Transcript Motivation As a painless means to probe into human brains, TMS

finalThreshold
E
538  18 Vm  0.05
T 
initial
Threshold  0.74
Magnetic stimulation of one dimensional neural cultures in-vitro
Assaf Rotem, Elisha Moses
Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot, Israel.
Abstract
Although Transcranial Magnetic Stimulation (TMS) is a widely used clinical tool, little is
known about its physical and neural mechanisms. In an effort to uncover these mechanisms
we culture hippocampal neurons in ring-like patterns and image their calcium transients while
pulsing magnetic fields via coils located concentrically above the rings. Preliminary results
demonstrate neuronal activity evoked in rings of 5-10mm in diameter with magnetic pulses of
1-5 Tesla. In consistency with theoretical predictions, the magnetic threshold of single neurons
decreases with the size of the patterns and with the number of stimulations but is not affected
by network connectivity (inhibitory or excitatory). With inhibitory connections blocked, 1Hz
repetitive TMS increases activity rate of the network while 3-10Hz stimulations decrease it.
Hopefully, this unique setup will solve some of the open issues of TMS such as the neural
origin of TMS, the effects of rTMS on neural activity and the effect of pharmacology on TMS.
rTMS
Methods
A
B
C
D
3mm
Motivation
As a painless means to probe into human brains, TMS continuously gains diagnostic and therapeutic applications [1] [3]. Despite the impressive progress, there is a wide agreement that the full clinical potential of TMS, mainly as an
alternative to Electroconvulsive Therapy (ECT - electric shocks) is still unrealized [4] - [6]. The main limitation
holding back a realization of the full potential of TMS is the incomplete comprehension of the physical and
neurophysiological mechanisms that underlie its operation.
In particular, it is not clear why certain regions in the cortex are excitable while others are not but brain geometry
and the anatomy of sulci and gyri seem to play a role in this mechanism [7] - [11]. The effectiveness of TMS was
shown to be affected by nerve morphology [12], Coil orientation [18]-[22], neuronal excitability [24],[23] and
myelination [17]. The interplay between TMS and pharmacology was demonstrated in a few studies [14], [15].
Repetitive TMS (rTMS) was shown to produce potentiation and depression in humans, depending on the TMS
rate [13], [16]. All these describe an intriguing parameter space in which TMS functions. Applying TMS without
mapping this space of possibilities leaves us puzzled as to unexpected results which are probably accounted for by
some hidden parameter which was left uncontrolled.
One way to explore this parameter space is using current methods of TMS in-vivo on human or animal brains.
Human experiments are limited in their use of pharmacology and of high frequency rTMS. Animal experiments are
technically difficult and both animal and human experiments are limited in their ability to explore the effects of
anatomy, morphology, excitability and myelination. TMS in-vitro is a perfect candidate for such explorations since it
offers full control over all parameters mentioned.
The experimental setup. A,B) Inverted microscope images fluorescent transient of “ring colonies” reacting to
magnetic pulses of a coil which is located 5mm concentrically above the neural dish. C) The dynamics of the TMS
coil (TMS load = 2000kV) as integrated from a pick up coil positioned concentrically and adjacent to the coil. D) A
bright field image of one of the 24mm coverslips. The bright spots are dense accumulation of neurons on the pattern.
Line width is ~200μm.
Results
Firing rate of two ROIs of a ring culture (after application of a saturating concentration of Bicuculline). Top: ROI #2
responded with a single population activity to single TMS pulses, while ROI #1 did not respond to TMS pulses. The beginning
of each TMS pulse train is marked with a vertical line. Bottom: differential plot of the rate of ROI #2 – the rate of ROI #1
displays a clear increase in activity rate after 3 trains of 1Hz pulses (10 pulses each).
Magnetic stimulation induced neural activity in 44 out of 345 “ring colonies” that were
stimulated. Using consecutive stimulations of decreasing intensity one can determine the Power
Threshold of each colony of neurons. The Power Threshold is defined as the stimulation intensity
which induces neural activity in 50% of the events.
ET  172  42.4 Vm
Discussion
Theoretical Background
ET  538  18 Vm
The fact that the threshold is dependant on the length of the arcs on which the culture is patterned combined with the minor
effects that synaptic blockers had on the threshold, suggests that the origin of magnetic stimulation in-vitro consist of a small
number of neurons, with specific anatomical and / or electrophysiological properties which are relevant to magnetic stimulation.
Although the threshold of these neurons is not affected by synaptic blockers, the resulting population activity might be strongly
affected by these blockers since the amount of excited neurons which is required for population activity is dependant on the
connectivity of the network.
Summary
TMS Power Threshold as a function of ring radius. The red
line fits the set of monotonically decreasing minimal values
of the data with 1/r model. ET is an estimate on the minimal
electric field required to elicit activity.
The probability that an electric field will elicit neural activity
in the experiments. μ is an estimate on the average electric field
required to elicit activity
Taken from Mark George’s Article on Brain Stimulation in Scientific American 09/03
Results - ctnd
When a strong pulse of current is discharged through the blue coil a proportional magnetic flux is pulsed, depicted by the red
streamlines. Following the law of induction, any change in this flux will induce an opposing electric field (color coded, arrows
indicate field direction) in a direction opposite to that of the current flowing in the coil. This field is proportional to the rate of
change of magnetic flux it encloses and therefore it is larger for larger amount of enclosed flux, i.e. the larger the rings, the
larger area of flux they enclose and the larger the induced electric field inside them. In the case of neurons, this electric field
induces ionic currents that destabilizes the distribution of ions inside the cell and which may result in super threshold
membrane potentials. In order for a magnetic pulse of 1T (a typical limit for TMS devices) to induce an electric field of 200V/m
(a typical value for activating CNS neurons) one needs a ring which is 13.5mm in radius. Moreover, only the electric field
component which is parallel to the axonal and dendritic tree will induce ionic currents inside the neuron so in order to obtain a
maximal charge current the neurites should be aligned with the electric field. Practically speaking, this means that one has to
implement aligned and patterned growth of neurons along a ring of 13.5mm radius.
Geometry: In order to explore the role of nerve geometry in magnetic stimulation we tested 3 nonpatterned coverslips with 2D cultures, 1 coverslip that was patterned into straight lines and 16 rings which
were cut to 1/2, 1/3rd and 1/6th of their perimeter. None of the 2D cultures reacted to TMS, None of the 1/6th
segments reacted to TMS, only one of the 1/3rd segments reacted to TMS (with VTMS=4kV) and 5 of the ½
segments reacted to TMS (with VTMS=3kV±0.5kV). Cultures that were patterned into straight lines did not
react to TMS when they were oriented perpendicular to the electric field, and reacted to TMS when
oriented parallel (VTMS=3.5kV) or with 45º inclination (VTMS=3.5kV) with respect to the electric field.
Plasticity: in part of the experiments Power Thresholds were measured initially by increasing the TMS
voltage load from 0 to the first value which stimulated the rings, and then decreased to the last value which
still stimulated the rings. In all of these experiments, the initial value was significantly higher than the final
value.
finalThreshold
initialThreshold  0.74  0.05
A novel setup of magnetic simulation in-vitro described in this paper is suggested as a benchmark model for TMS. Preliminary
observations of non-trivial results concerning the effect of geometry, pharmacology and plasticity on the magnetic threshold of
neurons in-culture suggests that this setup can serve an in-vitro model for the effects of TMS on nerves. An endless row of
experiments can be applied via this model. The role of morphology and electrophysiology of neurons in TMS can finally be
approached since theses properties are easily monitored and measured in-vitro. Pharmacology can be applied without any
limitations, to test its effect on TMS. The effects of rTMS on neural activity can be tested in an unlimited range of frequencies.
Safety issues in TMS can finally be approached in a sterile manner.
Magnetic Stimulation of neurons in-vitro has proved to be a challenge which required insight and new techniques in both physics
and neurobiology. While a lot is still missing in the understanding of TMS, many of the experiments which can unfold the
mystery were not adequate for in-vivo setups and may now be approached via a new door which accesses the intriguing field of
TMS.
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Pharmacology: Power threshold measured before addition of 50μM Bicuculline was not significantly
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Bicuculline. When applying saturating concentrations of CNQX, TMS induced activity could still be
observed although their thresholds increased significantly.
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