of our
1 Digital Signal Processing
system has been tested in two applications using frog hypophyseal melanotrope cells, which display a spontaneous electrical activity. The first experiment consists in a computational ablation of the fast inward sodium currents, and the second, a graft of artificial autaptic GABAergic connections.
2. Materials and methods
2.1 Melanotroph culture cells
Frog melanotrope cells were isolated and cultured as previously described [10]. Briefly, 8 neurointermediate lobes from frog (Rana ridibounda) pituitaries were dissected and enzymatically dissociated in a Leibovitz L-15 medium supplemented with 0,15 % collagenase for 20 min. The digested tissue was then mechanically disaggregated and dispersed cells were plated in the same culture medium supplemented with 10 % fetal calf serum at a density of 10 000 cells in plastic culture dishes. Electrophysiological recordings were conducted at 25-28°C using the standard patch-clamp technique in the whole-cell configuration and the current-clamp mode. Signals were filtered at 5 kHz and digitized at a sampling rate of 20 kHz.
2.2 Dynamic clamp realization
Fig. 1: A- signal acquisition and current calculations platform.
B- the prototype of our dynamic-clamp device.
Basically, the dynamic-clamp platform is composed of three elements:
• a patch-clamp setup, comprising an inverted microscope Nikon TE2000 equipped with a robotic micromanipulator Sutter MP285. Recordings were made with an Axopatch 200B patch-clamp amplifier interfaced to a 1.5 GHz computer via a Digidata 1322 and pClamp 8.0 software (Axon instruments, Foster City, CA),
• a dynamic-clamp device connected to the voltage OUTUP of the patch amplifier. This device contains, in a 19' rackable case, several circuits assuming the signal conditioning, the artificial conductance calculation and the corresponding current injection. The OUTUP of the dynamic-clamp device is connected to the current- clamp INPUT of the patch amplifier,
• a programming interface, used to write mathematical models of ionic conductances and to load the corresponding programs in the DSP. This part will be developped in §2.3.
The dynamic-clamp device is divided into 3 stages, corresponding to 3 independent boards:
• signal conditioning: this stage is used to reduce voltage membrane in the range of the ADC; the adaptations were performed by using analog circuits,
• digitalization and calculations are realized by a DSP (dsPIC 33F, Microchip INC). The dsPIC DSC is a Microchip's 16-bit family of Digital Signal Controllers;
it provides high performances (10-bit 2.2 Msps or 12- bit 1 Msps conversion, 2 or 4 simultaneously conversion) [11]
• digital to analog conversion: this stage is built around a 12-bit digital/analog converter (AD7541A, ANALOG DEVICES). In addition, a Sallen and Key filter is implemented in order to smooth signal.
Programming aspects of dsPIC are easier and cheaper than other solutions mentioned in [6]. Indeed, codes can be written in C, in addition the Microchip compiler C30 is free (student version).
2.3 Models and implementation
• Sodium currents
We used a Hodgkin-Huxley based approach [1]; it is given by the equation 1 where Vm is the membrane potential, VNa is the reversal potential for the Na+, gmax is the maximal conductance expressed in (mS).
) V V .(
h . m . g
INa = max 3 m− Na (1) m and h are activation and inactivation gating variables, they are described by first order, nonlinear differential equations
x . ) x dt .(
dx
x
x β
α − −
= 1 (x=m,n) (2)
where αm, αh,βm and βh are functions of Vm. In order to implement sodium current model in DSP and to optimize the computing time, we have developed a look-up table which contains values of all the rate constants (αm, αh,βm and βh) and solved the set of differential equations by using Euler’s method.
The cycle of reading the membrane potential and computing and injecting the sodium current is fixed by the Timer-1 of DSP (50µs).
• GABAergic autapses
The term autapse, coined by van der Loos & Glaser in 1972, refers to recurrent synaptic connections established between an axon and its own somatodendritic domain [12]. The autaptic GABAergic current Isyn is obtained from the relation (3).
ssiiggnnaall ccoonnddiittiioonniinngg
AAmmpplliiffiiccaattiioonn xx1100 OOffffeesstt ++11VV AAmmpplliiffiiccaattiioonn xx22
DSDSPP ddeevveellooppmmeenntt KKiitt
D
DAACC 1122bbiittss
Filtering & external commands Digidata
AADDCC((1122bbiittss
)) CCaallccuullaattiioonn I I==ff((VV)) +4+400 mmvv
00,,335500vv 2,2,88vv
U Uppddaattee p paarraammeetteerrss D
Daattaa aaccqquuiissiittiioonn,, ssttoocckkaaggee aanndd v
viissuuaalliizzaattiioonn
- -6655 mmvv
A
B
-20001 -1800 -1600 -1400 -1200 -1000 -800 -600 -400 -200 0 200
I Na (pA)
0 0.2 0.4 0.6 0.8 1 -80
0 0.2 0.4 0.6 0.8 1 -80
0 1 2 3 4 5
-80 -75 -70 -65 -60 -55 -50 -45 -40
Time(s)
IPSP (mv)
IPSP Timing of spike arrivals
) V V (
* ) t ( g
* g
Isyn = syn syn m− syn (3)
where gsyn is the maximal synaptic conductance, gsyn (t) is the temporal variation of conductance described by a first order differential equation, Vm is the postsynaptic membrane potential and Vsyn is the reversal potential.
= −
−
>
− +
=
otherwise
g
dt dg
mv V
if g U
dt dg
inact syn syn
m syn
act syn syn
τ
τ 40
(4)
GABAergic currents (shown in fig 2-B & C) have been simulated under Matlab (The Matworks Inc).
We used the Euler method as a solver which provides an acceptable approximation in our experimental conditions (sampling rate 20 kHz). Implementation of GABAergic were performed as described in [13].
Fig.2 : GABAergic current simulation : A- Inhibitory post-synaptic potential B-C - calculated GABAergic currents. D-
GABAergic current obtained from [14]
3. Results
Fig.3 : Sodium current ablation experimentation : A- Recorded membrane voltage. B- Calculated sodium currents in real time. C, D- Schematics of cell/computer interactions in dynamic-clamp with external stimulation, in open loop (C) or closed loop situations (D).
In Figure 3, are presented representative traces of a sodium current cancellation experiment. First, increasing external depolarizing current steps have been applied to the cell, in open loop as illustrated in Fig 3-C.
The electrical activity of the cell have been recorded and used to compute the sodium current in real time.
According to figure 3.A-B, it appears that the cell discharge frequency increases with the depolarizating currents. In addition, the computed sodium current is a function of the membrane potential, with a progressive
current inactivation, more marked at high firing rates.
This corresponds to long-lasting depolarized states.
When the dynamic-clamp circuit is turned ON (Fig 3.D), a feedback control is applied to the cell.
Therefore, an ablation of a sodium conductance is achieved, leading to spike cancellation.
The results of artificial autapses graft in a frog melanotrophs are shown in Figure 4.
Cell I=f(V)
Activated dynamic-clamp Deactivated dynamic-clamp
Cell I=f(V)
-801 -60 -40 -20 0 20 40 60
V membrane (mV)
1s
1s A
B
C D
D
0 1 2 3 4 5
0 20 40 60 80 100 120
Time (s) IGABA (pA)
B
2.3 2.4 2.5 2.6 2.7
0 20 40 60 80 100
Time (s) IGABA (pA)
C A
Dynamic clamp turned OFF Dynamic clamp turned ON
Stimulations 0 pA
80 pA
800 ms
Stimulations 0 pA
80 pA
800 ms
Vmembrane (mV) INa (pA) IPSP (mV) IGABA (pA)
Time (s) Time (s)
Time (s) IGABA (pA)
-801 -60 -40 -20 0 20 40 60
Vmembrane (mV)
0 0.2 0.4 0.6 0.8 1
0 0.2 0.4 0.6 0.8 1
2-100 -50 0 50
IGABA (pA)
-10001 -800 -600 -400 -200 0 200
IGABA (pA)
Fig.4 : GABAergic current adding experimentation : A- Recorded membrane voltage. B- Calculated GABAergic currents in real time.
Our results show that the spike frequency is strongly diminished whereas the post-hyperpolarization is significantly modified when GABAergic autapses are added to the recorded cell. In addition, the cell electrical activity itself dynamically modulates the amplitude and kinetic of the computed artificial autaptic currents, proving the efficiency of the close-loop dialogue established between the living cell and the numerical model.
4. Conclusions
The synergistic efforts and skills of both laboratories GREAH/LEMA have been at the origin of a new digital electrophysiology instrument. The prototype described herein (aC/DC) provides the researchers with an advanced toolbox to study the mechanisms governing the processing of information at the cell or small network level. Immediate applications are, for example, in silico pharmacology, permitting to study ad libitum the effects of the absence or presence of a particular conductance, simply by switching ON or OFF the dynamic-clamp device. This approach can also be used to identify kinetic constants and voltage-dependence of ion currents or to work on the influence of use- dependent plasticity or weight of synapses.
Further experiments are in progress to examine the role of the nature, amplitude and time-course of autaptic currents in the alteration of the cell spiking profile. The data should provide comprehensive informations concerning the influence of autaptic afferances on neural encoding.
References
[1] A. L. Hodgkin & A. F. Huxley, A quantitative description of membrane current and its application to conduction and excitation in nerve, Journal of Physiology, 117, 1952, 500-544.
[2] B. Sakmann & E. Neher, Geometric parameters of pipettes and membrane patches : Single Channel Recording, (First ed. New York: Plemnum press, 1983).
[3] F. J. Sigworth & E. Neher, Single Na+ channel currents observed in cultured rat muscle cells, Nature, 287, 1980, 447-449.
[4] H. P. Robinson & N. Kawai, Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons, J Neurosci Meth, 49, 1993, 157-165.
[5] A. A. Sharp, M. B. O'Neil, L. F. Abbott, & E.
Marder, The dynamic clamp: artificial conductances in biological neurons, Trends in Neurosciences, 16, 1993, 389-394.
[6] A. A. Prinz, L. F. Abbott, & E. Marder, The dynamic clamp comes of age, Trends in Neurosciences, 27, 2004, 218-224.
[7] I. Raikov, A. Preyer, & R. J. Butera, MRCI: a flexible real-time dynamic clamp system for electrophysiology experiments, Journal of Neuroscience Methods, 132, 2004, 109-123.
[8] T. Nowotny, A. Szucs, R. D. Pinto, & A. I.
Selverston, StdpC: A modern dynamic clamp, Journal of Neuroscience Methods, 158, 2006, 287-299.
[9] R. J. Butera & M. L. McCarthy, Analysis of real- time numerical integration methods applied to dynamic clamp experiments, J Neural Eng, 1, 2004, 187-94.
[10] F. Le Foll, H. Castel, O. Soriani, H. Vaudry, & L.
Cazin, Gramicidin-perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs, J Physiol, 507, 1998, 55-69.
[11] Microchip, dsPIC33F Family Data Sheet, High- Performance, 16-Bit Digital Signal Controllers, technical report.
[12] H. Van der Loos & E. M. Glaser, Autapses in neocortex cerebri: synapses between a pyramidal cell's axon and its own dendrites, Brain Res, 48, 1972, 355- 360.
[13] S. Boussa, M. Marin, F. Le Foll, A. Faure, & F.
Leboulenger, Création de connexions autaptiques artificielles dans des cellules pacemaker par dynamic clamp, Proc. 1st Neurocomp, Pont-à-mousson, France, 2006, 145-148.
[14] A. Destexhe, Z. F. Mainen, & T. J. Sejnowski, Kinetic models of synaptic transmission: MIT Press, 1998.
1s
1s A
B
Dynamic clamp turned OFF Dynamic clamp turned ON
Vmembrane (mV) IGABA (pA) IGABA (pA)
