Grit Mehnert
email: g.mehnert@igb-berlin.de

Franziska Leunert

Samuel Cirés

Klaus Jöhnk

Jacqueline Rücker

Brigitte Nixdorf

Claudia Wiedner

Competitiveness of invasive and native cyanobacteria from temperate freshwaters under various light and temperature conditions

Keywords: Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, climate change, competition, ecophysiology Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, climate change, 33 competition, ecophysiology 34

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Introduction

57

In the course of global warming, many species could expand their range from lower to higher 58 latitudes [START_REF] Parmesan | Ecological and evolutionary responses to recent climate change[END_REF]. In the case of prokaryotes including freshwater cyanobacteria it has 59 been stated that 'Everything is everywhere, but, the environment selects' [START_REF] Baas Becking | Geobiologie of inleiding tot de milieukunde[END_REF], 60 from which it was concluded that cyanobacteria were cosmopolitan [START_REF] Fogg | The blue-green algae[END_REF]. Nowadays, 61 it is well established that not only cosmopolitan cyanobacterial species, but also species with 62 holarctic or pantropical distributions exist [START_REF] Komárek | Current trends and species delimitation in cyanoprokaryote taxonomy[END_REF][START_REF] Hoffmann | Geographic distribution of freshwater blue-green algae[END_REF]. Cylindrospermopsis 63 raciborskii (Woloszynská) Seenayya & Subba Raju 1972 is a prime example of a cyanobacterial 64 species that has spread from tropical and subtropical to temperate latitudes of the northern and 65 southern hemisphere over the last decades [START_REF] Padisák | Cylindrospermopsis raciborskii (Wolozynska) Seenayya et Subba Raju, an 520 expanding, highly adaptive cyanobacterium: worldwide distribution and review of its ecology[END_REF]: Wiedner et al., 2007). In Germany, 66 C. raciborskii is now widespread [START_REF] Stüken | Distribution 567 of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis 568 raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides[END_REF] and accounts for a considerable proportion of 67 the phytoplankton biovolume [START_REF] Nixdorf | Phytoplankton assemblages and steady state in 515 deep and shallow eutrophic lakes -an approach to differentiate the habitat properties of 516 Oscillatoriales[END_REF][START_REF] Mischke | Cyanobacteria associations in shallow polytrophic lakes: influence of 503 environmental factors[END_REF]. Two other cyanobacteria, 68 Such invasive species are of general ecological interest as they can have a major impact on 75 community composition and ecosystem processes [START_REF] Sala | 552 Biodiversity -Global biodiversity scenarios for the year 2100[END_REF][START_REF] Strayer | Understanding the long-term 565 effects of species invasions[END_REF]. The 76 successful establishment of an invasive species in a new habitat depends on its invasive potential 77 and the invasibility of the native community [START_REF] Lonsdale | Global patterns of plant invasions and the concept of invasibility[END_REF]. In this context, the competitive 78 potential of invasive species against native species that are overlapping in an ecological niche is of 79 crucial importance. Since all three invasive species belong to the order Nostocales, their niche most 80 likely overlaps with that of native Nostocales species. In temperate freshwaters, the invasive species 81 are most abundant in polymictic shallow lakes [START_REF] Stüken | Distribution 567 of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis 568 raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides[END_REF], where they co-exist with a 82 great variety of native Nostocales of the genus Anabaena and Aphanizomenon, among which 83

Aphanizomenon gracile is the most frequent and abundant [START_REF] Mischke | Equilibrium phase conditions in shallow German lakes: How 505 Cyanoprokaryota species establish a steady state phase in late summer[END_REF]Rücker et 84 al., 2007). However, the invasive species can also be found, albeit less abundantly, in stratified 85 lakes [START_REF] Stüken | Distribution 567 of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis 568 raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides[END_REF], where the most frequent Nostocales are Aphanizomenon flos-aquae and 86 several taxa of the genus Anabaena. 87 Temperature and light intensity, the two abiotic factors that vary most between latitudes, are key 88 factors in the regulation of Nostocales growth and, presumably, of range expansion of tropical and 89 subtropical species. Previous studies have shown that light is the main growth-limiting factor of 90 Nostocales populations in temperate freshwaters, where they were out-competed by Oscillatoriales 91 under light limitation and high nutrient conditions [START_REF] Zevenboom | N2-Fixing Cyanobacteria: Why They Do Not Become Dominant In 587 Shallow Hypertrophic Lakes[END_REF][START_REF] Scheffer | Ecology in shallow lakes[END_REF]Wiedner et 92 al., 2002). Light is also a growth-limiting factor for C. raciborskii populations in temperate waters 93 [START_REF] Wiedner | Climate change affects timing and 581 size of populations of an invasive cyanobacterium in temperate regions[END_REF]. Ecophysiological studies show that C. raciborskii require high light 94 intensities (80 to 120 µmol photons m -2 s -1 ) for maximum growth [START_REF] Shafik | Factors effecting growth and cell 556 composition of cyanoprocaryote Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba 557 Raju[END_REF]Briand et al., 95 2004); this is similar to the light intensities (100 to 110 µmol photons m -2 s -1 ) required by 96Aphanizomenon species [START_REF] Gentile | Toxicity and Environmental Requirements of a Strain of 465 Aphanizomenon Flos-Aquae (L) Ralfs[END_REF][START_REF] Uehlinger | Experimental studies on the autecology of Aphanizomenon flos-aquae 573 (Experimentelle Untersuchungen zur Autökologie von Aphanizomenon flos-aquae)[END_REF]. However, Padisák and 97

Reynolds (Padisák and Reynolds, 1998) consider C. raciborskii a shade-tolerant species due to its 98 low light saturation parameter (I K ). 99 Cyanobacteria are known to have higher temperature optima than other phytoplankton species 100 [START_REF] Reynolds | Water-Blooms[END_REF][START_REF] Paerl | Climate change: a catalyst for global expansion of harmful 526 cyanobacterial blooms[END_REF]. However, our knowledge about species 101 specific differences in the temperature requirements within the cyanobacteria community is still 102 incomplete. Temperature plays an important role in the onset of Nostocales akinete germination in 103 spring [START_REF] Kim | Relationship between akinete 496 germination and vegetative population of Anabaena flos-aquae (Nostocales, Cyanobacteria) in 497 Seokchon reservoir (Seoul, Korea)[END_REF][START_REF] Rücker | Impact of the inoculum 549 size on the population of Nostocales cyanobacteria in a temperate lake[END_REF] and in their vegetative growth during the season. Earlier increases in water temperatures in spring as well as a rise in the entire temperature amplitude 105 were found to promote the development of C. raciborskii in temperate freshwaters (Wiedner et al., 106 2007). Minimum temperatures, for example, required for growth of C. raciborskii, as determined in 107 field and laboratory studies, range from 15°C to 22°C [START_REF] Dokulil | Population dynamics and photosynthetic rates of a 453 Cylindrospermopsis-Limnothrix association in a highly eutrophic urban lake, Alte Donau, Vienna, 454 Austria[END_REF]Shafik et al., 108 2001;[START_REF] Mischke | Equilibrium phase conditions in shallow German lakes: How 505 Cyanoprokaryota species establish a steady state phase in late summer[END_REF]. The optimum growth temperature (T opt ) for C. raciborskii, as 109 determined in culture experiments, ranges from 29 to 31°C [START_REF] Shafik | Factors effecting growth and cell 556 composition of cyanoprocaryote Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba 557 Raju[END_REF]Briand et al., 2004), 110 which is slightly higher than that for Aphanizomenon and Anabaena species, namely, 20 to 28°C 111 [START_REF] Foy | The influence of daylength, light intensity and 463 temperature on the growth rates of planktonic blue-green algae[END_REF][START_REF] Uehlinger | Experimental studies on the autecology of Aphanizomenon flos-aquae 573 (Experimentelle Untersuchungen zur Autökologie von Aphanizomenon flos-aquae)[END_REF]. However, as the published results are based on a mixture of 112 different field and culture studies and culture methods, data comparisons are difficult. So far, no 113 direct comparison of C. raciborskii with other Nostocales species in terms of temperature and light-114 dependent growth has been published. Moreover, there are only few studies available on the 115 ecology of A. aphanizomenoides [START_REF] Moisander | Salinity effects on growth, photosynthetic 510 parameters, and nitrogenase activity in estuarine planktonic cyanobacteria[END_REF][START_REF] Stefaniak | Occurrence Of Invasive Cyanobacteria Species In Polimictic 562 Lakes Of The Wielkopolska Region (Western Poland)[END_REF], and 116 none for A. bergii. Data about temperature-dependent growth of Aphanizomenon ovalisporum, a 117 potentially invasive Nostocales species currently restricted to the Mediterranean region of Europe 118 [START_REF] Gkelis | First report of the 467 cyanobacterium Aphanizomenon ovalisporum Forti in two Greek lakes and cyanotoxin occurrence[END_REF][START_REF] Quesada | Toxicity of Aphanizomenon ovalisporum (Cyanobacteria) in a Spanish water reservoir[END_REF], are restricted to strains from Lake Kinneret (Israel), 119

where Hadas and co-workers (Hadas et al., 2002) estimated that its T opt ranges between 26 and 120 Strains of three invasive species (Cylindrospermopsis raciborskii, Anabaena bergii, described by Preußel (Preußel et al., 2006), and the Spanish strain as described by Wormer 144 [START_REF] Wormer | Cylindrospermopsin is not degraded 585 by co-occurring natural bacterial communities during a 40-day study[END_REF]. 145

146

Culture experiments 147

Temperature dependent growth of one strain of each species (Table I) was determined at seven 148 temperatures between 10°C and 35°C at 80 µmol photons m -2 s -1 for all species, and additionally at 149 40°C for A. aphanizomenoides (22C4-9) and A. ovalisporum (UAM290). Light dependent growth 150 of all strains was determined at six or more light intensities between 17 and 57 µmol photons m -2 s -1 151 at 15°C and 20°C (Table I). 152

Growth experiments were performed using 100 mL culture volume in 250 mL Erlenmeyer flasks, 153 which were continuously shaken at 80-100 rpm. Z8-medium (Haande et al., 2008) modified with 154 164 nM biotin, 30 nM cyanocobalamine and 119 nM thiamine hydrochloride was used for 155 culturing. The experiments were carried out in growth chambers (BINDER, KBW 400, Germany) 156 with dimmable illumination (OSRAM, Lumilux 18W/865XT Cool Daylight, Germany) simulating 157 12 h:12 h light:dark photoperiods. The cultures were grown in a semi-continuous fashion according 158 to the turbidostat principle [START_REF] Paasche | Growth Of The Plankton Diatom Thalassiosira Nordenskioeldii Cleve At Low 518 Silicate Concentrations[END_REF][START_REF] Nicklisch | The interaction of irradiance and temperature on the growth rate of Limnothrix 513 redekei and its mathematical description[END_REF]. The biovolume was measured every 159 second day and the every second day increase of biovolume was removed by dilution with Z8. The 160 biovolume determination was carried out using a UV/VIS spectrophotometer (Lambda 2, Perkin 161 Elmer, USA) at 750 nm. The cultures were diluted every second day to an optical density (OD) of 162 0.1. 163 164

Growth rate and curve fitting 165

As biomass is correlated to OD, the specific growth rate (µ) was calculated from the change of 166 biomass over time (∆t): 167

t X X µ t ∆ ⋅         = ∆ 1 ln 0 ,
(1) 168 where X ∆t is biomass after cultivation time t, X 0 is biomass after dilution and ∆t is the time between 169 two dilutions. 170

If biomass increase was equal to biomass loss after dilution, steady state was assumed. The mean 171 specific growth rate as well as the standard deviation was calculated from at least three consecutive 172 measurements of the specific growth rates per steady state. This was done for each strain and at all

) ( ) ( ) ( exp 1 ) ( 0 0 I I A I I A I - -               - ⋅ + - - ⋅ = β β α µ (2) 177                       + ⋅ + + ⋅ = β α β β α β α ln max µ A , (3) 178
where µ is the specific growth rate, µ max the maximum specific growth rate, α the initial slope at I = I 0 with light intensity I and compensation light intensity I 0 . If the factor of photoinhibition, β, is 180 β = 0, then A = µ max and the enhanced exponential model reduces to the model of Mitscherlich.

181

Irradiance at onset of light saturation, I K , was calculated as

I K = µ max / α.

182

Curve fitting of the temperature-dependent growth rates was performed according to Jöhnk (Jöhnk 183 et al. 2008): 184

                - ⋅ - - + ⋅ = - - ) 1 ( ) ln( ) ln( ) 1 ( 1 ) ( , , 2 2 1 1 max i opt i opt T T i i i T T i i R R R R b T µ µ , ( 4

Microcosm experiment 195

The microcosm experiment was carried out using a mixed culture including three native and three 196 invasive species (Table I). Pre-cultures were grown as batch cultures in 500 mL Erlenmeyer flasks 197 with a culture volume of 300 mL. Z8 medium was used as described above. The pre-cultures were 198 incubated at 21°C under 59 ± 15 µmol photons m -2 s -1 provided by daylight fluorescent tubes 199 (Phillips TL-D 36W/25-740) with 10 h:14 h light:dark photoperiod. 200 Approximately equal parts of biovolume (assessed from correlations between OD and biovolume, 201 data not shown) of the pre-cultures were mixed and diluted to a final biovolume of 28 mm 3 L -1 . The 202 pooled sample was filled in 2-L bottles (Nalgene) and incubated for four days at depths of 0.1, 0.2, 203 0.4, 0.6, 0.8, 1.0 and 1.6 m in a shallow experimental pond in the Scharmützelsee region. The 204 bottles were aerated daily. Subsamples from the starting suspensions and from each bottle were 205 taken after 4 days of incubation and preserved for phytoplankton biovolume analyses. The 206 biovolume was determined according to the method of Utermöhl [START_REF] Utermöhl | Zur Vervollkommnung der quantitativen Phytoplankton-Methodik[END_REF]. 207

The mean water temperature during the experiment was 19.7 ± 0.4°C. Global radiation was 208 measured with a cosine-corrected quantum sensor (MQS-B, Heinz Walz GmbH, Germany). The 209 vertical attenuation coefficient (K d ) was calculated using photosynthetically active radiation (PAR) 210 measurements obtained at two defined depths with two spherical quantum sensors (LI-193 SA, LI-211 COR Bioscience, USA). PAR at different depths was calculated from K d , light intensity at the water 212 surface (I 0 ) and depth (z). 213

The specific growth rate (µ) was determined as the change in biovolume from beginning to end of 214 the experiment as described above (Eq. 1). Curve-fitting of light-dependent growth rates was 215 performed using Equation 2. The negative growth rate of A. flos-aquae at 254 µmol photons m -2 s -1 216 was not included in the fit. A. macrospora growth rates were not fitted as Eq. ( 2) was not suitable 217 for them. 218 219

Simulation model 220

We used a simple model to point out the effects of differences in growth rates for different 221 Nostocales species on the seasonal course of population biovolume. In contrast to a simulation 222 model describing the full life cycle of Nostocales cyanobacteria (Jöhnk et al., 2010, unpublished 223 data), we only included temperature-dependent growth (neglecting nutrient and light limitation) and 224 mortality rates from that model. Changes in pelagic population, C [mm 3 L -1 ], are thus described by 225 growth and loss processes: 226

C C m m C T t C ] [ ) ( d d dens + - = µ .
(5) 227 Temperature-dependent growth was simulated using growth functions µ(T) for three species, . Loss rates 231 are in good agreement with data published by [START_REF] Reynolds | The ecology of freshwater phytoplankton[END_REF]. Changing the mortality rates within 232 a feasible range does not significantly change the characteristics of the dynamics described 233 capacities. The populations were initialized at a threshold temperature of T threshold = 12°C, when 235 overwintering akinetes in the sediments are germinating [START_REF] Rücker | Impact of the inoculum 549 size on the population of Nostocales cyanobacteria in a temperate lake[END_REF]

T T t C t C ≥ = = . ( 6

) 238

Inoculum values, C 0 , for the three species were chosen as ratios of 1 to 1/3 to 1/10 of 10 -4 mm 3 L -1 , 239 representing spring conditions. 240

The model is driven by seasonal changes in water temperature of a lake. Here, we chose the mean 241 seasonal course (averaged over a period of 14 years, 1994 -2007) of Lake Melangsee (Table I). 242

Simulations for current mean water temperatures (CWT) were complemented by scenarios for 243 historic (CWT minus 2°C) and future mean water temperatures (CWT plus 4°C) representing 244 possible future climate change conditions. 245 246

Statistical analysis 247

The investigated species were divided into four groups, native (A. gracile, A. flos-aquae, 248 A. macrospora), invasive (C. raciborskii, A. bergii, A. aphanizomenoides), deep oligotrophic lake 249 (A. flos-aquae and A. macrospora) and shallow eutrophic to polytrophic lake species (A. gracile and 250 invasive species), for the statistical analysis. A. ovalisporum was not included in the statistical 251 analysis. Statistically significant differences between the groups were assessed using the t-test for 252 independent samples and the Welch test of non-homogeneous variance. The Mann-Whitney U-test 253 was used to compare growth rates at 35°C. All statistical tests were two-tailed. 254 255

Results

256

Culture experiments 257

Temperature-dependent growth 258

Native and invasive species differed significantly in their growth rates at very low (10°C) and very 259 high (35°C) temperatures (Fig. 1). At 10°C, native cyanobacteria grew at rates of 0.06 to 0.15 d -1 , 260 whereas invasive cyanobacteria did not grow. At 35°C, native cyanobacteria ceased to grow, 261 whereas the invasive cyanobacteria grew at rates of 0.09 to 0.32 d -1 . 262

The growth curves of A. aphanizomenoides, C. raciborskii and A. bergii exceeded those of 263 A. gracile, the main competitor of these invasive species in shallow lakes, at 16°C, 20°C and 31°C 264 (Fig. 1). Below these temperatures A. gracile grew faster than invasive species. Aphanizomenon 265 aphanizomenoides and C. raciborskii had clearly higher maximum growth rates than native species 266 (Table II). 267

The invasive cyanobacteria had significantly higher Q10 values between 15 to 25°C ranging from 268 2.0 to 3.2 than native cyanobacteria with values of 1.4 to 1.7 (df = 4; t = 3.025; p ≤ 0.05). Their 269 declined at high irradiance levels. This kind of grouping was not observed in the laboratory 305 experiments. Growth rates of all strains determined in the microcosm experiment were lower than 306 those determined in the laboratory culture experiments. 307 308

Simulation of seasonal dynamics 309

Simulations show that compared to A. gracile, the biovolume of C. raciborskii is much lower at 310 lower historic temperatures (Fig. 5, upper panel), nearly equal at current temperatures (Fig. 5, 311 middle panel), and higher at higher future temperatures (Fig. 5, lower panel). The potential invader 312 A. ovalisporum is virtually absent in the colder scenario, reaching (undetectable) low values at 313 current temperatures and blooms during future temperature increases, as can be expected based on 314 its higher T opt (Table II). In the future scenario, the vegetation time of all three species is increased. 315 316

Discussion

317

The aim of this study was to determine which of the native, invasive and potentially invasive 318 species are stronger competitors under various temperature and light conditions. Our culture 319 experiments clearly demonstrated that native cyanobacterial species were superior competitors at 320 low temperatures and invasive cyanobacterial species at high temperatures. 321

The T opt of native and invasive Nostocales species do not differ significantly. The T opt of 27.8°C for 322 C. raciborskii coincided with values of 28 to 31°C reported for C. raciborskii isolates from different 323 latitudes [START_REF] Briand | 447 Cylindrospermopsis raciborskii (Cyanobacteria) invasion at mid-latitudes: Selection, wide 448 physiological tolerance, or global warming?[END_REF]. This also holds true for the two native Aphanizomenon species, the 324 T opt of which were 24.7°C and 27.1°C in this study compared 25°C reported for A. flos-aquae 325 RALFS f. gracile LEMM. [START_REF] Foy | The influence of daylength, light intensity and 463 temperature on the growth rates of planktonic blue-green algae[END_REF]) and 28°C reported for A. flos-aquae var. klebahnii 326 ELENK. [START_REF] Uehlinger | Experimental studies on the autecology of Aphanizomenon flos-aquae 573 (Experimentelle Untersuchungen zur Autökologie von Aphanizomenon flos-aquae)[END_REF] in other studies. In this study, A. ovalisporum had the highest T opt of 327 32.8°C, which is also higher than T opt of 26 to 30°C reported for strains from Lake Kinneret (Hadas 328 et al., 2002). This high T opt is advantageous as ponds and reservoirs in the Mediterranean region can 329 reach temperatures over 30°C during summer months (A. Quesada,Madrid,personal 330 communication). 331

Overall, high T opt values suggest that Nostocales tolerate the low temperatures of their habitats 332 rather than are adapted to them. Tang and co-workers [START_REF] Tang | Cyanobacterial dominance of polar 570 freshwater ecosystems: Are high-latitude mat-formers adapted to low temperature?[END_REF] published similar 333 observations for cyanobacteria from polar freshwater ecosystems. The T opt of all Nostocales were 334 higher than the mean water temperatures of their habitats during their vegetation period. Therefore, 335 all Nostocales would benefit from rising temperatures. This is in agreement with the recently 336 confirmed concept of global expansion of cyanobacterial blooms favoured by global and regional 337 warming [START_REF] Paerl | Climate change: a catalyst for global expansion of harmful 526 cyanobacterial blooms[END_REF]. Cylindrospermospis raciborskii, A. aphanizomenoides and 338 values for C. raciborskii (38 to 101 µmol photons m -2 s -1 ) were much higher in the present study 345 than those (15 to 26 µmol photons m -2 s -1 ) reported in the literature [START_REF] Shafik | Factors effecting growth and cell 556 composition of cyanoprocaryote Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba 557 Raju[END_REF]Briand et 346 al., 2004). This might be due to differences in culturing methods since the low I K values in the 347 literature resulted from the exponential growth phase of batch cultures started at a much lower 348 trichome density (OD 750 nm ~ 0.002) and determined at higher temperatures (25°C and 27°C) 349 than those in the semi-continuous culture experiments of our study. As we considered the relative 350 differences between native and invasive species, these absolute differences played a minor role in 351 our overall conclusions. 352

Aphanizomenon flos-aquae and A. macrospora, the two species isolated from a deep oligotrophic 353 lake, had a significantly lower optimal light intensity and were more stenoecious in regard to light 354 than the species from the eutrophic shallow lakes. 355

The results of the microcosm experiment (at 19°C) are consistent with the main results of the 356 culture experiments (at 20°C): The most powerful competitors were A. aphanizomenoides, followed 357 by C. raciborskii, A. gracile and A. bergii; A. flos-aquae and A. macrospora could not compete with 358 the other species. This shows that results obtained for single strains using artificial irradiance in the 359 laboratory can be transferred to community level where species co-occur under natural light 360 conditions. Although this study is restricted to the effects of light and temperature on cyanobacterial 361 growth and competition, it can be noted that buoyancy enables the co-occurrence of Nostocales 362 species with different light requirements. For example, under stratified conditions, Anabaena 363 species can adjust their buoyancy to maintain a near surface position, while C. raciborskii can 364 compete more effectively by remaining at greater depths. In shallow turbid waters, Nostocales 365 species can regulate their buoyancy only under calm weather conditions. 366

Since there is increasing evidence that cyanobacterial populations are composed of different 367 ecotypes or subpopulations [START_REF] Becker | Quantitative tracing, by Taq nuclease 444 assays, of a Synechococcus ecotype in a highly diversified natural population[END_REF][START_REF] Johnson | Niche partitioning among Prochlorococcus ecotypes along ocean-scale environmental 494 gradients[END_REF]Rohrlack et al., 2008), we 368 included different strains of each species (except for A. macrospora) in the light experiment to 369 estimate the variance in growth between strains. Figure 2 clearly illustrates that strains of some 370 species such as A. aphanizomenoides or A. flos-aquae show a nearly identical growth response at 371 different light intensities while others, such as A. gracile vary considerably in their growth, 372 especially at high light intensities. In our study differences between species were more pronounced 373 than differences between strains. However, variability between strains should be taken into account 374 when extending results obtained for one strain to the whole population. 375

An interesting result of our study is the fact that A. aphanizomenoides reached the highest growth 376 rates in all experiments. This is contradictory to its low abundance in the field [START_REF] Stüken | Distribution 567 of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis 568 raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides[END_REF]. 377 Therefore, the development of A. aphanizomenoides field populations may be limited by other 378 factors as nutrients, or it may just be at the beginning of its population development in German 379

freshwaters. 380

Overall, our study showed that temperature has a major impact on the competition between native 381 and invasive cyanobacteria. Our results suggest that native species can begin to build up 382 populations earlier during the season. This is confirmed by field studies showing that populations of 383 A. gracile occur earlier in the season from temperatures of 13°C (J. Rücker,Bad Saarow,personal 384 communication), while populations of C. raciborskii occur later in the season when the water 385 temperature exceeds 17°C [START_REF] Mischke | Cyanobacteria associations in shallow polytrophic lakes: influence of 503 environmental factors[END_REF][START_REF] Wiedner | Climate change affects timing and 581 size of populations of an invasive cyanobacterium in temperate regions[END_REF][START_REF] Dokulil | Population dynamics and photosynthetic rates of a 453 Cylindrospermopsis-Limnothrix association in a highly eutrophic urban lake, Alte Donau, Vienna, 454 Austria[END_REF]. Our 386 results also suggest that C. raciborskii could outcompete A. gracile in summer when temperatures 387 rise above 20°C. So far the population size of C. raciborskii does not exceed that of A. gracile 388 [START_REF] Wiedner | Seasonal dynamics of 583 cylindrospermopsin and cyanobacteria in two German lakes[END_REF]. However, this could change if the water temperature increases by a further 389 4.0°C in the future (scenario A1F1, IPCC 2007). Our simulation clearly demonstrates a shift from 390 past dominance by A. gracile to C. raciborskii in the future. The simulation also indicates that the 391 Mediterranean species A. ovalisporum could undergo massive population growth under future 392 climate conditions. Moreover, the simulation illustrates that the seasonal growth period of all 393

Nostocales will be prolonged in the future scenario. 394

Other factors, such as nutrient concentration must also be considered in regulating the growth and 395 competition of cyanobacteria. For example during eutrophication of Lake Balaton (Hungary) in the 396 1970s, populations of A. gracile disappeared while first populations of C. raciborskii and 397 A. aphanizomenoides had been established. After re-eutrophication A. gracile reappeared and now 398 co-occurs with the two invasive species (Padisák and Reynolds, 1998). In general, Nostocales are 399 superior competitors under nitrogen shortage, because they can fix atmospheric nitrogen. However, 400 it is unknown whether they differ in their ability to fix N 2 or to use dissolved nitrogen. Concerning 401 the efficiency of phosphate use, C. raciborskii is known to be a strong competitor under phosphate 402 limitation (Isvánovics et al., 2000;[START_REF] Spröber | Nitrogen uptake and 559 fixation in the cyanobacterium Cylindrospermopsis raciborskii under different nitrogen conditions[END_REF][START_REF] Posselt | Pulses of Phosphate Promote Dominance of the 530 Toxic Cyanophyte Cylindrospermopsis Raciborskii in a Subtropical Water Reservoir[END_REF]. However, to complete 403 the picture on the competitiveness of invasive and native cyanobacteria their growth under various 404 nutrient concentrations must be compared in further laboratory studies. 405

Finally changes in the cyanobacterial community are of concern, because cyanobacteria can 406 produce different toxins. For example, a survey in German lakes showed that hepatotoxic 407 microcystins, which are manly produced by Microcystis or Planktothrix occur in high 408 occur less frequent and in much lower concentrations [START_REF] Fastner | Microcystins (hepatotoxic heptapeptides) in German fresh water bodies[END_REF]. More recently it was 410 shown that also the toxin cylindrospermopsin is widely distributed in German lakes (Fastner et al., 411 2007), where it is produced by A. flos-aquae and A. gracile (Preußel et al., 2006) and occurs in 412 concentrations that are in the same order of magnitude as those of microcystins (Rücker et al., 413 2007). An increase in the abundance of Nostocales species could result in an increase of anatoxin, 414 saxitoxin and cylindrospermopsin concentrations. Moreover, an introduction of the Mediterranean 415 A. ovalisporum could massively enhance cylindrospermopsin concentrations in temperate lakes, 416 because all isolated strains of this species produce cylindrospermopsin [START_REF] Banker | Identification of 441 cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, 442 Israel[END_REF]417 Quesada et al., 2006) I: Studied strains: their origin (poly = polymictic; di = dimictic) and the experiments in 627 which they were used (T = temperature-dependent growth, L = light-dependent growth, M = 628 microcosm experiment). 629

Species

  current knowledge about the temperature and light-dependent growth of invasive and 122 potentially invasive species in relation to native Nostocales species is insufficient to assess their 123 competitive potential and predict their development in temperate freshwaters as a consequence of 124 climate change. Therefore, the aim of the present study was to determine the temperature and light-125 dependent growth of three invasive and three native Nostocales species from German freshwater as 126 well as of one potentially invasive species. Three approaches were used: 127 (i) Semi-continuous culture experiments to study temperature and light-dependent growth of 128 one to four different strains of each species 129 (ii) Microcosm experiment to assess the growth of one strain of each native and invasive species 130 along a natural light gradient in a shallow lake 131 (iii) A simple mathematical model based on growth as a function of temperature (as determined 132 in the culture experiments) was created to simulate the seasonal course of biovolume 133 development and community composition for one native species (A. gracile), one invasive 134 species (C. raciborskii) and one potentially invasive species (A. ovalisporum) at three 135 different temperature scenarios: past, present and future.

  ) 185 where µ (T) is the specific growth rate at temperature T, max µ the maximum specific growth rate

  228A. gracile, C. raciborskii, and A. ovalisporum, determined by laboratory experiments (Eq. 4). 229Model calibration provided a mortality rate m = 0.06 d -1 and a density-dependent loss rate, m dens C, 230 with m dens = 0.01 d -1 L mm -3 , which is equivalent to a carrying capacity of dens / m K µ =

  benefit most from any increase in water temperature. Below 20°C the lowest Values of their light-dependent growth parameters (α, I K ) were similar to those of A. gracile. I K 344

  neurotoxic as saxitoxins or anatoxins, which are produced by Nostocales, 409

(

  Fig. 1 Temperature-dependent growth rates and growth curves at 80 µmol photons m -2 s -1 . The 605 growth rate of A. aphanizomenoides at 40°C was not included in curve fitting. Error bars represent 606 standard deviation of the mean growth rate. Each symbol represents one strain. 607 608 Fig. 2 Light-dependent growth rates and growth curves at 15°C (A) and 20°C (B). Error bars 609 represent standard deviation of the mean growth rate. Each symbol represents one strain. 610 611 Fig. 3 Maximum growth rate (µ max ), initial slope (α) and light saturation parameter (I K ) for 612 individual strains of each species from light-dependent growth curves at 15°C (left) and 20°C 613 (right). 614 615 Fig. 4 (A) Microcosm experiment: Growth rates and growth curves (dashed lines) along a natural 616 light gradient. In the case of A. macrospora, growth rates without curve fitting are shown. (B) 617 Comparison of light-dependent growth curves derived from microcosm (dashed lines) versus 618 laboratory experiment at 20°C (solid lines). 619 620 Fig. 5 Simulated population dynamics. Upper panel: Historical scenario with seasonal water 621 temperature 2°C below current mean values. Middle panel: Presence scenario using seasonal mean 622

Table II :

 II Isolated as described byPreußel (2007). (b) Isolated as A. flos-aquae but designated here 631 A. gracile as its morphology in culture is similar to that of 16D11 and ZIE23AFA. (c) 632 Maximum depth. (d) Northern Bay of Scharmützelsee, which can be considered a separated 633 Parameters of temperature-dependent growth calculated using Equation (4), where 636 R 2 is the coefficient of determination, µ max the maximum growth rate, T opt the optimum 637 growth temperature, α the slope at 10°C [d -1 µmol photons -1 m 2 s], and Q10 the temperature 638 coefficient between 15 to 25°C and 20 to 30°C, respectively. 639

	635
		Strain	Lake of origin	Mean lake	Mixis	Exp. per-
	630 634	depth [m] α (10°C) Q10 (15:25°C) Q10 (20:30°C) type formed α α α Melangsee (13°59′18″ E, µ max [d -1 ] T opt [°C] 1.7 Poly 0.34 ± 0.01 27.8 ± 0.9 0.024 3.2 1.1 L 0.25 ± 0.01 26.5 ± 1.3 0.026 2.8 1.3 52°09′40″ N) ZIE11CR R 2 19F6 a 0.99 0.96 Zierker See (13°02′12″ E, 1.6 Poly 0.98 0.36 ± 0.01 28.8 ± 2.0 0.035 2.0 1.2 L 0.99 0.29 ± 0.02 27.1 ± 1.2 0.010 1.6 1.0 53°21′44″ N) 24G7 a Werbellinsee (12°56′37″ E, 52°55′27″ N) 3.5 c Poly 0.96 0.27 ± 0.01 24.7 ± 0.8 0.011 1.4 0.2 L, T, M 26D9 a Rangsdorfer See (13°24′14″ E, 52°17′19″ N) 1.5 Poly L LIE02AB Lieps (13°09′30″ E, 53°27′07″ N) 2.3 Poly L, T, M ZIE26AB Zierker See 1.6 Poly L 22C4-9 a Heiliger See (13°04′18″ E, 52°24′44″ N) 6.6 Di/ Poly L, T, M 22F6-1 a Heiliger See 6.6 Di/ Poly L ZIE23AFA Zierker See 1.6 Poly L 30D11 a, b Petersdorfer See (14°04′27″ E, 52°18′52″ N) 1.9 Poly L 16D11 a Scharmützelsee, Northern Bay d (14°02′46″ E, 52°14′22″ N) 7.9 c Di/ Poly L, T, M ST115AF Stechlin (13°01′40″ E, 53°09′08″ N) 22.8 Di L, T, M ST86AF Stechlin 22.8 Di L ST195AS Stechlin 22.8 Di L, T, M UAM290 Juan Carlos First Park, Spain (3°36′59″ W, 40°27′42″ N) 1.5 Poly T F 0.99 0.19 ± 0.01 25.5 ± 0.5 0.011 1.7 0.9 0.99 0.36 ± 0.02 32.8 ± 0.9 0.014 3.6 2.1 F o r F o r F o r F o r F o r o r P P P P P P e e e r e e r e e r e e r e e r e r R e v i e R e v i e w 228x185mm (300 x 300 DPI) R e v i e w R e v i e w R e v i e w R e v i e w w 237x270mm (300 x 300 DPI) (a) water body (Christen 2006) Species Invasive species C. raciborskii C. raciborskii A. bergii C. raciborskii A. aphanizomenoides A. gracile C. raciborskii A. flos-aquae C. raciborskii A. bergii A. bergii A. aphanizomenoides A. aphanizomenoides Native species A. gracile A. gracile A. gracile A. flos-aquae A. flos-aquae A. macrospora Potentially invasive species A. ovalisporum A. macrospora A. ovalisporum 150x195mm (300 x 300 DPI) 149x194mm (300 x 300 DPI) 187x275mm (300 x 300 DPI)

(Hindák, 2000) and Poland[START_REF] Stefaniak | Occurrence Of Invasive Cyanobacteria Species In Polimictic 562 Lakes Of The Wielkopolska Region (Western Poland)[END_REF]. These three invasive cyanobacteria

of the order Nostocales are able to fix molecular nitrogen and can survive unfavourable conditions

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

invasive Spanish species (A. ovalisporum) were used (TableI). German strains were isolated as

native cyanobacteria (α = 0.010 to 0.011) (df = 4; t = 5.151; p ≤ 0.01). Native and invasive species

did not differ significantly in their Q10 values between 20 to 30°C (df = 2.210; t = 1.936; p > 0.05)

and their T opt (df = 4; t = 1.999; p > 0.05). Optimal growth temperatures of native and invasive

present in German freshwaters yet.

Contrary to our assumption, the invasive Nostocales species are not superior competitors for light.

(2002) Physiological variables determined under laboratory conditions may explain the bloom of

Planktothrix can form subpopulations with dissimilar ecological traits. Limnol Oceanogr 53, 1279-

Regulation of cyanobacteria and microcystin dynamics in polymictic shallow lakes. Arch Hydrobiol

Acknowledgments


Alexandre Eusebio

Wesley Thevathasan

Louise Doyle Gaynor

Alek Pogosyan

Ed Bye

Tom Foltynie

Ludvic Zrinzo

Keymour Ashkan

Tipu Aziz

Peter Brown
email: peter.brown@clneuro.ox.ac.uk

Doyle Gaynor

Prof P Brown

Deep Brain Stimulation can suppress pathological synchronization in parkinsonian patients

Keywords: Parkinson's disease, Basal ganglia, Deep Brain Stimulation, Oscillations, Neurophysiology Parkinson's disease, Basal ganglia, Deep Brain Stimulation, Oscillations, Neurophysiology

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Although pharmacological treatment in Parkinson's disease is initially satisfactory, many patients suffer from severe fluctuations in their clinical state, involuntary movements and prolonged periods of bradykinesia and rigidity after a few years. These problems are difficult to manage and have led to a renaissance of invasive treatment strategies for late stage Parkinson's disease, particularly deep brain stimulation (DBS) of the subthalamic nucleus (STN). Many potential mechanisms of action of DBS in Parkinson's disease have been suggested. [START_REF] Hammond | Latest view on the mechanism of action of deep brain stimulation[END_REF] One possibility, explored further here, is that DBS suppresses or overrides pathologically synchronized oscillatory activity which acts as a noisy disruptive signal (reviewed in [START_REF] Brown | Paradoxes of functional neurosurgery: clues from basal ganglia recordings[END_REF][START_REF] Gatev | Oscillations in the basal ganglia under normal conditions and in movement disorders[END_REF][START_REF] Hammond | Pathological synchronization in Parkinson's disease: networks, models and treatments[END_REF][START_REF] Uhlhaas | Neural synchrony in brain disorders: relevance for cognitive dysfunctions and pathophysiology[END_REF]). One type of activity in particular has received attention in recordings from patients with Parkinson's disease and consists of exaggerated synchronization at about 20 Hz, -in the beta frequency band. This is evident in the crosscorrelation of neuronal discharges, oscillations of the local field potential (LFP) and in spike-triggered averages of LFP activity (reviewed in [START_REF] Gatev | Oscillations in the basal ganglia under normal conditions and in movement disorders[END_REF][START_REF] Hammond | Pathological synchronization in Parkinson's disease: networks, models and treatments[END_REF][START_REF] Uhlhaas | Neural synchrony in brain disorders: relevance for cognitive dysfunctions and pathophysiology[END_REF][START_REF] Weinberger | Pathological subthalamic nucleus oscillations in PD: Can they be the cause of bradykinesia and akinesia?[END_REF]).

The evidence that DBS may modulate beta activity in patients with Parkinson's disease is mixed and mostly indirect. The latter is because simultaneous recordings of STN LFPs during DBS were, until recently, obviated by stimulation-induced electrical artefacts which are several orders of magnitude larger than the spontaneous fluctuations of the LFP. [START_REF] Rossi | An electronic device for artefact suppression in human local field potential recordings during deep brain stimulation[END_REF] So investigators have either recorded at projection sites of the STN, where stimulation artefact is less of a problem, or recorded the immediate after-effects of STN DBS in those patients with a delayed return of bradykinesia upon cessation of DBS. The findings have been mixed, with most studies reporting suppression of beta activity, [START_REF] Bronte-Stewart | The STN beta-band profile in Parkinson's disease is stationary and shows prolonged attenuation after deep brain stimulation[END_REF][START_REF] Brown | Effects of stimulation of the subthalamic area on oscillatory pallidal activity in Parkinson's disease[END_REF][START_REF] Kuhn | High-frequency stimulation of the subthalamic nucleus suppresses oscillatory beta activity in patients with Parkinson's disease in parallel with improvement in motor performance[END_REF][START_REF] Silberstein | Patterning of globus pallidus local field potentials differs between Parkinson's disease and dystonia[END_REF][START_REF] Wingeier | Intra-operative STN DBS attenuates the prominent beta rhythm in the STN in Parkinson's disease[END_REF] but one study failing to find such an effect. [START_REF] Foffani | Subthalamic oscillatory activities at beta or higher frequency do not change after high-frequency DBS in Parkinson's disease[END_REF] The authors of the latter study have since also recorded from the STN directly during DBS and again failed to show significant suppression of LFP power in the beta band. [START_REF] Rossi | Subthalamic local field potential oscillations during ongoing deep brain stimulation in Parkinson's disease[END_REF] Although this study was a technological feat, not all recordings had peaks in the beta band prior to DBS so that power suppression may have been difficult to detect in these cases, a problem compounded by the recording of four patients on medication and four patients withdrawn from levodopa. Here we use similar methodology to study the effects of STN DBS in a larger sample of 16 patients with evidence of pathological synchrony in the subthalamic region at baseline, prior to stimulation. Our aim was to address whether STN DBS suppresses local beta activity when this is present.

METHODS

A complete description of the methods is available as supplementary material.

Patients and surgery

Sixteen patients participated with informed written consent and the permission of the local ethics committees, and in compliance with national legislation and the Declaration of Helsinki. All had advanced idiopathic Parkinson's disease. Implantation of bilateral STN DBS electrodes was performed sequentially in the same operative session under local anaesthesia, as previously described, [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] in all but one patient (case 10; see Table in supplementary material). The patients reported here are distinct from those reported by Kühn et al [START_REF] Kuhn | High-frequency stimulation of the subthalamic nucleus suppresses oscillatory beta activity in patients with Parkinson's disease in parallel with improvement in motor performance[END_REF].

Recordings

Recordings were performed in the few days between electrode implantation and their connection to the pulse generator. The Medtronic electrodes used have four equally spaced contacts. Contact 0 was the lowermost and contact 3 was the uppermost (see supplementary material). Out of the 16 patients recordings were possible on 28 sides (see Table in supplementary material). We used a single channel, isolated high gain (100 dB) amplifier [START_REF] Rossi | An electronic device for artefact suppression in human local field potential recordings during deep brain stimulation[END_REF] with pass band (4-40 Hz) to record LFP signals from the contacts of an electrode whilst another contact of the same electrode was stimulated. All patients were recorded while they sat in a comfortable chair after overnight withdrawal of their usual antiparkinsonian medication. Patients were instructed to rest quietly and absence of voluntary movement was confirmed by continuous visual inspection. Initially, about 100 s was recorded from contacts 1/3 and 0/2 on each side with the patient at rest and with no stimulation. The spectral pattern was then analyzed immediately off-line in Spike 2 using spectral averages and time-evolving spectral displays. Three of the 28 sides had no discrete spectral peaks, regardless of frequency (see Table in supplementary material), and were not studied further. In the remaining 25 sides we selected the contact pair with the highest peak power (contact pair 1/3 on 19 sides and contact pair 0/2 on six sides) for recording during subsequent stimulation. This was done to maximise our chances of detecting power suppression during DBS (i.e. of avoiding a floor effect). The contact pair with the highest beta activity has also been previously documented to be well positioned in STN and to concur with the site selected for chronic therapeutic stimulation. [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF][START_REF] Alonso-Frech | Slow oscillatory activity and levodopa-induced dyskinesias in Parkinson's disease[END_REF][START_REF] Devos | Predominance of the contralateral movement-related activity in the subthalamo-cortical loop[END_REF][START_REF] Miyagi | Spectral analysis of field potential recordings by deep brain stimulation electrode for localization of subthalamic nucleus in patients with Parkinson's disease[END_REF][START_REF] Yoshida | Value of subthalamic nucleus local field potentials recordings in prediciting stimulation parameters for deep brain stimulation in Parkinson´s Disease[END_REF] Thereafter, patients were recorded at the selected contact pair on a given side for a further two minutes without stimulation, the latter having been discontinued at least 20 minutes earlier. Then unilateral DBS was begun at 1.0 V (19 sides), 1.5 V (five sides) or 2.0 V (one side), depending on time constraints and prior clinical information regarding efficacy. Stimulation was subsequently increased in step increments of 0.5 V (or 1.0 V on two sides) up to 3.5 V, or until side-effects were encountered. Each new voltage was maintained for ~100 s. When time allowed (12 sides in eight patients) at the end of the above slow ramping of stimulation, the latter was then discontinued for 100 s and thereafter stimulation re-presented for a further 100 s at a clinically effective voltage.

This was performed so that we could determine whether any suppression of LFP activity during voltage ramping depended on a certain duration of stimulation or on a certain threshold intensity (see results).

Monopolar simulation was delivered by a Medtronic external stimulator (type 3625) between active contacts 1 (when recording from 0/2) or 2 (when recording from 1/3) and a subclavicular surface electrode (pulse-width 60µs; frequency 130 Hz). Clinical assessment, other than visual inspection, was not made until after all recordings were completed so as to avoid any influence on the LFP. Clinical assessments (items 20, 22 and 24 of UPDRS III) were not blinded. The lowest voltage (threshold) for clinically effective stimulation was determined separately on each side contralateral to the stimulation (see Supplementary Methods in supplementary material for further details).

Analysis

Spectral analysis was performed in Spike2 v6, using serial FFT blocks of 256 data points (frequency resolution 0.78 Hz, Hanning window, windows not overlapped). These were visualised as time-averaged power spectra (Fig 1 and2A) and time-frequency plots (Fig 2B). Peaks were defined as local elevations of power in which the five contiguous bins centred on the peak had to be significantly different (p < 0.05) to the mean of the two adjacent bins below and three adjacent bins above. One or more discrete peaks were seen in the power spectra from 25 sides. These tended to vary in frequency (5-29 Hz, see Table in supplementary material). For this reason we analysed the amplitude of the LFP in each peak rather than the sum of the LFP power over the whole frequency band of interest. Nineteen sides had a discrete spectral peak in the beta 11-30 Hz band, and these were selected for further analysis.

The beta-band was considered in its entirety and also subdivided into 11-20 Hz and 21-30 Hz bands, following the suggestion that these may have different functional characteristics. [START_REF] Priori | Movement-related modulation of neural activity in human basal ganglia and its L-DOPA dependency: recordings from deep brain stimulation electrodes in patients with Parkinson's disease[END_REF][START_REF] Priori | Rhythm-specific pharmacological modulation of subthalamic activity in Parkinson's disease[END_REF][START_REF] Williams | Dopamine-dependent changes in the functional connectivity between basal ganglia and cerebral cortex in humans[END_REF][START_REF] Foffani | Movement-related frequency modulation of beta oscillatory activity in the human subthalamic nucleus[END_REF] Power in a selected peak was calculated from the sum of the five contiguous bins centred on the peak for each period of stimulation (0 to 3.5 V). Peak power levels were expressed as percentages of the baseline peak power (in the absence of stimulation). This was performed so as to limit the effects of variability in baseline power between sides, which may, in part, be due to small variations in the positioning of recording contact with respect to the source of the beta activity between sides and patients. A similar normalisation procedure is a standard step in the analysis of event related power changes in the EEG, where baseline variability between subjects is also significant. Statistical analyzes were performed in SPSS 15 for Windows. As data were not normally distributed non-parametric statistics were used as detailed in the results (and outliers included). Median values are given, together with the 25 th and 75 th percentiles (Inter-quartile range or IQ), except when the range was illustrated in a figure. All posthoc tests specified in the text as p < 0.05 survived correction for multiple testing using the false discovery rate procedure. [START_REF] Curran-Everett | Multiple comparisons: philosophies and illustrations[END_REF]

RESULTS

The custom-built amplifier performed well so that between 4-40 Hz there was little difference between autospectra of LFPs recorded from the subthalamic area with this and commercial devices, provided no DBS was delivered (black and green traces in Post-hoc Mann Whitney U tests revealed that stimulation at ≥ 1.5 V suppressed the power in peaks over the 11-30 Hz band (Fig 3) and at ≥ 2.0 V for its subdivisions when these were considered separately (all p <0.05). This threshold compared favourably with the median threshold for the clinical effect of stimulation, determined later using the same contact for stimulation (2.0 V, range 1.5 to 3.5 V). Moreover, the voltage threshold for peak suppression on individual sides was always 0.5 V less than or equal to the threshold for inducing contralateral foot dyskinesias (case 5 R STN 2V and case 16 L STN 2 V) or tremor suppression (case 2 R STN 2.5V and case 4 R STN 2.5V and L STN 3.0V), where these were evident during the initial simultaneous stimulation and recording session.

With stimulation intensities of 1.0 V, 1.5 V, 2.0 V, 2.5 V, 3.0 V and 3.5 V the median power of peaks in the 11-30 Hz band fell to 97.5 %, 90.5 %, 69.6 %, 59.6 %, 54.2 % and 40.5 % of baseline values (see Fig 3 for ranges and significance levels). Note that this graded suppression in the averaged data partly arose because individual thresholds for beta suppression varied. Indeed, four sides showed a temporary increase in peak power at lower stimulation voltages, before peak power was suppressed as voltage was increased still further (Fig 2 and3).

The general picture described above did not change if the frequency band of interest was defined slightly differently as 13-30 Hz, if a broader area of 13 (rather than 5) bins centred on each peak in the beta band was analyzed or if the two sides in which stimulation induced contralateral dyskinesias were excluded (see supplemental results).

Moreover, similar results were got if we only analyzed those sides in which stimulation was performed at both 1.5 V and 2.0 V (regardless of any other voltages tested), thereby permitting analysis using Friedman's test for related samples (see supplemental results).

Finally, we addressed a potential ambiguity in the interpretation of the increasing suppression of LFP peaks in the 11-30 Hz band with increasing voltage. As presented above the increasing suppression could be a direct consequence of the voltage being stepped up or could arise form the progressively longer duration of stimulation.

Accordingly, on 12 sides stimulation was discontinued for 100s at the end of each series of voltage steps to allow spontaneous activity to recover and then a single clinically effective voltage step re-presented (Fig 2). These single steps of higher voltage (median 2.5 V, IQ range 2.0 -3.0 V) remained effective at suppressing peaks between 11-30 Hz, even though they were not preceded by any prolonged ramping of stimulation (median suppression to 45% of preceding 50 s without stimulation, IQ range 26% -97%; z = 2.48, p = 0.013, Wilcoxon signed ranks test). However, the level of suppression was still less than that observed with the same stimulation intensity after the progressive stepping up of voltages on the same sides (Median 35 %, IQ range 25% -63%; z = 2.229, p = 0.026, Wilcoxon signed ranks test), suggesting that duration of stimulation as well as voltage also had some effect. Relevant in this regard is the finding that longer STN stimulation durations produce longer lasting after-effects [START_REF] Bronte-Stewart | The STN beta-band profile in Parkinson's disease is stationary and shows prolonged attenuation after deep brain stimulation[END_REF].

DISCUSSION

We have shown that DBS of the STN area at frequencies and voltages in the therapeutic range leads to a concurrent suppression of locally synchronized activity between 11-30 Hz, where the latter is evidenced by a peak in baseline spectra of LFP activity over this frequency range. Such beta activity is believed to be pathological [START_REF] Hammond | Pathological synchronization in Parkinson's disease: networks, models and treatments[END_REF] and is much attenuated in healthy animals, and, in implanted dystonic patients is only seen in those made Parkinsonian by the use of chronic tetrabenazine [START_REF] Kühn | Beta activity in dystonia patients after drug-induced dopamine deficiency[END_REF]. Thus the present study bears out the assumption that the suppression of pathological oscillatory activity seen temporarily after cessation of DBS is a reflection of suppression during DBS [START_REF] Bronte-Stewart | The STN beta-band profile in Parkinson's disease is stationary and shows prolonged attenuation after deep brain stimulation[END_REF][START_REF] Kuhn | High-frequency stimulation of the subthalamic nucleus suppresses oscillatory beta activity in patients with Parkinson's disease in parallel with improvement in motor performance[END_REF][START_REF] Wingeier | Intra-operative STN DBS attenuates the prominent beta rhythm in the STN in Parkinson's disease[END_REF].

The LFP is currently believed to mainly reflect slow sub-threshold currents, primarily post-synaptic potentials, of a large local neuronal population [START_REF] Ecceles | Interpretation of action potentials evoked in the cerebral cortex[END_REF] and is considered to reflect mainly the 'input' to the local network. [START_REF] Logothetis | The neural basis of the blood-oxygen-level-dependent functional magnetic resonance imaging signal[END_REF] This interpretation is supported in the case of the beta band LFP activity recorded in the STN by the fact that it is coherent with, but lags, similar oscillatory activity in the cerebral cortex, in line with cortical driving. [START_REF] Williams | Dopamine-dependent changes in the functional connectivity between basal ganglia and cerebral cortex in humans[END_REF][START_REF] Fogelson | Different functional loops between cerebral cortex and the subthalmic area in Parkinson's disease[END_REF][START_REF] Lalo | Patterns of bidirectional communication between cortex and basal ganglia during movement in patients with Parkinson disease[END_REF] Viewed in this light our results are compatible with the recent demonstration in a rodent model that it is the afferents to the subthalamic nucleus that must be stimulated at high frequency, rather than the local neurons themselves, to over come parkinsonism. [START_REF] Gradinaru | Optical deconstruction of parkinsonian neural circuitry[END_REF] The above rodent study also showed that stimulation of cortical afferents at 20 Hz greatly exacerbated parkinsonism. [START_REF] Gradinaru | Optical deconstruction of parkinsonian neural circuitry[END_REF] Indeed, synchronization in the beta band has been implicated in parkinsonian bradykinesia and rigidity in both correlative and interventional studies (reviewed in [START_REF] Gatev | Oscillations in the basal ganglia under normal conditions and in movement disorders[END_REF][START_REF] Hammond | Pathological synchronization in Parkinson's disease: networks, models and treatments[END_REF][START_REF] Uhlhaas | Neural synchrony in brain disorders: relevance for cognitive dysfunctions and pathophysiology[END_REF][START_REF] Weinberger | Pathological subthalamic nucleus oscillations in PD: Can they be the cause of bradykinesia and akinesia?[END_REF]), so its suppression by subthalamic stimulation using standard therapeutically effective parameters raises the possibility that this suppression of local beta activity may underlie some of the therapeutic actions of DBS. This is not to say that the effects of this suppression are limited to the STN region, or that this is the sole consequence of stimulation. It is possible that DBS influences neuronal activity both locally at the site of stimulation, i.e. in and around STN, and also over other functionally connected elements of the cortex -basal ganglia network. [START_REF] Hammond | Latest view on the mechanism of action of deep brain stimulation[END_REF][START_REF] Brown | Effects of stimulation of the subthalamic area on oscillatory pallidal activity in Parkinson's disease[END_REF][START_REF] Kuhn | High-frequency stimulation of the subthalamic nucleus suppresses oscillatory beta activity in patients with Parkinson's disease in parallel with improvement in motor performance[END_REF][START_REF] Dorval | Deep brain stimulation reduces neuronal entropy in the MPTP-primate model of Parkinson's disease[END_REF][START_REF] Montgomery | Mechanisms of Action of Deep Brain Stimulation[END_REF] This could arise either directly through the suppression of synchronized activity at the stimulation site so that this activity is no longer propagated or through the effects of stimulationinduced high frequency activity of STN neurons and axons in the vicinity. [START_REF] Hammond | Latest view on the mechanism of action of deep brain stimulation[END_REF][START_REF] Montgomery | Mechanisms of Action of Deep Brain Stimulation[END_REF][START_REF] Garcia | High-frequency stimulation in Parkinson's disease: more or less?[END_REF] The latter would not have been detected by our amplifier, which necessarily had a frequency cut-off well below this. Any induced high frequency activity might then be transmitted to the output structures of the basal ganglia [START_REF] Hashimoto | Stimulation of the subthalamic nucleus changes the firing pattern of pallidal neurons[END_REF][START_REF] Maurice | Spontaneous and evoked activity of substantia nigra pars reticulata neurons during high-frequency stimulation of the subthalamic nucleus[END_REF][START_REF] Hilker | STN-DBS activates the target area in Parkinson disease: an FDG-PET study[END_REF] where it might have further effects, including suppression of pathological activity at these and later relay stages.

Additionally, stimulation may lead to antidromic spikes that collide with ongoing spontaneous pathological activity, such as might come from cortex. [START_REF] Hammond | Latest view on the mechanism of action of deep brain stimulation[END_REF] This could contribute to the suppression of synchronization in the subthalamic region, given that much of this may be driven by cerebral cortex. [START_REF] Williams | Dopamine-dependent changes in the functional connectivity between basal ganglia and cerebral cortex in humans[END_REF][START_REF] Fogelson | Different functional loops between cerebral cortex and the subthalmic area in Parkinson's disease[END_REF][START_REF] Lalo | Patterns of bidirectional communication between cortex and basal ganglia during movement in patients with Parkinson disease[END_REF] Our study was constructed to test whether deep brain stimulation can suppress local beta activity when this is present. We argued that there was little point proceeding to stimulation if there was no peak at rest that could potentially be suppressed. Possible reasons for an absent beta peak at rest include suboptimal targeting, [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] stun effect, [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF][START_REF] Hilker | STN-DBS activates the target area in Parkinson disease: an FDG-PET study[END_REF][START_REF] Koop | Improvement in a quantitative measure of bradykinesia after microelectrode recording in patients with Parkinson's disease during deep brain stimulation surgery[END_REF][START_REF] Rosa | Time dependent subthalamic local field potential changes after DBS surgery in Parkinson's disease[END_REF] and incomplete withdrawal of antiparkinsonian medications (either through the long-lasting nature of some dopamine agonists or covert levodopa ingestion). Still, those few cases in which spectral peaks are absent in the beta band during the post-operative period, raise the possibility that DBS-induced suppression of such activity may not explain clinical improvement in every patient. In line with this, although suppression of beta activity correlates with treatment induced improvement in bradykinesia and rigidity, it does not correlate with improvement in parkinsonian tremor, so that even in patients with clear beta activity, this may not relate to all motor impairments. [START_REF] Kuhn | Reduction in subthalamic 8-35 Hz oscillatory activity correlates with clinical improvement in Parkinson's disease[END_REF][START_REF] Kuhn | Pathological synchronisation in the subthalamic nucleus of patients with Parkinson's disease relates to both bradykinesia and rigidity[END_REF][START_REF] Ray | Local field potential beta activity in the subthalamic nucleus of patients with Parkinson's disease is associated with improvements in bradykinesia after dopamine and deep brain stimulation[END_REF] However, it is also possible that pathological synchrony may, in the minority of our cases, have predominated at sites in the basal ganglia-cortical circuit other than the STN. We only measured synchronization in the subthalamic region and yet, as discussed above, high frequency stimulation of the STN area may have effects at several sites in the distributed basal ganglia-cortical circuit.

Although the present data indicate that DBS of the STN at clinically effective voltages is associated with the simultaneous suppression of oscillatory activity in the beta band, they do not address the question of the precise site of origin of such LFP activity. In particular, whether beta activity is confined to the dorsal STN or extends for a few millimetres above its dorsal border is unclear. Support has been provided for both possibilities, depending on whether LFPs [START_REF] Miyagi | Spectral analysis of field potential recordings by deep brain stimulation electrode for localization of subthalamic nucleus in patients with Parkinson's disease[END_REF][START_REF] Kühn | The relationship between local field potential and neuronal discharge in the subthalamic nucleus of patients with Parkinson's disease[END_REF] or population spiking activity [START_REF] Zaidel | Subthalamic span of beta oscillations predicts deep brain stimulation efficacy for patients with Parkinson's disease[END_REF] are studied. Both DBS of the dorsal STN and caudal zona incerta have clear antiparkinsonian effects [START_REF] Saint-Cyr | Localization of clinically effective stimulating electrodes in the human subthalamic nucleus on magnetic resonance imaging[END_REF][START_REF] Voges | Bilateral high-frequency stimulation in the subthalamic nucleus for the treatment of Parkinson disease: correlation of therapeutic effect with anatomical electrode position[END_REF][START_REF] Plaha | Stimulation of the caudal zona incerta is superior to stimulation of the subthalamic nucleus in improving contralateral parkinsonism[END_REF].

The present study suffers from several limitations. In order to recruit a large patient sample we studied patients at several different surgical centres, which may have introduced additional variance in our data set. Another limitation was the absence of kinematic assessment that might have allowed us to compare the threshold stimulation voltage for significant power suppression with the voltage that objectively improved motor performance in all patients. This issue should be addressed in future studies.

Nevertheless, our results demonstrate a potentially important association between the suppression of local oscillatory synchrony and DBS in the therapeutic range. [START_REF] Moro | The impact on Parkinson's disease of electrical parameter settings in STN stimulation[END_REF] Note, that there was no significant difference in the DBS-induced suppression of peak power in the lower and upper frequency bands, when the beta band was split for analysis. This indicates that although the functional significance of the subdivisions within the beta band remains debated they are both suppressed by STN DBS.

The present study provides direct evidence that STN DBS can reduce beta frequency activity in the region of the STN. It is interesting to note that levodopa has a similar effect, [16, 38-40, 48, 49] raising the possibility that suppression of such oscillatory synchrony by several treatment modalities may contribute to the amelioration of some aspects of parkinsonism. Moreover, even if the suppression of beta activity were found to be epiphenomenal rather than causally important in parkinsonism, the present results are important in reinforcing the suitability of subthalamic LFP activity in the beta band as a feedback signal for closed loop deep brain stimulation systems. [START_REF] Stanslaski | An implantable bi-directional brainmachine interface system for chronic neuroprosthesis research[END_REF] This activity correlates with bradykinesia and rigidity [START_REF] Rosa | Time dependent subthalamic local field potential changes after DBS surgery in Parkinson's disease[END_REF][START_REF] Kuhn | Reduction in subthalamic 8-35 Hz oscillatory activity correlates with clinical improvement in Parkinson's disease[END_REF][START_REF] Kuhn | Pathological synchronisation in the subthalamic nucleus of patients with Parkinson's disease relates to both bradykinesia and rigidity[END_REF][START_REF] Chen | Complexity of subthalamic 13-35 Hz oscillatory activity directly correlates with clinical impairment in patients with Parkinson's disease[END_REF] and is suppressed by DBS.

  Fig 1).Similarly, there was little difference between autospectra of LFPs recorded with the custom-built device without DBS and with therapeutically subthreshold DBS (black and blue traces inFig 1). However, stronger stimulation voltages suppressed peaks in the 11-30 Hz band (red trace inFig 1). In vitro experiments suggested that DBS induced artifact was unlikely to be responsible for this suppression (see supplementary materials). Note that recordings were made sequentially, rather than simultaneously, which likely accounts for the small differences in the autospectra recorded without DBS in Fig1. Time-evolving spectra also suggested suppression of LFP peaks between 11-30 Hz once a certain threshold voltage of stimulation was surpassed (Fig 2). At the group level, there was an effect of stimulation intensity for peaks in the 11-30 Hz band (n = 144, χ 2 = 49.1, p < 0.00001; Kruskal Wallis test; Fig 3). If the latter band was subdivided into lower and upper ranges, then there was also an effect of stimulation intensity for peaks in the 11-20 Hz (n = 86, χ 2 = 35.1, p < 0.00001) and 21-30 Hz bands (n = 58, χ 2 = 15.4, p = 0.017; Kruskal Wallis tests).

Fig 1 .

 1 Fig 1. Power autospectra of LFP recorded between contacts 1 and 3 from left STN

Fig 2 .

 2 Fig 2. Effect of DBS of right STN on LFP in case 5. [A] Power autospectrum of LFP

Fig 3 .

 3 Fig 3. Summary box plot of power suppression of beta band peaks during

ACKNOWLEDGEMENTS

We thank Patricia Limousin and Marwan I. Hariz from the Unit of Functional Neurosurgery (Institute of Neurology, 33 Queen Square London, WCIN 3BG) for giving us permission to study their patients. This work was supported by the Medical Research Council, Wellcome Trust, the Oxford NIHR Biomedical Research Centre, Parkinson Disease UK and Fondation pour la Recherche Médicale.