Motivation

1. A B C D 3mm Taken from Mark George’s Article on Brain Stimulation in Scientific American 09/03 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. rTMS 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. Methods 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 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. 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). Discussion 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. Theoretical Background Summary 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. 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. μis an estimate on the average electric field required to elicit activity • Results - ctnd • Geometry: In order to explore the role of nerve geometry in magnetic stimulation we tested 3 non-patterned 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. • Pharmacology: Power threshold measured before addition of 50μM Bicuculline was not significantly different than after addition. However, 19 rings responded to TMS only after the application of Bicuculline. When applying saturating concentrations of CNQX, TMS induced activity could still be observed although their thresholds increased significantly. • References • M S George, E M Wassermann and R M Post. “Transcranial magnetic stimulation: a neuropsychiatric tool for the 21st century,” J Neuropsychiatry Clin Neurosci, 8(4):373–382, Fall 1996. • M Hallett, “Transcranial magnetic stimulation and the human brain,” Nature, 406:147-150, July 2000. • A. Pascual-Leone et al. Handbook of transcranial magnetic stimulation. London, England, Arnold, 2002. • S. Pridmore, “Substitution of rapid transcranial magnetic stimulation treatments for electroconvulsive therapy treatments in a course of electroconvulsive therapy,” Depression and Anxiety, 12(3):118-123, Nov 2000. • A. Post and M. E. Keck, “Transcranial magnetic stimulation as a therapeutic tool in psychiatry: what do we know about the neurobiological mechanisms?,” Journal of Psychiatric Research 35:193–215, 2001. • M. K.obayashi and A Pascual-Leone, “Transcranial magnetic stimulation in neurology,” Lancet Neurol, 2(3):145-56, Mar 2003. • J. P. Brasil-Neto, L. G. Cohen, M. Panizza, J. Nilsson, B. J. Roth and M. Hallett, “Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity,” J Clin Neurophysiol 9(1):132–136, Jan 1992. • K. R. Mills, S. J. Boniface and M. Schubert, “Magnetic brain stimulation with a double coil: the importance of coil orientation,” Electroenceph clin Neurophysiol, 85(1): 17–21, Feb 1992 • A. Pascual-Leone, L. G. Cohen, J. P. Brasil-Neto and M. Hallett, “Non-invasive differentiation of motor cortical representation of hand muscles by mapping of optimal current directions,” Electroenceph clin Neurophysiol 93(1):42–48, Feb 1994. • A.G. Guggisberg, P. Dubach, C.W. Hess, C. Wüthrich, and J. Mathis, “Motor evoked potentials from masseter muscle induced by transcranial magnetic stimulation of the pyramidal tract: the importance of coil orientation,” Clin Neurophysiol 112(12):2312–2319, Dec 2001. • P. Dubach , A. G. Guggisberg , K. M. Rösler , C. W. Hess and J. Mathis, “Significance of coil orientation for motor evoked potentials from nasalis muscle elicited by transcranial magnetic stimulation,” Clin Neurophysiol, 115(4):862-870, Apr 2004. • Rotem A, Moses E. Magnetic stimulation of curved nerves, IEEE Trans Biomed Eng. Accepted Jun 2005. • Paulus W. “Toward establishing a therapeutic window for rTMS by theta burst stimulation,” Neuron. 2005 Jan 20;45(2):181-3. • Ziemann U. TMS and drugs. Clin Neurophysiol. 2004 Aug;115(8):1717-29. • Kahkonen S, Ilmoniemi RJ. Transcranial magnetic stimulation: applications for neuropsychopharmacology. J Psychopharmacol. 2004 Jun;18(2):257-61. • Huang YZ, Edwards MJ, Rounis E, Bhatia KP, Rothwell JC. “Theta burst stimulation of the human motor cortex,” Neuron. 2005 Jan 20;45(2):201-6. • Caramia MD, Palmieri MG, Desiato MT, Boffa L, Galizia P, Rossini PM, Centonze D, Bernardi G. Brain excitability changes in the relapsing and remitting phases of multiple sclerosis: a study with transcranial magnetic stimulation. Clin Neurophysiol. 2004 Apr;115(4):956-65. • J. P. Brasil-Neto, L. G. Cohen, M. Panizza, J. Nilsson, B. J. Roth and M. Hallett, “Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity,” J Clin Neurophysiol 9(1):132–136, Jan 1992. • K. R. Mills, S. J. Boniface and M. Schubert, “Magnetic brain stimulation with a double coil: the importance of coil orientation,” Electroenceph clin Neurophysiol, 85(1): 17–21, Feb 1992 • A. Pascual-Leone, L. G. Cohen, J. P. Brasil-Neto and M. Hallett, “Non-invasive differentiation of motor cortical representation of hand muscles by mapping of optimal current directions,” Electroenceph clin Neurophysiol 93(1):42–48, Feb 1994. • A.G. Guggisberg, P. Dubach, C.W. Hess, C. Wüthrich, and J. Mathis, “Motor evoked potentials from masseter muscle induced by transcranial magnetic stimulation of the pyramidal tract: the importance of coil orientation,” Clin Neurophysiol 112(12):2312–2319, Dec 2001. • P. Dubach , A. G. Guggisberg , K. M. Rösler , C. W. Hess and J. Mathis, “Significance of coil orientation for motor evoked potentials from nasalis muscle elicited by transcranial magnetic stimulation,” Clin Neurophysiol, 115(4):862-870, Apr 2004. • Moliadze V, Zhao Y, Eysel U, Funke K. “Effect of transcranial magnetic stimulation on single-unit activity in the cat primary visual cortex,” J Physiol. 553(2):665-79 December 2003 • Kanno M, Matsumoto M, Togashi H, Yoshioka M, Mano Y. "Effects of acute repetitive transcranial magnetic stimulation on extracellular serotonin concentration in the rat prefrontal cortex," J Pharmacol Sci. 2003 Dec;93(4):451-7. 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.