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High-frequency stimulation of the subthalamic nucleus silences subthalamic neurons: a possible cellular mechanism in Parkinson’s disease
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Neuroscience Vol. 115, No. 4, pp. 1109^1117, 2002 L 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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HIGH-FREQUENCY STIMULATION OF THE SUBTHALAMIC NUCLEUS SILENCES SUBTHALAMIC NEURONS: A POSSIBLE CELLULAR MECHANISM IN PARKINSON’S DISEASE OS-ASCONE,a J. H. PAZO,b O. MACADARb and W. BUN Ob C. MAGARIN a
Neurolog|¤a Experimental (Unidad Asociada al Instituto Cajal, CSIC), Depto. de Investigacio¤n, Hospital Ramo¤n y Cajal, Ctra. Colmenar, Madrid 28034, Spain b
Instituto Cajal, CSIC, Madrid, Spain
Abstract
to parkinsonian symptoms (Benazzouz et al., 1992; Bergman et al., 1994). The recent di¡usion of high-frequency electrical stimulation (i.e., ‘tetanic’) of the STN in the treatment of parkinsonian motor symptoms in humans and MPTP-treated primates (Benazzouz et al., 1992; DeLong, 1990; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998) has renewed interest in this structure, but the underlying cellular mechanisms of this stimulation-induced remission of parkinsonian symptoms are not totally understood. Tetanic stimulation and destruction of the STN have similar ameliorating e¡ects on parkinsonian motor symptoms (e.g., Benazzouz et al., 1992; Bergman et al., 1994), suggesting that both manipulations act by blocking the electrical activity of the nucleus. A prolonged post-stimulus frequency-dependent silencing e¡ect of STN stimulation induced by a brief high-intensity electrical stimulation has recently been demonstrated in an in vitro rat preparation by Beurrier et al. (2001). The authors suggested that the same silencing e¡ect may occur during stimulation, but they could not verify the suppression of action potential (AP) activity in their preparation due to stimulus artifacts. Parkinsonian symptoms in patients only fade during STN stimulation and resume rapidly once stimulation is
The subthalamic nucleus (STN) plays an important role in motor control and in the genesis of Parkinson’s disease (e.g., DeLong, 1990; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000). Loss of substantia nigra dopaminergic neurons in Parkinson’s disease results in a reduction of activity in the thalamus partly due to an increased bursting activity of STN cells (DeLong, 1990). The abnormally augmented burst ¢ring of STN cells was correlated with tremor in Parkinson patients (Hutchison et al., 1998; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998) and in primates treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Benazzouz et al., 1992; Bergman et al., 1994), a drug that selectively destroys the dopaminergic cells of the substantia nigra. This correlation has suggested that the bursting of STN neurons could contribute
*Corresponding author. Tel. : +34-91-336-8320. E-mail address: [email protected] (C. Magarin‹os-Ascone). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; AP, action potential ; EPSP, excitatory postsynaptic potential ; IPSP, inhibitory postsynaptic potential; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; PPF, paired-pulse facilitation; Rin , input resistance; STN, subthalamic nucleus; TTX, tetrodotoxin; Vm, membrane potential. 1109
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stopped (Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Therefore, to test the suggestion made by Beurrier et al. (2001), it is highly important to record STN cell activity during stimulation. Moreover, the STN cell types that are involved in the reversion of parkinsonian symptoms should be de¢ned because their functional properties are still open to question. Indeed, the neuronal pool attributed to the STN varies from one (Afsharpour, 1985; Hammond and Yelnik, 1983), two (Rafols and Fox, 1976) or three morphologically di¡erent cell types (Iwahori, 1978). In addition, extracellular recordings in rats indicate three ¢ring patterns de¢ned as ‘normal’, ‘bursting’ and ‘mixed’ (Hollerman and Grace, 1992), but only a single tonic type was reported in in vitro preparations (e.g., Bevan and Wilson, 1999; Nakanishi et al., 1987) that could switch to a bursting mode (Beurrier et al., 1999). Therefore, we reinvestigated the electrophysiological characteristics of STN neurons, paying special attention to the e¡ects observed during prolonged tetanic stimulation of the STN as used in humans to treat parkinsonian symptoms.
EXPERIMENTAL PROCEDURES
Brown Norway rats (9^14 days old) were anesthetized with ether and decapitated immediately after disappearance of the pinch re£ex. The brain was rapidly removed and submerged in a vial with cold (4‡C) arti¢cial cerebrospinal £uid (ACSF) that contained (in mM) 124 NaCl, 2.69 KCl, 1.25 KH2 PO4 , 2.0 MgSO4 , 26.0 NaHCO3 , 2.0 CaCl2 , and 10.0 glucose. The pH was adjusted to 7.4 by gassing with 95% O2 and 5% CO2 . A slab that contained the brainstem at the level of the STN was cut transversely and ¢xed with cyanoacrylate to the base of a chamber containing cold ACSF. Transverse 350-Wm slices including the STN were cut with a vibratome (Pelco 101, Series 1000, St. Louis, MO, USA). After incubation for s 1 h in gassed ACSF at 21^23‡C in separate chambers, slices were moved to a recording chamber (2 ml), viewed under a dissecting microscope (Nikon/SMZ-1, Tokyo, Japan) and superfused at 2^3 ml/min with gassed ACSF at 30^32‡C. In a few experiments the control solution was substituted by a nominally Ca2þ -free ACSF in which CaCl2 was substituted with MgCl2 (2.0 mM). In some cases voltage-gated Naþ conductances were blocked with tetrodotoxin (TTX, 1.0 WM) added to the control ACSF. All chemicals were purchased from Sigma (St. Louis, MO, USA). The STN was identi¢ed by its position within the slice according to the Paxinos and Watson (1998) atlas. Recording pipettes were fabricated from borosilicate glass capillaries (R-Series 1B150F-4, WPI, Sarasota, FL, USA) with a Brown^Flamming model P-80 micropipette puller (Sutter Instruments, Novato, CA, USA). Pipettes back-¢lled either with potassium acetate (3.0 M; 90^120 M6) or with 2% carboxy£uorescein in potassium acetate (1.0 M; 150^200 M6) were connected to an Axoclamp-2B ampli¢er (Axon Instruments, Foster City, CA, USA). Pipette tips were placed under direct visualization of the STN and recordings were in the bridge mode. Carboxy£uorescein was injected by passing 0.1^0.3 nA, 300^400 ms hyperpolarizing pulses at 1 Hz (Nun‹ez and Bun‹o, 1999). Electrical stimulation (GRASS S88, Quincy, USA) was through a pair of 80-Wm-diameter Nichrome wires insulated except at the tip and placed on the STN under direct visualization. This ‘intranuclear’ placement of stimulation electrodes was aimed at reproducing the stimulation used in the treatment of parkinsonian symptoms (e.g., Benabid et al., 1996; Benazzouz et al., 1992; FigueirasMe¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al.,
1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Stimulation intensity (0.2^1.0 WA) was adjusted to evoke excitatory postsynaptic potentials (EPSPs) that were subthreshold for eliciting APs when presented in isolation. Exceptionally, suprathreshold stimuli were also tested. Since we aimed at analyzing synaptic responses, stimulations that triggered ‘direct’ or antidromic responses were rejected. To exclude an artifact caused by the stimulating electrodes (failing to generate APs in the axons or not propagating to the terminal during high-frequency stimulation), recordings that revealed repeated EPSP failures were rejected and the stimulation intensity readjusted until stimulations devoid of failures were obtained. Data were low-pass ¢ltered (3 kHz) and fed to a 486/PC computer through a TL-1DMA interface board. The pClamp programs (Axon Instruments, Foster City, CA, USA) were used to record signals, to control stimulation, and to analyze data. Prolonged transmembrane current pulses were generated with the Grass stimulator. All values are expressed as the mean X S.D. Young rats were used because the proportion of successful cell impalements with the required membrane potential (Vm), overshooting APs and input resistance (Rin ) was much higher than in preliminary experiments using older rats (see Results). The slices containing carboxy£uorescein-¢lled cells were ¢xed in a mixture of 4% paraformaldehyde in 0.1 M phosphate bu¡er (pH 7.4). A Leica TCS 4D (Wetzlar, Germany) confocal laser-scanning microscope with an argon/kryptonmixed gas laser with excitation peak at 488 nm was used to determine the morphological characteristics of the carboxy£uorescein-labelled neurons. The confocal microscope was associated with a Leitz DMIRB £uorescence inverted microscope (Wetzlar, Germany) equipped with oil immersion objectives and a ¢lter block for carboxy£uorescein. Accumulating stacks of 12 successive images, separated by 3 or 4 Wm, allowed image reconstruction (e.g., Fig. 1D, E). To estimate morphological di¡erences between the basal dendritic ¢elds we counted the number of dendritic intersections with concentric circles centered on the cell body with 25-Wm-increasing radii (Elston et al., 1999). Statistical comparisons between samples were made using the Mann^Whitney U-test. All experiments in this study conformed to International Guidelines on the ethical use of animals and every e¡ort was made to minimize the su¡ering and number of animals used.
RESULTS
Recordings with a Vm s 348 mV and overshooting APs were obtained from 104 STN neurons. Spontaneous and pulse-evoked AP activity The majority of the neurons (71/104, 68.3%) ¢red APs rhythmically and continuously either spontaneously or when depolarized with sustained injection of outward current. Most of these neurons were silent at rest (53/ 71, 75%) and showed a Vm of 355.6 mV X 3.5. Spontaneously silent cells ¢red rhythmically at rates of 9.2 Hz X 7.8 when depolarized above a 346 mV X 3.8 threshold (see below, however). The rest of the cells (18/71, 25%) were spontaneously active and ¢red rhythmically (14.9 Hz X 6.3). The Rin (98.4 M6 X 18.9) and the AP amplitude-duration (59.6 mV X 2.1, at ‘take-o¡’ and 1.1 ms X 0.2, at half amplitude) were not statistically di¡erent in active and silent cells. Moreover, other response properties described below were essentially identical in spontaneously silent and active cells. The responses evoked by depolarizing current pulses showed prolonged
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Fig. 1. Pulse-evoked response and cell morphology. (A) Pulse-evoked responses of a tonic cell showing continuous rhythmic ¢ring when depolarized and repolarizing sag when strongly hyperpolarized followed by a prolonged plateau-like rebound. A di¡erent type of rebound (i.e., slow spike) was evoked in another tonic neuron (a) that was insensitive to 1 WM TTX (b). The Vm was 354 mV and the current pulses were 30.4, 30.2 and 0.4 nA in A. In a, b the Vm was 383 mV and the current pulses were 0.2 nA in a and 0.4 nA in a and b. (B) Pulse-evoked responses of a phasic cell showing initial burst and oscillations (arrows) when depolarized, no sag when hyperpolarized, and a slow spike at the rebound. The Vm was 355 mV and the current pulses were 30.4, 30.2 and 0.2 nA. (C) Pulse-evoked responses of a phasic^tonic neuron showing initial burst and adapted response. Large sag and no rebound were evoked when hyperpolarized. (D, E) Computer reconstruction of ‘tonic’ and ‘phasic’ cells, respectively. Phasic cells were dye-coupled. (F) Superimposed representative APs of tonic and phasic neurons scaled to identical peak amplitude. Dotted lines indicate Vm in all ¢gures.
after hyperpolarization (Fig. 1A). Inward recti¢cation and a depolarizing sag characterized the responses evoked by hyperpolarizing pulses s 380 mV (Fig. 1A). A prolonged plateau (394.3 ms X 108.5; 12.1 mV X 6.7) followed at hyperpolarizing pulse o¡ (17/66, 26%; Fig. 1A), or a depolarizing current pulse evoked a low threshold spike when the cell was hyperpolarized (49/66, 74%; s 30.2 nA; Fig. 1Aa). The slow spike and the plateau
were not modi¢ed by 1.0 WM TTX (n = 4; Fig. 1Ab), but were abolished in Ca2þ -free solution (not shown; cf. Nakanishi et al., 1987; Osuka et al., 2001). High-intensity current pulses ( s 0.8 nA) could transiently induce high AP rates of W300 Hz (9/19, 47%; Fig. 2B), but maximum sustained ¢ring rates were usually under 100 Hz (Fig. 2C). Even higher-intensity pulses ( s 1.2 nA) induced a marked initial depolarization
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Fig. 2. E¡ect of strong depolarization ; tonic cells. (A) High-intensity depolarizing pulses evoked AP bursts and subthreshold Vm oscillations (inset). (B) A high, sustained AP rate was evoked in the same neuron by a depolarizing pulse of lower intensity. The Vm was 362 mV and the depo1arizing currents were 1.2 and 0.8 nA in A and B, respectively. (C) A sustained rhythmic ¢ring evoked by depolarization. (D) Higher pulse intensity evoked a high-rate initial burst, followed by oscillations, then a burst, and eventually the cell was silenced. The Vm was 352 mV and the depo1arizing currents were 0.8 and 1.4 nA in C and D, respectively. In C and D the recording is interrupted 40 s. A and B, C and D, two cells.
(30^40 mV) topped by a brief AP burst at a very high rate (80.5 ms X 12.3;W300 Hz; n = 15). The Vm rapidly decayed ( 6 500 ms) and stabilized at a depolarization of 10^20 mV that evoked low-amplitude (3^8 mV) rhythmic Vm oscillations at 10^30 Hz and periodic AP bursts (rate 3^4 Hz, duration 30^300 ms, 3^10 APs) as those reported in other systems (Garc|¤a-Mun‹oz et al., 1993; Fig. 2A, D; n = 15). During the initial burst there was a rapid and pronounced reduction of the amplitude of successive APs and a gradual recovery of AP amplitudes in the following burst (Fig. 2A, D). Sustained depolarization of s 15 mV (n = 9) initially evoked bursting (as above) and then, after 10^20 s, silenced the cell. The silence persisted until the pulse was o¡ 40 s after the onset (Fig. 2D). The pre-stimulus membrane potential or spontaneous activity and the characteristic responses to low-intensity currents recovered rapidly ( 6 5 s) after the end of the high-intensity stimulation. Other neurons (26/104, 25%) were silent at rest, showed a Vm of 356.4 mV X 2.8, and ¢red a single
brief AP burst when depolarized above a 341 mV X 4.1 threshold (Fig. 1B). The burst response changed little with pulse intensity, increasing between 150^500 ms and discharging 4^15 APs. With large depolarization above 336.9 mV X 8.2 the burst was followed by dampened Vm oscillations (39 X 6 Hz; Fig. 1B). Sustained ¢ring was never evoked by prolonged depolarization in these cells. The absence of inward recti¢cation, and in particular the typical single high-rate initial AP burst without subsequent ¢ring when depolarized with prolonged pulses, characterized this cell type that will be termed ‘phasic’. The delayed inward recti¢cation and especially the sustained rhythmic ¢ring characterized the other cell group of spontaneously active and silent cells that will be termed ‘tonic’. Other di¡erences between phasic and tonic neurons were the Rin (79.9 M6 X 20.1; P 6 0.005) and the amplitude-duration of the AP (42.7 mV X 1.7, 2.1 ms X 0.8, respectively; P 6 0.05; Fig. 1F). Di¡erences in the mor-
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(n = 17) were tested with depolarizing and hyperpolarizing current pulses and assigned as tonic (n = 10) or phasic (n = 7), according to their electrophysiological response. Computer-reconstructed images of these carboxy£uorescein-¢lled neurons revealed clear morphological di¡erences between both cell soma and dendrites (Fig. 1D, ‘tonic’). Tonic cells had a small round soma (13.4 Wm X 3) with 6^8 thin radiating primary dendrites and long secondary and tertiary dendrites. Phasic cells had signi¢cantly bigger triangular or elongated somas (38.4 Wm X 7; P 6 0.05) with 2^4 thick primary dendrites usually emerging from the soma and thin long secondary dendrites. More than one phasic cell was sometimes dye-¢lled during a single recording-injection (2 pairs), suggesting the presence of gap junctions and electrical coupling between some phasic cells (Fig. 1E, ‘phasic’). Both types of cells also demonstrated di¡erences in the dendritic ¢eld. The area counted revealed that tonic cells had on average V2.6 times more branches than phasic cells (P 6 0.05) when measured at a comparable distance from the cell body. Synaptic responses
Fig. 3. Excitatory responses evoked by STN stimulation; tonic cells. (A) EPSP evoked by a single subthreshold stimulus. (B^D) As in A, but pulse pairs at gradually decreasing delay. (E, F) Responses evoked by 100-ms barrage at 30 Hz and 80 Hz, respectively. (G) E¡ect of longer-lasting high-frequency barrage (1-s duration, 120 Hz). Inset shows plateau depolarization and AP burst outlasting stimulation. EPSPs were subthreshold in A^G. A^D : same cell (Vm = 353 mV); E, F: another neuron (Vm = 357 mV); and G : a di¡erent cell (Vm = 350 mV). A^D : same scale bars; E, F: same scale bars.
phology and synaptic response also helped in the classi¢cation (see below). In a preliminary study performed in older rats (15^23 days old; see Experimental Procedures) the responses of STN neurons evoked by depolarizing and hyperpolarizing current pulses did not vary with those obtained in younger rats except for a signi¢cantly higher proportion of tonic (16/19, 84%; P 6 0.01) than phasic cells (3/19, 16%) and lower Vm (345.2 mV X 18.12; P 6 0.05) and Rin (53.8 M6 X 21.2; P 6 0.05). These results were not included. A small group of cells (7/104, 6.8%; Vm 359.1 mV X 5.3; Rin 122.6 M6 X 25.3; duration 1.9 ms X 0.3) showed a di¡erent response typi¢ed by an initial highrate burst of APs followed by a discharge at a much lower rate (Fig. 1C). These cells will be called ‘phasic^ tonic’ and displayed marked inward recti¢cation when hyperpolarized but showed no rebound. Due to their scarcity we could not dye-¢ll or analyze the synaptic response.
Both tonic and phasic neurons could exhibit EPSPs, but synaptic responses were signi¢cantly di¡erent in phasic (n = 12) and tonic (n = 22) cells. Estimated tonic single EPSP’s duration was 33.3 ms X 7.3, amplitude 6.8 mV X 1.6, and the onset latency 1.1 ms X 0.5, while for phasic cells the duration was 15.0 X 9.0 ms (P 6 0.01), the amplitude 3.5 X 1.8 mV (P 6 0.01) and the latency of 1.6 X 0.3 ms (not statistically di¡erent). In tonic neurons single subthreshold stimuli evoked a slowly rising and decaying EPSP (Fig. 3A) and paired-pulse facilitation (PPF) was always present (Fig. 3B). Reducing the interval between a pair of stimuli markedly increased PPF, and a burst followed by a 50^80-ms duration depolarization could be evoked (Fig. 3C, D). Responses evoked by longer duration 100-ms barrages showed frequencydependent facilitation and summation of successive EPSPs followed by a gradual decay to the resting Vm (30 Hz; Fig. 3E). A higher frequency (80 Hz) barrage evoked a larger depolarization that reached AP threshold and elicited 1-to-1 ¢rings. This response was followed by a prolonged (80^150 ms) depolarization that outlasted the stimulation (Fig. 3F ; Nakanishi et al., 1987; Osuka et al., 2001). When long 1-s-duration barrages were used tonic neurons could transiently follow stimulation frequency of up to 300 Hz (3/20, 15%), but usually the upper frequency limit for frequent AP failures was 100^130 Hz (17/20, 85%; Fig. 3G). The response was characterized by a sustained depolarization that outlasted the synaptic stimulation (10^20 mV; W800 ms) in the form of a plateau topped by a high-rate AP burst (Fig. 3G, inset; cf. Nakanishi et al., 1987; Osuka et al., 2001). E¡ects of sustained high-frequency STN stimulation
Morphology Cells labeled intracellularly with carboxy£uorescein
Tonic STN neurons (n = 9) followed sustained highfrequency stimulation (100^130 Hz), as those used to
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Fig. 4. E¡ects of high-frequency STN stimulation. (A) AP bursts riding on summated EPSPs that generated a sustained depolarization were elicited 10 s, 20 s and 40 s after the onset of stimulation at 70 Hz. (B) Responses evoked 0 s, 15 s and 20 s after stimulation ‘on’ at 120 Hz. The initial sustained activity with few AP failures (0 s) switched to a bursting mode (15 s), and the neuron was then silenced (20 s). The responses rode on a sustained depolarization larger than in A. (C) Summary data from tonic neurons (n = 6) during STN stimulation at 120 Hz. (D) Response of phasic cell evoked by a prolonged highfrequency barrage (100 Hz), starting at the beginning of the record as in A and B. A, B and D: three di¡erent neurons. Dotted lines indicate Vm 359 mV in A, 355 mV in B, and 362 mV in D.
treat parkinsonian symptoms (Benabid et al., 1996; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998), with very few AP failures during 5^15 s, then switched to a bursting mode and eventually, 10^25 s later, ceased ¢ring (Fig. 4B, C). The suppression of AP activity by tetanic STN stimulation was essentially identical in spontaneously active (n = 3) and previously silent depolarized (n = 6) tonic neurons. The EPSP summation induced a sustained depolarization (18.1 mV X 4.3; n = 6) that changed little throughout the continuous stimulation, implying no presynaptic frequencydependent depression and suggesting that failures to evoke APs were due to a postsynaptic e¡ect. The prestimulus resting potential or activity and the characteristic responses to low-frequency stimulation recovered rapidly ( 6 10 s) after the end of the tetanic stimulation that lasted up to 60 s.
Sustained stimulation at a lower frequency ( 6 90 Hz) also induced a persistent depolarization (13.4 mV X 1.8; n = 12) that was followed with few AP failures during 5^15 s and then switched to a bursting mode (as above) that persisted until the stimulation was stopped 40 s afterwards (Fig. 4A). Higher stimulation intensities that evoked suprathreshold EPSP had similar bursting and silencing e¡ects when applied at low (n = 2) and high frequency (n = 1), respectively. In phasic neurons the PPF was not observed and stimulation with barrages did not show the frequency- and time-dependent EPSP e¡ects observed in tonic cells. Moreover, they responded with a single initial burst to tetanic stimulation of the STN (Fig. 4D). A group of tonic cells (n = 8) only responded to isolated stimuli with brief (20.4 ms X 6.9) inhibitory postsynaptic potentials (IPSPs) that were never recorded in
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Fig. 5. IPSPs evoked by intranuclear stimulation in tonic neurons. (A) Brief low-amplitude IPSP in an active tonic neuron. (B) Same as above, but higher-intensity stimulation evoked a larger IPSP that silenced the cell. The IPSP was followed by subthreshold oscillations. (C) Imposed depolarizing and hyperpolarizing currents revealed that the IPSP reversed at 363 mV. Resting potentials were 351 mV in A and 360 mV in B. A, B and C, three cells.
phasic neurons. The IPSPs could transiently inhibit ¢rings (Fig. 5A) and even silence tonic cells (Fig. 5B). Imposed Vm modi¢cations by current injection revealed that the IPSP reverted at W363 mV in this cell (Fig. 5C); measurements in ¢ve cells showed a mean reversal at 370.1 mV X 9.9.
DISCUSSION
We show that tonic STN neurons cease ¢ring during prolonged sustained high-frequency electrical stimulation of the STN ( s 25 s; s 100 Hz), a cellular mechanism that may be responsible for the alleviation of parkinsonian symptoms in human patients and in MPTP-treated primates when a similar electrical stimulation is applied (Benazzouz et al., 1992; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Beurrier et al. (2001) have shown a prolonged post-stimulation frequency-dependent silencing e¡ect of brief high-intensity tetanus in STN cells, but technical prob-
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lems precluded their recordings during tetanic stimulation. These authors suggest that the post-stimulus silence re£ects the after-e¡ects of the tetanic stimulation that also silenced the neuron, thus possibly explaining the clinical e¡ects. We now show that the prolonged STN tetanic stimulation induces a sustained depolarization with an initial high-frequency discharge, followed by bursts and ¢nally total inhibition of ¢rings throughout the stimulation. Interestingly, high-frequency stimulation, lesion and selective drug STN cell inactivation have comparable therapeutic e¡ects on parkinsonian symptoms (e.g., Baron et al., 2002; Benazzouz et al., 1992; Bergman et al., 1994), suggesting that they act by suppressing the output of the STN. Therefore, the suppression of AP activity we report ¢ts in well with those ¢ndings. Indeed, tetanic stimulation induces inhibition of activity in the nuclei where the STN projects (Benazzouz and Hallet, 2000). However, a recent contradictory report demonstrates an increase of glutamate levels in the globus pallidus of anaesthetized in vivo rats (Windels et al., 2000), probably implying an excitatory action of the STN stimulation. Nevertheless, the stimuli used by Windels et al. (2000) could have activated regions outside the STN because the current intensity was about one order of magnitude in excess of that used in Parkinson’s patients (Benabid et al., 1996; Figueiras-Me¤ndez et al., 2002; Magarin‹os-Ascone et al., 2000) and much higher than those used in the present experiments. Although circuital interactions cannot be ruled out (see below), the capacity of the tetanic stimulation to silence STN neurons is probably postsynaptic as the size, duration and summation of the EPSPs did not change during successive stimuli, thus it can be considered mainly due to an intrinsic property of tonic cells. Indeed, the late response evoked by tetanic stimulation and current injection was essentially identical, suggesting that similar postsynaptic cellular mechanisms were at work and that a presynaptic frequency-dependent depression did not contribute (present results). However, there were also marked di¡erences in the initial responses evoked by injected current and tetanic STN stimulation. Pulses evoked a large initial depolarization that was absent in the responses evoked by tetanic stimulation; eventually the Vm recovered and stabilized after the large initial depolarization at values as those reached during tetanic STN stimulation. These di¡erences could be due to the dissimilar conductances activated by the dendritic versus somatic site of origin of the synaptic and somatic depolarization, respectively, as occurs in other systems (e.g., Wong and Stewart, 1992). Although we have not investigated the postsynaptic mechanisms underlying the silencing e¡ect of the highfrequency STN stimulation, they probably involve the gradual inactivation of Naþ - and Ca2þ -mediated voltage-gated conductances that underlie the intrinsic rhythmic discharge of STN neurons (e.g., Beurrier et al., 1999; Beurrier et al., 2000; Bevan and Wilson, 1999), as has been shown to underlie the silencing post-e¡ect of STN stimulation by Beurrier et al. (2001). These authors show that synaptic interactions do not contribute to the silenc-
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ing e¡ect because they used intensities that directly activated STN neurons and could thus evoke the e¡ect in conditions of synaptic transmission block. We now show that similar e¡ects may be obtained with stimulation intensities that generate subthreshold EPSPs in the recorded neuron when presented in isolation, suggesting that the depolarization and initial high-frequency activity evoked by the EPSP facilitation and summation induced by the subthreshold tetanic stimulation probably trigger the same postsynaptic mechanism discovered by Beurrier et al. (2001). Stimulation of the STN at lower rates, 6 80 Hz, that depolarized STN cells and induced bursting discharges (present results), has been shown to increase tremor when tested in Parkinson’s patients with implanted electrodes (Struppler, 1989). Moreover, the capacity to switch to a bursting mode may have clinical importance because a similar bursting discharge typi¢es the abnormal activity recorded in the STN in rats (Beurrier et al., 1999) and in Parkinson’s patients (e.g., Hutchison et al., 1998; Magarin‹os-Ascone et al., 2000). A switch to bursting mode was also induced by sustained membrane hyperpolarization in tonic STN neurons (Beurrier et al., 1999), a type of activity we did not try to test. We cannot exclude that other mechanisms contribute to the suppression of AP activity by high-frequency stimulation of the STN. Indeed, synaptic inhibition (i.e., single IPSPs) could abort the ongoing activity of some tonic STN neurons (Bevan et al., 2002; present results), suggesting that they could also contribute to the stopping of tonic cell ¢rings during high-frequency stimulation. We found a W370-mV reversal potential for the IPSPs, a value that is between the equilibrium potentials of W379 mV and W352 mV estimated by Bevan et al. (2000, 2002), using the perforated and the whole-cell patch techniques, respectively, and recorded at 37‡C. The di¡erences may be attributed to the recording methods, to di¡erences in the ionic composition of the intraand extracellular solutions (Bevan and Wilson, 1999; Bevan et al., 2000; Bevan et al., 2002) or to temperature di¡erence in the recording chamber (Barnes, 1984; Biswas and Poddar, 1990; Korn, 1987). The demonstration of three cell types with distinct ¢ring behavior is a new ¢nding that may be roughly compared with the three ¢ring patterns called ‘normal’, ‘bursting’ and ‘mixed’ by Hollerman and Grace (1992). Moreover, the confocal analysis (present results) suggests distinct morphological characterization and agrees with the cell types reported in primates (Rafols and Fox, 1976) and cats (Iwahori, 1978). The principal ‘radiating’ neurons of primates (Rafols and Fox, 1976) seem to correspond to the ‘type I medium-sized’ described in cats (Iwahori, 1978) and are the main constituents of the STN. Their polygonal or oval cell body, soma size, dendrite origin from the soma and spines match the morphology of tonic cells (present results). The ‘elongated fusiform’ neurons described in primates (Rafols and Fox, 1976) are probably equivalent to the ‘type II’ described in cats (Iwahori, 1978) and seem to correspond to phasic neurons (present results) because they o¡er similar soma size and dendrite morphology. However,
our ¢ndings contrasts with reports obtained in rats that showed just one type of STN cell (Afsharpour, 1985). The only conceivable explanation could be the younger rats we used. In a preliminary study performed in a small sample of older rats we recorded a smaller percentage of phasic cells. Nevertheless, Afsharpour (1985) did not rule out the existence of other cell types. He also reported a small sample of STN neurons (11.1%) in old rats (30^75 days old) showing two main dendrites originating from opposite poles, as our phasic cells, and attributed the di¡erence to the probable limitations in the Golgi technique he used (cf., Iwahori, 1978; Rafols and Fox, 1976). A distinguishing electrophysiological feature of tonic cells that may be important functionally is that they can reach very high and sustained ¢ring rates when depolarized by current injection (e.g., Bevan and Wilson, 1999; Nakanishi et al., 1987; present results) and when stimulated synaptically at high frequency (present results). Indeed, sustained high AP rates were recorded from STN neurons during passive and voluntary limb movements in monkeys and humans (Bergman et al., 1994; Magarin‹os-Ascone et al., 2000), suggesting that those cells were of the tonic type and that they contribute to the control of movement. Phasic cells may participate during the execution of brief and fast (ballistic) movements. They only ¢re a brief burst and thereafter do not respond to sustained STN stimulation, indicating that their contribution to the remission of parkinsonian symptoms by tetanic stimulation of the STN may not be as important as that of tonic cells. The dye coupling of some phasic cells revealed an intercommunication system, probably mediated by gap junctions, that could aid in synchronizing the activity of these cells (Dudek et al., 1986), as demonstrated in other systems (e.g., Galarreta and Hestrin, 1998).
CONCLUSION
The STN in young rats is a complex structure composed mainly of two neuron types with tonic and phasic ¢ring modes and a di¡erent morphology. The most abundant cell, the tonic type, may determine the main functional characteristics of the STN and, when ¢ring in abnormal bursts, may govern the contribution of the STN to parkinsonian symptoms. Moreover, the remission of Parkinsonism by treatment with high-frequency electrical stimulation of the STN in humans may primarily reside on the response properties of tonic neurons and especially in their capacity to stop ¢ring during tetanic stimulation of the STN.
Acknowledgements$Work supported by ‘Fondo de Investigaciones Sanitarias de la Seguridad Social’, Grant 94/0527 to C.M.-A. and ‘Direccio¤n General de Investigacio¤n Cient|¤¢ca y Tecnolo¤gica’ and ‘Comunidad Auto¤noma de Madrid’, grants PB95-0031 and 08.5/0038/98 to W.B. J.H.P. and O.M., on leave from ‘Facultad de Medicina, Universidad de Buenos Aires, Argentina’ and from ‘Instituto de Investigaciones Biolo¤gicas Clemente Estable, Montevideo, Uruguay’, were supported
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by ‘Consejo Superior de Investigacio¤n Cient|¤¢ca’, and ‘Ministerio de Educacio¤n y Ciencia’ grants with W.B. We thank P.R.
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Williams for revision of the English language and V. Abraira Santos for helping in the statistical analysis.
REFERENCES
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Bergman, H., Wichmann, T., Karmon, B., DeLong, M.R., 1994. The primate subthalamic nucleus. II. Neuronal activity in the MPTP model of parkinsonian. J. Neurophysiol. 72, 507^520. Beurrier, C., Bioulac, B., Audin, J., Hammond, C., 2001. High-frequency stimulation produces a transient blockade of voltage-gated currents in subthalamic neurons. J. Neurophysiol. 85, 1351^1356. Beurrier, C., Bioulac, B., Hammond, C., 2000. Slowly inactivating sodium current (INaP ) underlies single-spike activity in rat subthalamic neurons. J. Neurophysiol. 83, 1951^1957. Beurrier, C., Congar, P., Bioulac, B., Hammond, C., 1999. Subthalamic nucleus neurons switch from single-spike activity to burst-¢ring mode. J. Neurosci. 19, 599^609. Bevan, M.D., Magill, P.J., Hallworth, N.E., Bolam, J.P., Wilson, C.J., 2002. Regulation of the timing and pattern of action potential generation in rat subthalamic neurons in vitro by GABA-A IPSPs. J. Neurophysiol. 87, 1348^1362. Bevan, M.D., Wilson, C.J., 1999. Mechanism underlying spontaneous oscillation and rhytmic ¢ring in rat subthalamic neurons. J. Neurosci. 19, 7617^7628. Bevan, M.D., Wilson, C.J., Bolam, J.P., Magill, P.J., 2000. Equilibrium potential of GABAA current and implications for rebound burst ¢ring in rat subthalamic neurons in vitro. J. Neurophysiol. 83, 3169^3172. Biswas, S., Poddar, M.K., 1990. E¡ect of short-and-long term exposure to low environmental temperature on brain regional GABA metabolism. Neurochem. Res. 15, 815^820. DeLong, M.R., 1990. Primate models of movement disorders of basal ganglia origin. Trends Neurosci. 13, 281^285. Dudek, F.E., Snow, R.W., Taylor, C.P., 1986. Role of electrical interactions in synchronization of epileptiform bursts. Adv. Neurol. 44, 593^617. Elston, G.N., Tweedale, R., Rosa, M.G.P., 1999. Cellular heterogeneity in cerebral cortex: a study of the morphology of pyramidal neurones in visual areas of the marmoset monkey. J. Comp. Neurol. 415, 33^51. Figueiras-Me¤ndez, R., Regidor, I., Riva-Meana, C., Magarin‹os-Ascone, C.M., 2002. Further supporting evidence of bene¢cial subthalamic stimulation in Parkinson’s patients. Neurology 58, 469^470. Galarreta, M., Hestrin, S., 1998. Frequency-dependent synaptic depression and the balance of excitation and inhibition in the neocortex. Nat. Neurosci. 1, 587^594. Garc|¤a-Mun‹oz, A.L., Barrio, L., Bun‹o, W., 1993. Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and £uctuation entrained by sinusoidal current. Exp. Brain Res. 97, 325^333. Hammond, C., Yelnik, J., 1983. Intracellular labeling of rat subthalamic neurones with horseradish peroxidase : computer analysis of dendrites and characterization of axon arborization. Neuroscience 8, 781^790. Hollerman, J.R., Grace, A.A., 1992. Subthalamic nucleus cell ¢ring in the 6-OHDA-treated rat: basal activity and response to haloperidol. Brain Res. 590, 291^299. Hutchison, W.D., Allan, R.J., Opitz, H., Levy, R., Dostrovsky, J.O., Lang, A.E., Lozano, A.M., 1998. Neurophysiological identi¢cation of the subthalamic nucleus in surgery for Parkinson’s disease. Ann. Neurol. 44, 622^628. Iwahori, N., 1978. A Golgy study on the subthalamic nucleus of the cat. J. Comp. Neurol. 182, 383^398. Korn, S.J., 1987. Epileptiform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition. J. Neurophysiol. 57, 325^340. Limousin, P., Pollak, P., Benazzouz, A., Ho¡mann, D., Le Bas, J.F., Broussolle, E., Perret, J.E., Benabid, A.L., 1995. E¡ect on Parkinsonian signs and symptoms of bilateral subthalamic nucleus stimulation. Lancet 345, 91^95. Magarin‹os-Ascone, C., Figueiras-Me¤ndez, R., Riva-Meana, C., Co¤rdoba-Ferna¤ndez, A., 2000. Subthalamic neuron activity related to tremor and movement in Parkinson’s disease. Eur. J. Neurosci. 12, 2597^2607. Nakanishi, H., Kita, H., Kitai, S.T., 1987. Electrical membrane properties of rat subthalamic neurons in an in vitro slice preparation. Brain Res. 437, 35^44. Nun‹ez, A., Bun‹o, W., 1999. In vitro electrophysiological properties of rat dorsal column nuclei neurons. Eur. J. Neurosci. 11, 1865^1876. Osuka, T., Murakamy, F., Song, W.J., 2001. Excitatory postsynaptic potentials a trigger plateau potential in rat subthalamic neurons at hyperpolarized states. J. Neurophysiol. 86, 1816^1825. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego. Rafols, J.A., Fox, C., 1976. The neurons in the primate subthalamic nucleus: a Golgi and electron microscopic study. J. Comp. Neurol. 168, 75^ 91. Rodr|¤guez, M.C., Oroz, J., Guridi, J., Alvarez, M., Mewes, K., Macias, R., Vitek, J., DeLong, M.R., Obeso, J.A., 1998. The subthalamic nucleus and tremor in Parkinson’s disease. Mov. Disord. 13, 111^118. Struppler, A., 1989. Stereoencephalotomy and control of skeletal muscle tone. Proc. Microelectrode Meet. Stereotact. Funct. Neurosurg. 52, 205^ 218. Windels, F., Bruet, N., Poupard, A., Urbain, N., Chouvet, G., Feuerstein, C., Savasta, M., 2000. E¡ect of high frequency stimulation of subthalamic nucleus on extracellular glutamate and GABA in substantia nigra and globus pallidus in the normal rat. Eur. J. Neurosci. 12, 4141^4146. Wong, R.K., Stewart, M., 1992. Di¡erent ¢ring patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus. J. Physiol. 457, 675^687. (Accepted 1 August 2002)
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Neuroscience Vol. 115, No. 4, pp. 1109^1117, 2002 L 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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HIGH-FREQUENCY STIMULATION OF THE SUBTHALAMIC NUCLEUS SILENCES SUBTHALAMIC NEURONS: A POSSIBLE CELLULAR MECHANISM IN PARKINSON’S DISEASE OS-ASCONE,a J. H. PAZO,b O. MACADARb and W. BUN Ob C. MAGARIN a
Neurolog|¤a Experimental (Unidad Asociada al Instituto Cajal, CSIC), Depto. de Investigacio¤n, Hospital Ramo¤n y Cajal, Ctra. Colmenar, Madrid 28034, Spain b
Instituto Cajal, CSIC, Madrid, Spain
Abstract
to parkinsonian symptoms (Benazzouz et al., 1992; Bergman et al., 1994). The recent di¡usion of high-frequency electrical stimulation (i.e., ‘tetanic’) of the STN in the treatment of parkinsonian motor symptoms in humans and MPTP-treated primates (Benazzouz et al., 1992; DeLong, 1990; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998) has renewed interest in this structure, but the underlying cellular mechanisms of this stimulation-induced remission of parkinsonian symptoms are not totally understood. Tetanic stimulation and destruction of the STN have similar ameliorating e¡ects on parkinsonian motor symptoms (e.g., Benazzouz et al., 1992; Bergman et al., 1994), suggesting that both manipulations act by blocking the electrical activity of the nucleus. A prolonged post-stimulus frequency-dependent silencing e¡ect of STN stimulation induced by a brief high-intensity electrical stimulation has recently been demonstrated in an in vitro rat preparation by Beurrier et al. (2001). The authors suggested that the same silencing e¡ect may occur during stimulation, but they could not verify the suppression of action potential (AP) activity in their preparation due to stimulus artifacts. Parkinsonian symptoms in patients only fade during STN stimulation and resume rapidly once stimulation is
The subthalamic nucleus (STN) plays an important role in motor control and in the genesis of Parkinson’s disease (e.g., DeLong, 1990; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000). Loss of substantia nigra dopaminergic neurons in Parkinson’s disease results in a reduction of activity in the thalamus partly due to an increased bursting activity of STN cells (DeLong, 1990). The abnormally augmented burst ¢ring of STN cells was correlated with tremor in Parkinson patients (Hutchison et al., 1998; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998) and in primates treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Benazzouz et al., 1992; Bergman et al., 1994), a drug that selectively destroys the dopaminergic cells of the substantia nigra. This correlation has suggested that the bursting of STN neurons could contribute
*Corresponding author. Tel. : +34-91-336-8320. E-mail address: [email protected] (C. Magarin‹os-Ascone). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; AP, action potential ; EPSP, excitatory postsynaptic potential ; IPSP, inhibitory postsynaptic potential; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; PPF, paired-pulse facilitation; Rin , input resistance; STN, subthalamic nucleus; TTX, tetrodotoxin; Vm, membrane potential. 1109
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stopped (Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Therefore, to test the suggestion made by Beurrier et al. (2001), it is highly important to record STN cell activity during stimulation. Moreover, the STN cell types that are involved in the reversion of parkinsonian symptoms should be de¢ned because their functional properties are still open to question. Indeed, the neuronal pool attributed to the STN varies from one (Afsharpour, 1985; Hammond and Yelnik, 1983), two (Rafols and Fox, 1976) or three morphologically di¡erent cell types (Iwahori, 1978). In addition, extracellular recordings in rats indicate three ¢ring patterns de¢ned as ‘normal’, ‘bursting’ and ‘mixed’ (Hollerman and Grace, 1992), but only a single tonic type was reported in in vitro preparations (e.g., Bevan and Wilson, 1999; Nakanishi et al., 1987) that could switch to a bursting mode (Beurrier et al., 1999). Therefore, we reinvestigated the electrophysiological characteristics of STN neurons, paying special attention to the e¡ects observed during prolonged tetanic stimulation of the STN as used in humans to treat parkinsonian symptoms.
EXPERIMENTAL PROCEDURES
Brown Norway rats (9^14 days old) were anesthetized with ether and decapitated immediately after disappearance of the pinch re£ex. The brain was rapidly removed and submerged in a vial with cold (4‡C) arti¢cial cerebrospinal £uid (ACSF) that contained (in mM) 124 NaCl, 2.69 KCl, 1.25 KH2 PO4 , 2.0 MgSO4 , 26.0 NaHCO3 , 2.0 CaCl2 , and 10.0 glucose. The pH was adjusted to 7.4 by gassing with 95% O2 and 5% CO2 . A slab that contained the brainstem at the level of the STN was cut transversely and ¢xed with cyanoacrylate to the base of a chamber containing cold ACSF. Transverse 350-Wm slices including the STN were cut with a vibratome (Pelco 101, Series 1000, St. Louis, MO, USA). After incubation for s 1 h in gassed ACSF at 21^23‡C in separate chambers, slices were moved to a recording chamber (2 ml), viewed under a dissecting microscope (Nikon/SMZ-1, Tokyo, Japan) and superfused at 2^3 ml/min with gassed ACSF at 30^32‡C. In a few experiments the control solution was substituted by a nominally Ca2þ -free ACSF in which CaCl2 was substituted with MgCl2 (2.0 mM). In some cases voltage-gated Naþ conductances were blocked with tetrodotoxin (TTX, 1.0 WM) added to the control ACSF. All chemicals were purchased from Sigma (St. Louis, MO, USA). The STN was identi¢ed by its position within the slice according to the Paxinos and Watson (1998) atlas. Recording pipettes were fabricated from borosilicate glass capillaries (R-Series 1B150F-4, WPI, Sarasota, FL, USA) with a Brown^Flamming model P-80 micropipette puller (Sutter Instruments, Novato, CA, USA). Pipettes back-¢lled either with potassium acetate (3.0 M; 90^120 M6) or with 2% carboxy£uorescein in potassium acetate (1.0 M; 150^200 M6) were connected to an Axoclamp-2B ampli¢er (Axon Instruments, Foster City, CA, USA). Pipette tips were placed under direct visualization of the STN and recordings were in the bridge mode. Carboxy£uorescein was injected by passing 0.1^0.3 nA, 300^400 ms hyperpolarizing pulses at 1 Hz (Nun‹ez and Bun‹o, 1999). Electrical stimulation (GRASS S88, Quincy, USA) was through a pair of 80-Wm-diameter Nichrome wires insulated except at the tip and placed on the STN under direct visualization. This ‘intranuclear’ placement of stimulation electrodes was aimed at reproducing the stimulation used in the treatment of parkinsonian symptoms (e.g., Benabid et al., 1996; Benazzouz et al., 1992; FigueirasMe¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al.,
1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Stimulation intensity (0.2^1.0 WA) was adjusted to evoke excitatory postsynaptic potentials (EPSPs) that were subthreshold for eliciting APs when presented in isolation. Exceptionally, suprathreshold stimuli were also tested. Since we aimed at analyzing synaptic responses, stimulations that triggered ‘direct’ or antidromic responses were rejected. To exclude an artifact caused by the stimulating electrodes (failing to generate APs in the axons or not propagating to the terminal during high-frequency stimulation), recordings that revealed repeated EPSP failures were rejected and the stimulation intensity readjusted until stimulations devoid of failures were obtained. Data were low-pass ¢ltered (3 kHz) and fed to a 486/PC computer through a TL-1DMA interface board. The pClamp programs (Axon Instruments, Foster City, CA, USA) were used to record signals, to control stimulation, and to analyze data. Prolonged transmembrane current pulses were generated with the Grass stimulator. All values are expressed as the mean X S.D. Young rats were used because the proportion of successful cell impalements with the required membrane potential (Vm), overshooting APs and input resistance (Rin ) was much higher than in preliminary experiments using older rats (see Results). The slices containing carboxy£uorescein-¢lled cells were ¢xed in a mixture of 4% paraformaldehyde in 0.1 M phosphate bu¡er (pH 7.4). A Leica TCS 4D (Wetzlar, Germany) confocal laser-scanning microscope with an argon/kryptonmixed gas laser with excitation peak at 488 nm was used to determine the morphological characteristics of the carboxy£uorescein-labelled neurons. The confocal microscope was associated with a Leitz DMIRB £uorescence inverted microscope (Wetzlar, Germany) equipped with oil immersion objectives and a ¢lter block for carboxy£uorescein. Accumulating stacks of 12 successive images, separated by 3 or 4 Wm, allowed image reconstruction (e.g., Fig. 1D, E). To estimate morphological di¡erences between the basal dendritic ¢elds we counted the number of dendritic intersections with concentric circles centered on the cell body with 25-Wm-increasing radii (Elston et al., 1999). Statistical comparisons between samples were made using the Mann^Whitney U-test. All experiments in this study conformed to International Guidelines on the ethical use of animals and every e¡ort was made to minimize the su¡ering and number of animals used.
RESULTS
Recordings with a Vm s 348 mV and overshooting APs were obtained from 104 STN neurons. Spontaneous and pulse-evoked AP activity The majority of the neurons (71/104, 68.3%) ¢red APs rhythmically and continuously either spontaneously or when depolarized with sustained injection of outward current. Most of these neurons were silent at rest (53/ 71, 75%) and showed a Vm of 355.6 mV X 3.5. Spontaneously silent cells ¢red rhythmically at rates of 9.2 Hz X 7.8 when depolarized above a 346 mV X 3.8 threshold (see below, however). The rest of the cells (18/71, 25%) were spontaneously active and ¢red rhythmically (14.9 Hz X 6.3). The Rin (98.4 M6 X 18.9) and the AP amplitude-duration (59.6 mV X 2.1, at ‘take-o¡’ and 1.1 ms X 0.2, at half amplitude) were not statistically di¡erent in active and silent cells. Moreover, other response properties described below were essentially identical in spontaneously silent and active cells. The responses evoked by depolarizing current pulses showed prolonged
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Fig. 1. Pulse-evoked response and cell morphology. (A) Pulse-evoked responses of a tonic cell showing continuous rhythmic ¢ring when depolarized and repolarizing sag when strongly hyperpolarized followed by a prolonged plateau-like rebound. A di¡erent type of rebound (i.e., slow spike) was evoked in another tonic neuron (a) that was insensitive to 1 WM TTX (b). The Vm was 354 mV and the current pulses were 30.4, 30.2 and 0.4 nA in A. In a, b the Vm was 383 mV and the current pulses were 0.2 nA in a and 0.4 nA in a and b. (B) Pulse-evoked responses of a phasic cell showing initial burst and oscillations (arrows) when depolarized, no sag when hyperpolarized, and a slow spike at the rebound. The Vm was 355 mV and the current pulses were 30.4, 30.2 and 0.2 nA. (C) Pulse-evoked responses of a phasic^tonic neuron showing initial burst and adapted response. Large sag and no rebound were evoked when hyperpolarized. (D, E) Computer reconstruction of ‘tonic’ and ‘phasic’ cells, respectively. Phasic cells were dye-coupled. (F) Superimposed representative APs of tonic and phasic neurons scaled to identical peak amplitude. Dotted lines indicate Vm in all ¢gures.
after hyperpolarization (Fig. 1A). Inward recti¢cation and a depolarizing sag characterized the responses evoked by hyperpolarizing pulses s 380 mV (Fig. 1A). A prolonged plateau (394.3 ms X 108.5; 12.1 mV X 6.7) followed at hyperpolarizing pulse o¡ (17/66, 26%; Fig. 1A), or a depolarizing current pulse evoked a low threshold spike when the cell was hyperpolarized (49/66, 74%; s 30.2 nA; Fig. 1Aa). The slow spike and the plateau
were not modi¢ed by 1.0 WM TTX (n = 4; Fig. 1Ab), but were abolished in Ca2þ -free solution (not shown; cf. Nakanishi et al., 1987; Osuka et al., 2001). High-intensity current pulses ( s 0.8 nA) could transiently induce high AP rates of W300 Hz (9/19, 47%; Fig. 2B), but maximum sustained ¢ring rates were usually under 100 Hz (Fig. 2C). Even higher-intensity pulses ( s 1.2 nA) induced a marked initial depolarization
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Fig. 2. E¡ect of strong depolarization ; tonic cells. (A) High-intensity depolarizing pulses evoked AP bursts and subthreshold Vm oscillations (inset). (B) A high, sustained AP rate was evoked in the same neuron by a depolarizing pulse of lower intensity. The Vm was 362 mV and the depo1arizing currents were 1.2 and 0.8 nA in A and B, respectively. (C) A sustained rhythmic ¢ring evoked by depolarization. (D) Higher pulse intensity evoked a high-rate initial burst, followed by oscillations, then a burst, and eventually the cell was silenced. The Vm was 352 mV and the depo1arizing currents were 0.8 and 1.4 nA in C and D, respectively. In C and D the recording is interrupted 40 s. A and B, C and D, two cells.
(30^40 mV) topped by a brief AP burst at a very high rate (80.5 ms X 12.3;W300 Hz; n = 15). The Vm rapidly decayed ( 6 500 ms) and stabilized at a depolarization of 10^20 mV that evoked low-amplitude (3^8 mV) rhythmic Vm oscillations at 10^30 Hz and periodic AP bursts (rate 3^4 Hz, duration 30^300 ms, 3^10 APs) as those reported in other systems (Garc|¤a-Mun‹oz et al., 1993; Fig. 2A, D; n = 15). During the initial burst there was a rapid and pronounced reduction of the amplitude of successive APs and a gradual recovery of AP amplitudes in the following burst (Fig. 2A, D). Sustained depolarization of s 15 mV (n = 9) initially evoked bursting (as above) and then, after 10^20 s, silenced the cell. The silence persisted until the pulse was o¡ 40 s after the onset (Fig. 2D). The pre-stimulus membrane potential or spontaneous activity and the characteristic responses to low-intensity currents recovered rapidly ( 6 5 s) after the end of the high-intensity stimulation. Other neurons (26/104, 25%) were silent at rest, showed a Vm of 356.4 mV X 2.8, and ¢red a single
brief AP burst when depolarized above a 341 mV X 4.1 threshold (Fig. 1B). The burst response changed little with pulse intensity, increasing between 150^500 ms and discharging 4^15 APs. With large depolarization above 336.9 mV X 8.2 the burst was followed by dampened Vm oscillations (39 X 6 Hz; Fig. 1B). Sustained ¢ring was never evoked by prolonged depolarization in these cells. The absence of inward recti¢cation, and in particular the typical single high-rate initial AP burst without subsequent ¢ring when depolarized with prolonged pulses, characterized this cell type that will be termed ‘phasic’. The delayed inward recti¢cation and especially the sustained rhythmic ¢ring characterized the other cell group of spontaneously active and silent cells that will be termed ‘tonic’. Other di¡erences between phasic and tonic neurons were the Rin (79.9 M6 X 20.1; P 6 0.005) and the amplitude-duration of the AP (42.7 mV X 1.7, 2.1 ms X 0.8, respectively; P 6 0.05; Fig. 1F). Di¡erences in the mor-
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(n = 17) were tested with depolarizing and hyperpolarizing current pulses and assigned as tonic (n = 10) or phasic (n = 7), according to their electrophysiological response. Computer-reconstructed images of these carboxy£uorescein-¢lled neurons revealed clear morphological di¡erences between both cell soma and dendrites (Fig. 1D, ‘tonic’). Tonic cells had a small round soma (13.4 Wm X 3) with 6^8 thin radiating primary dendrites and long secondary and tertiary dendrites. Phasic cells had signi¢cantly bigger triangular or elongated somas (38.4 Wm X 7; P 6 0.05) with 2^4 thick primary dendrites usually emerging from the soma and thin long secondary dendrites. More than one phasic cell was sometimes dye-¢lled during a single recording-injection (2 pairs), suggesting the presence of gap junctions and electrical coupling between some phasic cells (Fig. 1E, ‘phasic’). Both types of cells also demonstrated di¡erences in the dendritic ¢eld. The area counted revealed that tonic cells had on average V2.6 times more branches than phasic cells (P 6 0.05) when measured at a comparable distance from the cell body. Synaptic responses
Fig. 3. Excitatory responses evoked by STN stimulation; tonic cells. (A) EPSP evoked by a single subthreshold stimulus. (B^D) As in A, but pulse pairs at gradually decreasing delay. (E, F) Responses evoked by 100-ms barrage at 30 Hz and 80 Hz, respectively. (G) E¡ect of longer-lasting high-frequency barrage (1-s duration, 120 Hz). Inset shows plateau depolarization and AP burst outlasting stimulation. EPSPs were subthreshold in A^G. A^D : same cell (Vm = 353 mV); E, F: another neuron (Vm = 357 mV); and G : a di¡erent cell (Vm = 350 mV). A^D : same scale bars; E, F: same scale bars.
phology and synaptic response also helped in the classi¢cation (see below). In a preliminary study performed in older rats (15^23 days old; see Experimental Procedures) the responses of STN neurons evoked by depolarizing and hyperpolarizing current pulses did not vary with those obtained in younger rats except for a signi¢cantly higher proportion of tonic (16/19, 84%; P 6 0.01) than phasic cells (3/19, 16%) and lower Vm (345.2 mV X 18.12; P 6 0.05) and Rin (53.8 M6 X 21.2; P 6 0.05). These results were not included. A small group of cells (7/104, 6.8%; Vm 359.1 mV X 5.3; Rin 122.6 M6 X 25.3; duration 1.9 ms X 0.3) showed a di¡erent response typi¢ed by an initial highrate burst of APs followed by a discharge at a much lower rate (Fig. 1C). These cells will be called ‘phasic^ tonic’ and displayed marked inward recti¢cation when hyperpolarized but showed no rebound. Due to their scarcity we could not dye-¢ll or analyze the synaptic response.
Both tonic and phasic neurons could exhibit EPSPs, but synaptic responses were signi¢cantly di¡erent in phasic (n = 12) and tonic (n = 22) cells. Estimated tonic single EPSP’s duration was 33.3 ms X 7.3, amplitude 6.8 mV X 1.6, and the onset latency 1.1 ms X 0.5, while for phasic cells the duration was 15.0 X 9.0 ms (P 6 0.01), the amplitude 3.5 X 1.8 mV (P 6 0.01) and the latency of 1.6 X 0.3 ms (not statistically di¡erent). In tonic neurons single subthreshold stimuli evoked a slowly rising and decaying EPSP (Fig. 3A) and paired-pulse facilitation (PPF) was always present (Fig. 3B). Reducing the interval between a pair of stimuli markedly increased PPF, and a burst followed by a 50^80-ms duration depolarization could be evoked (Fig. 3C, D). Responses evoked by longer duration 100-ms barrages showed frequencydependent facilitation and summation of successive EPSPs followed by a gradual decay to the resting Vm (30 Hz; Fig. 3E). A higher frequency (80 Hz) barrage evoked a larger depolarization that reached AP threshold and elicited 1-to-1 ¢rings. This response was followed by a prolonged (80^150 ms) depolarization that outlasted the stimulation (Fig. 3F ; Nakanishi et al., 1987; Osuka et al., 2001). When long 1-s-duration barrages were used tonic neurons could transiently follow stimulation frequency of up to 300 Hz (3/20, 15%), but usually the upper frequency limit for frequent AP failures was 100^130 Hz (17/20, 85%; Fig. 3G). The response was characterized by a sustained depolarization that outlasted the synaptic stimulation (10^20 mV; W800 ms) in the form of a plateau topped by a high-rate AP burst (Fig. 3G, inset; cf. Nakanishi et al., 1987; Osuka et al., 2001). E¡ects of sustained high-frequency STN stimulation
Morphology Cells labeled intracellularly with carboxy£uorescein
Tonic STN neurons (n = 9) followed sustained highfrequency stimulation (100^130 Hz), as those used to
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C. Magarin‹os-Ascone et al.
Fig. 4. E¡ects of high-frequency STN stimulation. (A) AP bursts riding on summated EPSPs that generated a sustained depolarization were elicited 10 s, 20 s and 40 s after the onset of stimulation at 70 Hz. (B) Responses evoked 0 s, 15 s and 20 s after stimulation ‘on’ at 120 Hz. The initial sustained activity with few AP failures (0 s) switched to a bursting mode (15 s), and the neuron was then silenced (20 s). The responses rode on a sustained depolarization larger than in A. (C) Summary data from tonic neurons (n = 6) during STN stimulation at 120 Hz. (D) Response of phasic cell evoked by a prolonged highfrequency barrage (100 Hz), starting at the beginning of the record as in A and B. A, B and D: three di¡erent neurons. Dotted lines indicate Vm 359 mV in A, 355 mV in B, and 362 mV in D.
treat parkinsonian symptoms (Benabid et al., 1996; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998), with very few AP failures during 5^15 s, then switched to a bursting mode and eventually, 10^25 s later, ceased ¢ring (Fig. 4B, C). The suppression of AP activity by tetanic STN stimulation was essentially identical in spontaneously active (n = 3) and previously silent depolarized (n = 6) tonic neurons. The EPSP summation induced a sustained depolarization (18.1 mV X 4.3; n = 6) that changed little throughout the continuous stimulation, implying no presynaptic frequencydependent depression and suggesting that failures to evoke APs were due to a postsynaptic e¡ect. The prestimulus resting potential or activity and the characteristic responses to low-frequency stimulation recovered rapidly ( 6 10 s) after the end of the tetanic stimulation that lasted up to 60 s.
Sustained stimulation at a lower frequency ( 6 90 Hz) also induced a persistent depolarization (13.4 mV X 1.8; n = 12) that was followed with few AP failures during 5^15 s and then switched to a bursting mode (as above) that persisted until the stimulation was stopped 40 s afterwards (Fig. 4A). Higher stimulation intensities that evoked suprathreshold EPSP had similar bursting and silencing e¡ects when applied at low (n = 2) and high frequency (n = 1), respectively. In phasic neurons the PPF was not observed and stimulation with barrages did not show the frequency- and time-dependent EPSP e¡ects observed in tonic cells. Moreover, they responded with a single initial burst to tetanic stimulation of the STN (Fig. 4D). A group of tonic cells (n = 8) only responded to isolated stimuli with brief (20.4 ms X 6.9) inhibitory postsynaptic potentials (IPSPs) that were never recorded in
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A cellular mechanism in Parkinson’s disease
Fig. 5. IPSPs evoked by intranuclear stimulation in tonic neurons. (A) Brief low-amplitude IPSP in an active tonic neuron. (B) Same as above, but higher-intensity stimulation evoked a larger IPSP that silenced the cell. The IPSP was followed by subthreshold oscillations. (C) Imposed depolarizing and hyperpolarizing currents revealed that the IPSP reversed at 363 mV. Resting potentials were 351 mV in A and 360 mV in B. A, B and C, three cells.
phasic neurons. The IPSPs could transiently inhibit ¢rings (Fig. 5A) and even silence tonic cells (Fig. 5B). Imposed Vm modi¢cations by current injection revealed that the IPSP reverted at W363 mV in this cell (Fig. 5C); measurements in ¢ve cells showed a mean reversal at 370.1 mV X 9.9.
DISCUSSION
We show that tonic STN neurons cease ¢ring during prolonged sustained high-frequency electrical stimulation of the STN ( s 25 s; s 100 Hz), a cellular mechanism that may be responsible for the alleviation of parkinsonian symptoms in human patients and in MPTP-treated primates when a similar electrical stimulation is applied (Benazzouz et al., 1992; Figueiras-Me¤ndez et al., 2002; Hutchison et al., 1998; Limousin et al., 1995; Magarin‹os-Ascone et al., 2000; Rodr|¤guez et al., 1998). Beurrier et al. (2001) have shown a prolonged post-stimulation frequency-dependent silencing e¡ect of brief high-intensity tetanus in STN cells, but technical prob-
1115
lems precluded their recordings during tetanic stimulation. These authors suggest that the post-stimulus silence re£ects the after-e¡ects of the tetanic stimulation that also silenced the neuron, thus possibly explaining the clinical e¡ects. We now show that the prolonged STN tetanic stimulation induces a sustained depolarization with an initial high-frequency discharge, followed by bursts and ¢nally total inhibition of ¢rings throughout the stimulation. Interestingly, high-frequency stimulation, lesion and selective drug STN cell inactivation have comparable therapeutic e¡ects on parkinsonian symptoms (e.g., Baron et al., 2002; Benazzouz et al., 1992; Bergman et al., 1994), suggesting that they act by suppressing the output of the STN. Therefore, the suppression of AP activity we report ¢ts in well with those ¢ndings. Indeed, tetanic stimulation induces inhibition of activity in the nuclei where the STN projects (Benazzouz and Hallet, 2000). However, a recent contradictory report demonstrates an increase of glutamate levels in the globus pallidus of anaesthetized in vivo rats (Windels et al., 2000), probably implying an excitatory action of the STN stimulation. Nevertheless, the stimuli used by Windels et al. (2000) could have activated regions outside the STN because the current intensity was about one order of magnitude in excess of that used in Parkinson’s patients (Benabid et al., 1996; Figueiras-Me¤ndez et al., 2002; Magarin‹os-Ascone et al., 2000) and much higher than those used in the present experiments. Although circuital interactions cannot be ruled out (see below), the capacity of the tetanic stimulation to silence STN neurons is probably postsynaptic as the size, duration and summation of the EPSPs did not change during successive stimuli, thus it can be considered mainly due to an intrinsic property of tonic cells. Indeed, the late response evoked by tetanic stimulation and current injection was essentially identical, suggesting that similar postsynaptic cellular mechanisms were at work and that a presynaptic frequency-dependent depression did not contribute (present results). However, there were also marked di¡erences in the initial responses evoked by injected current and tetanic STN stimulation. Pulses evoked a large initial depolarization that was absent in the responses evoked by tetanic stimulation; eventually the Vm recovered and stabilized after the large initial depolarization at values as those reached during tetanic STN stimulation. These di¡erences could be due to the dissimilar conductances activated by the dendritic versus somatic site of origin of the synaptic and somatic depolarization, respectively, as occurs in other systems (e.g., Wong and Stewart, 1992). Although we have not investigated the postsynaptic mechanisms underlying the silencing e¡ect of the highfrequency STN stimulation, they probably involve the gradual inactivation of Naþ - and Ca2þ -mediated voltage-gated conductances that underlie the intrinsic rhythmic discharge of STN neurons (e.g., Beurrier et al., 1999; Beurrier et al., 2000; Bevan and Wilson, 1999), as has been shown to underlie the silencing post-e¡ect of STN stimulation by Beurrier et al. (2001). These authors show that synaptic interactions do not contribute to the silenc-
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C. Magarin‹os-Ascone et al.
ing e¡ect because they used intensities that directly activated STN neurons and could thus evoke the e¡ect in conditions of synaptic transmission block. We now show that similar e¡ects may be obtained with stimulation intensities that generate subthreshold EPSPs in the recorded neuron when presented in isolation, suggesting that the depolarization and initial high-frequency activity evoked by the EPSP facilitation and summation induced by the subthreshold tetanic stimulation probably trigger the same postsynaptic mechanism discovered by Beurrier et al. (2001). Stimulation of the STN at lower rates, 6 80 Hz, that depolarized STN cells and induced bursting discharges (present results), has been shown to increase tremor when tested in Parkinson’s patients with implanted electrodes (Struppler, 1989). Moreover, the capacity to switch to a bursting mode may have clinical importance because a similar bursting discharge typi¢es the abnormal activity recorded in the STN in rats (Beurrier et al., 1999) and in Parkinson’s patients (e.g., Hutchison et al., 1998; Magarin‹os-Ascone et al., 2000). A switch to bursting mode was also induced by sustained membrane hyperpolarization in tonic STN neurons (Beurrier et al., 1999), a type of activity we did not try to test. We cannot exclude that other mechanisms contribute to the suppression of AP activity by high-frequency stimulation of the STN. Indeed, synaptic inhibition (i.e., single IPSPs) could abort the ongoing activity of some tonic STN neurons (Bevan et al., 2002; present results), suggesting that they could also contribute to the stopping of tonic cell ¢rings during high-frequency stimulation. We found a W370-mV reversal potential for the IPSPs, a value that is between the equilibrium potentials of W379 mV and W352 mV estimated by Bevan et al. (2000, 2002), using the perforated and the whole-cell patch techniques, respectively, and recorded at 37‡C. The di¡erences may be attributed to the recording methods, to di¡erences in the ionic composition of the intraand extracellular solutions (Bevan and Wilson, 1999; Bevan et al., 2000; Bevan et al., 2002) or to temperature di¡erence in the recording chamber (Barnes, 1984; Biswas and Poddar, 1990; Korn, 1987). The demonstration of three cell types with distinct ¢ring behavior is a new ¢nding that may be roughly compared with the three ¢ring patterns called ‘normal’, ‘bursting’ and ‘mixed’ by Hollerman and Grace (1992). Moreover, the confocal analysis (present results) suggests distinct morphological characterization and agrees with the cell types reported in primates (Rafols and Fox, 1976) and cats (Iwahori, 1978). The principal ‘radiating’ neurons of primates (Rafols and Fox, 1976) seem to correspond to the ‘type I medium-sized’ described in cats (Iwahori, 1978) and are the main constituents of the STN. Their polygonal or oval cell body, soma size, dendrite origin from the soma and spines match the morphology of tonic cells (present results). The ‘elongated fusiform’ neurons described in primates (Rafols and Fox, 1976) are probably equivalent to the ‘type II’ described in cats (Iwahori, 1978) and seem to correspond to phasic neurons (present results) because they o¡er similar soma size and dendrite morphology. However,
our ¢ndings contrasts with reports obtained in rats that showed just one type of STN cell (Afsharpour, 1985). The only conceivable explanation could be the younger rats we used. In a preliminary study performed in a small sample of older rats we recorded a smaller percentage of phasic cells. Nevertheless, Afsharpour (1985) did not rule out the existence of other cell types. He also reported a small sample of STN neurons (11.1%) in old rats (30^75 days old) showing two main dendrites originating from opposite poles, as our phasic cells, and attributed the di¡erence to the probable limitations in the Golgi technique he used (cf., Iwahori, 1978; Rafols and Fox, 1976). A distinguishing electrophysiological feature of tonic cells that may be important functionally is that they can reach very high and sustained ¢ring rates when depolarized by current injection (e.g., Bevan and Wilson, 1999; Nakanishi et al., 1987; present results) and when stimulated synaptically at high frequency (present results). Indeed, sustained high AP rates were recorded from STN neurons during passive and voluntary limb movements in monkeys and humans (Bergman et al., 1994; Magarin‹os-Ascone et al., 2000), suggesting that those cells were of the tonic type and that they contribute to the control of movement. Phasic cells may participate during the execution of brief and fast (ballistic) movements. They only ¢re a brief burst and thereafter do not respond to sustained STN stimulation, indicating that their contribution to the remission of parkinsonian symptoms by tetanic stimulation of the STN may not be as important as that of tonic cells. The dye coupling of some phasic cells revealed an intercommunication system, probably mediated by gap junctions, that could aid in synchronizing the activity of these cells (Dudek et al., 1986), as demonstrated in other systems (e.g., Galarreta and Hestrin, 1998).
CONCLUSION
The STN in young rats is a complex structure composed mainly of two neuron types with tonic and phasic ¢ring modes and a di¡erent morphology. The most abundant cell, the tonic type, may determine the main functional characteristics of the STN and, when ¢ring in abnormal bursts, may govern the contribution of the STN to parkinsonian symptoms. Moreover, the remission of Parkinsonism by treatment with high-frequency electrical stimulation of the STN in humans may primarily reside on the response properties of tonic neurons and especially in their capacity to stop ¢ring during tetanic stimulation of the STN.
Acknowledgements$Work supported by ‘Fondo de Investigaciones Sanitarias de la Seguridad Social’, Grant 94/0527 to C.M.-A. and ‘Direccio¤n General de Investigacio¤n Cient|¤¢ca y Tecnolo¤gica’ and ‘Comunidad Auto¤noma de Madrid’, grants PB95-0031 and 08.5/0038/98 to W.B. J.H.P. and O.M., on leave from ‘Facultad de Medicina, Universidad de Buenos Aires, Argentina’ and from ‘Instituto de Investigaciones Biolo¤gicas Clemente Estable, Montevideo, Uruguay’, were supported
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by ‘Consejo Superior de Investigacio¤n Cient|¤¢ca’, and ‘Ministerio de Educacio¤n y Ciencia’ grants with W.B. We thank P.R.
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Williams for revision of the English language and V. Abraira Santos for helping in the statistical analysis.
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