Receptive field properties are computed independently for excitatory and inhibitory cells and compared within and across columns. Within digit representational zones intracolumnar excitatory and inhibitory receptive field extents are concentric, single-digit, small, and unimodal. Exclusively in representational boundary-adjacent zones, intracolumnar excitatory and inhibitory receptive field properties diverge: excitatory cell receptive fields are single-digit, small, and unimodal; and the paired inhibitory cell receptive fields are bimodal, double-digit, and large. In simulated syndactyly (webbed fingers), boundary-adjacent intracolumnar receptive field properties reorganize to within-representation type; divergent properties are reacquired following syndactyly release. This study generates testable hypotheses for assessment of cortical laminar-dependent receptive field properties and plasticity within and between cortical representational zones. For computational studies, present results suggest that concurrent excitatory and inhibitory plasticity may underlie novel emergent properties.Mechanisms underlying the emergence and plasticity of representational discontinuities in the mammalian primary somatosensory cortical representation of the hand are investigated in a computational model. The model consists of an input lattice organized as a three-digit hand forward-connected to a lattice of cortical columns each of which contains a paired excitatory and inhibitory cell. Excitatory  Representational discontinuity is a striking feature of the normal somatotopic map of the hand in primary sensory cortical area 3b in adult owl monkeys  in determining individual and collective excitatory receptive field properties. Second the basic model unit is that of a cortical column that consists of excitatory and inhibitory cell(s) with characteristic interconnectivity, including mutual excitatory-excitatory cell connections and recurrent inhibition.not firing action potentials in the latter due to action potential firing by the former the adaptation rule is not strictly Hebbian, but is covariance-driven. Other than an initial local random weight distribution, there is no external overlay of synaptic patterning, for example, to effect details of lateral inhibition. Second, the study reports the results of separate analyses of individual and collective receptive field properties observed in the population of excitatory cells (excitatory receptive fields) and in the population of inhibitory cells (inhibitory receptive fields). This enables a more detailed understanding particularly of the role of inhibition in cortical somatotopy. Spatial patterns of synaptic strengths are observed that appear not to conform simply to lateral inhibition .This study is unique in two ways. First, all synaptic types are adapted subject to a simple covariance rule. These include synapses between sensory afferents to cortical excitatory cells, lateral cortical excitatory to excitatory cells, lateral cortical excitatory to inhibitory cells, and lateral cortical inhibitory to excitatory cells. Adaptation of the excitatory to excitatory connection type is Hebbian-like: spiking rate activity in the former is facilitative of changes in spiking rate in the latter. The same holds for the excitatory to inhibitory connection type. Since the inhibitory to excitatory connection type is facilitative of The study concludes that in the present neural network model, representational continuities and discontinuities and their reorganization are a consequence of the emergence of characteristic spatial patterns of synaptic weights in both excitatory and inhibitory neural populations. The study illustrates these spatial patterns and tracks their evolution in a simulated three-digit somatotopic map from initial random conditions, to a baseline refinement, through simulated digital syndactyly, and release from syndactyly.The effects typically attributed to and which result as a consequence of lateral inhibition are shown to be an emergent property of neural networks whose connection weights are subject to a simple covariance adaptation rule. These may represent a novel emergent neural mechanism contributing to cortical somatotopy.The model consists of a pair of forward connected two-dimensional lattices each containing a 45 × 45 array of nodes. An input layer (S) node consists of a single “input unit” with exclusively ascending connections to its corresponding topographically centered 7 × 7 neighborhood of cortical layer (C) nodes. There is no skin surface model and no mechanoreceptor-specific model. Individual input layer afferents are experimentally activated as ON or OFF (with additive noise). The activity on these afferents is considered to model individual and groups of fast conduction velocity low-threshold cutaneous mechanoreceptors positioned in glabrous skin  and ~400,000 synapses.A cortical layer node consists of an excitatory (E) and inhibitory (I) cell pair. These are interpreted as a lumped representation of a cortical column. Cortical layer E-cells are the exclusive target of S layer ascending connections. A cortical layer E-cell makes collateral excitatory connections with itself, with a corresponding topographically centered 7 × 7 neighborhood of E-cells, and with a neighborhood of I-cells. A C layer I-cell makes collateral inhibitory connections with a corresponding topographically centered 7 × 7 neighborhood of E-cells. See Tables vi) subject to depolarization and hyperpolarization through weighted input connections. The S layer input unit is, in addition, driven by an external ON/OFF stimulus. Average spiking rate (ri) for the neurons and input units are modeled by passing the corresponding membrane potential (vi) through a sigmoidal compression function. See Table The model neuron is an RC-time constant membrane potential  are initially assigned values from a uniform distribution. These are subsequently adapted according to exponential decay, a simple covariance rule, and normalization. Weight adaptation (plasticity) is controlled by an external ON or OFF signal. When ON, connection weight adaptation occurs at each time step △The present study is organized as an “experimental” track consisting of three phases  and a corresponding “control” track . Each phase consists of a set of 15 cycles. Each cycle consists of a set of 0.350 s duration trials. Model inputs are presented and model outputs measured during each such trial. See Table while respecting digit “boundaries.” The number of 7 × 7 patches in a 45 × 45 baseline network is 351 per digit . The baseline refinement phase consists of 15 cycles . Weight adaptation is ON during baseline refinement. A receptive field map is derived after each cycle. See Table Phase I “baseline refinement” is the simulation that models the emergence and maintenance of somatotopy from random initial conditions. A single baseline refinement cycle consists of stimulating all possible patches of a fixed size one at a time per trial while respecting the D2-D3 boundary, but ignoring the D1-D2 boundary. The number of 7 × 7 patches in a 45 × 45 syndactyly network is 351 for D3 and 936 for the syndactyly D1+2 . The syndactyly refinement phase consists of 15 cycles . Weight adaptation is ON during syndactyly cycles. A receptive field map is derived after each cycle. During the receptive field mapping procedure weight adaptation is OFF.Phase II “syndactyly refinement” is the simulation that models the response of a baseline refined network to simulated digital syndactyly. The driving stimuli are spatially localized, correlated inputs on layer S. A single syndactyly refinement cycle consists of stimulating all possible patches of a fixed size one at a time per trial Phase III “syndactyly release” repeats Phase I, but uses the syndactyly network as its starting point. Weight adaptation is ON during syndactyly release. A receptive field map is derived after each cycle. During the receptive field mapping procedure weight adaptation is OFF.The Experimental track evolves a single network of fixed size and fixed input patch size through Phase I (baseline refinement), Phase II (syndactyly) and Phase III (syndactyly release). On the Experimental track, digits 1 and 2 are the “experimental” digits; D3 provides within-network control.The Control track starts with the Experimental Phase I baseline refined network and subjects it to two additional complete baseline refinement runs. In this protocol D3 undergoes the same total number of stimulation trials overall on both the Control and Experimental tracks. Similarly, all areas of digits 1 and 2 (sufficiently distant from the D1–D2 border) undergo the same number of stimulation trials.Figure N = 2025) and the cortical layer E and I cell activity stored. See Table and I cell. See Table Simulated receptive field mapping is a two-step process. A Receptive Field Probe trial is run one at a time once for each layer S node  and between digits 2 and 3 (the control discontinuity). In the unrefined network the distribution of centroids is largely uniform and random. In the baseline refined network centroids cluster to either side of the digit midline. There are no centroids located on the nodes on either side of and immediately adjacent to digit boundaries (with the exception of a few nodes at network edge).Following a single syndactyly refinement cycle the D1–D2 boundary is nearly obliterated. The border between digits 2 and 3 remains intact (with the exception of a few nodes at the network edges). Additional syndactyly refinement cycles fine-tune the centroid map.Syndactyly release cycles reverse the effects of simulated syndactyly. Following a single cycle, a D1–D2 representational boundary begins to reemerge, though there remain centroids located on nodes immediately adjacent to the boundary. The D1–D2 boundary is fully restored with additional refinement cycles. The effects observed at the edges of the D2–D3 border during syndactyly are reversed as well. Additional syndactyly release cycles produce a map of excitatory receptive field centroids indistinguishable from a baseline refined map.The results for inhibitory receptive field centroids are at once similar and strikingly different from those for excitatory receptive field centroids. Figure In the unrefined network the distribution of centroids is largely uniform and random. In the baseline refined network, in similar fashion to excitatory receptive fields, the inhibitory receptive field centroids cluster to either side of the digit midline. The E cell and I cell within these single cortical columns have coincident centroids.A dramatic difference between excitatory and inhibitory receptive field centroid distributions is the observation of inhibitory receptive field centroids positioned on the nodes on either side of and immediately adjacent to digit boundaries. The E cell and I cell within these single cortical columns have divergent centroids.Following a single syndactyly refinement cycle the D1–D2 boundary is nearly eliminated. The D2–D3 boundary remains intact. The consequence of additional syndactyly refinement cycles is a fine-tuning of the D1–D2 centroid map.Syndactyly release cycles reverse the effects of simulated syndactyly. The border between D1–D2 begins to reemerge following a single baseline refinement cycle. Inhibitory receptive field clusters begin to migrate back toward their respective digit midlines and inhibitory receptive field centroids reappear located on the nodes on either side of and immediately adjacent to digit boundaries.With additional syndactyly release cycles the D1–D2 boundary is fully restored. Inhibitory receptive field centroids reappear located on the nodes on either side of and immediately adjacent to D1–D2 boundary. At the D2–D3 boundary the inhibitory receptive field centroids located on the nodes on either side persist and undergo fine-tuning. The overall result is a map of inhibitory receptive field centroids nearly indistinguishable from a baseline refined map.Figure In the unrefined network, control track excitatory receptive fields are of similar though not uniform size, orientation and overlap. Experimental track excitatory receptive fields are of similar size, orientation and overlap. They are all double-digit D1, D2 in extent.Baseline refinement yields features of normal somatotopy on both the experimental and control tracks. First, receptive fields are continuous, single-peaked, and roughly uniform in size (with possible though limited edge effects). Second, topographic order is preserved within digit representations. Third, receptive field overlap is greatest for immediately adjacent within-digit recording sites, and decreases monotonically with distance along the digit longitudinal axis. Last, representational discontinuities emerge. With the exception of a few sites at the extreme edges of the network, there are no longer any double-digit excitatory receptive fields on the experimental recording track. On the experimental track, receptive field centroids have shifted away from the D1–D2 border and toward the D1 midline, and receptive field sizes are smaller than those on the control track.Following a single syndactyly refinement cycle the D1–D2 boundary is nearly obliterated. All of the excitatory receptive fields recorded on the experimental track have shifted their centroid away from the D1 midline and have acquired “double-digit” receptive field extent. Other features of normal somatotopy persist on both the experimental and control tracks. The consequence of additional syndactyly refinement cycles on the experimental track is a fine-tuning and greater uniformity of excitatory receptive field size. Differences in excitatory receptive field size between extreme edge and interior on the experimental track mitigate.The effects of simulated syndactyly are reversible. Following a single cycle of baseline stimulation, excitatory receptive fields on the experimental track begin to migrate back in the direction of the D1 midline (not shown). Receptive field sizes, orientations and extent of overlap are more variable. Additional syndactyly release cycles result in a map of excitatory receptive field centroids indistinguishable from a baseline refined map.The results for inhibitory receptive field extents and overlap are at once similar and strikingly different from those for excitatory receptive field extents and overlap.Figure In the unrefined network, control track inhibitory receptive fields are of similar though not uniform size, orientation and overlap. Experimental track inhibitory receptive fields are of similar size, orientation and overlap. Experimental track inhibitory receptive fields are double-digit in extent and larger than corresponding excitatory receptive fields.Baseline refinement yields features of normal somatotopy on both the experimental and control tracks. First, receptive fields are continuous, single-peaked, and roughly uniform in size. Second, topographic order is preserved within digit representations. Last, receptive field overlap is greatest for immediately adjacent within-digit recording sites, and decreases monotonically with distance along the digit longitudinal axis.There are differences between the control and experimental tracks. On the control track, inhibitory receptive field locations have not changed, but they are reduced in size and are single digit in extent. On the experimental track, inhibitory receptive field positions have not significantly translocated, but extent is much larger.Comparison of the excitatory and inhibitory control tracks shows that inhibitory receptive field extent and overlap are greater than those of excitatory receptive fields in the same cortical column.Comparison of the excitatory and inhibitory experimental tracks reveals profound differences in receptive field extent, orientation and degree of overlap. By visual inspection, inhibitory receptive field extent is much greater, there is less or no apparent orientation, and there is greater overlap. Whereas E cell receptive fields are single-digit in extent, I cell receptive field extents are double-digit and as will be shown below bimodal. A consequence of baseline refinement is a subset of cortical columns whose E cell and I cell receptive field characteristics profoundly differ. This divergence is located exclusively along (both sides of) representational discontinuities.Following a single syndactyly refinement cycle changes are observed in both the control and experimental tracks. On the control track, by visual inspection the single cycle syndactyly inhibitory receptive field extents appear to be more variable, with some possible effects at the network edges, compared with the baseline refined results. It is likely that the changes observed on the control track are driven by a combination of network edge effects, frequency of stimulation and the profound changes occurring on the experimental track. On the experimental track, again by visual inspection, the inhibitory receptive fields are smaller and show relatively more orientation, compared with the baseline refined results. The consequence of additional syndactyly refinement cycles on the experimental track is a further reduction in inhibitory receptive field extent and increased orientation.Following a single post-syndactyly release cycle of normal stimulation, inhibitory receptive fields on the experimental track resemble those of a baseline refined network. Additional syndactyly release cycles result in a map of inhibitory receptive field centroids largely indistinguishable from that of a baseline refined map. The exceptions are minor in the form of a few inhibitory receptive fields whose size or orientation appear somehow “in between” that of the syndactyly refined and baseline refined network.Figure These numerical results confirm the visual observation to be shown below that in the regions near discontinuities the E cells are unimodal, single-digit and small, and the I cells are bi-modal, double-digit and large.Figure Detailed analysis of individual simulated cortical columns reveals characteristic differences between columns located within a cortical representational zone and those immediately adjacent to and on either side of a cortical representational discontinuity.Column (#333) is within the D1 representation adjacent to the D1 representational midline. Figure Figure The spatial pattern of input layer to excitatory cell synaptic weights following baseline refinement is a single-peaked, continuous distribution concentrated within a sub-zone slightly offset from center. The configuration remains stable throughout syndactyly refinement. The spatial pattern of excitatory to excitatory synaptic weights is single-peaked and remains stable from baseline refinement through syndactyly refinement. Notably, following baseline refinement, the spatial pattern of inhibitory to excitatory weights is nearly uniform with little or no orientation. The range of values of the weights is wider following syndactyly, though still narrow (ratio of maximum to minimum is 1.16 in contrast to excitatory to excitatory connection type maximum to minimum ratio of 2.7). The inhibitory cell in cortical column #333 receives weighted inputs from a 7 × 7 grid of excitatory cells. Following baseline refinement, the spatial pattern of weights is single-peaked, through broad, continuous, and slightly offset from center away from the discontinuity between digits 2 and 3, and slightly proximal. Following syndactyly refinement, the spatial pattern remains single-peaked, continuous, but is more concentrated in a zone distal to center and slightly toward the border between digits 2 and 3.The above analysis was repeated on all applicable cortical columns with similar results. These results establish a profile of characteristic spatial patterns of incoming excitatory and inhibitory synaptic weights within cortical representational zones. The stage is set for a comparative analysis with cortical columns straddling a cortical representational discontinuity.Cortical column (#648) lies immediately adjacent to the D1-D2 cortical representational discontinuity. Figure The inhibitory receptive field—from the same cortical column #648—undergoes a dramatically different evolution. The progression from initial conditions to a baseline refined map results in a large, double-digit receptive field. The progression from baseline refined map to a syndactyly refined map—wherein the D1-D2 representational boundary is obliterated—results in an inhibitory receptive field single-digit profile characteristic of a normal within-representational inhibitory cell. Not shown is the further result that following syndactyly release, the double-digit inhibitory receptive field characteristic is restored and is indistinguishable from that recorded at baseline refinement.Figure The spatial pattern of input layer to excitatory cell synaptic weights undergoes shifts that are highlighted in comparison with the position of the D1-D2 border indicated by the green line. The result of baseline refinement is a tight cluster of weights exclusive to D1 locations spanning the region between the D1 midline and the D1-D2 representational boundary. As a result of syndactyly there is a significant shift such that there is a large, single-peaked continuous pattern spanning locations on both D1-D2. The resulting distribution is indistinguishable from that of a normal baseline within-representational zone profile. Not shown is the further result that following syndactyly release refinement the baseline refined map spatial pattern is largely, if not completely, recovered.The changes in spatial pattern of excitatory to excitatory cell synaptic weights across experimental conditions mirrors those of the sensory afferent to excitatory cells. The result of baseline refinement is a broad nearly flat distribution of weights originating from a subset of connected excitatory cells whose receptive fields, in turn, are located exclusively on D1 locations spanning the region between the D1 midline and the D1-D2 boundary. The ratio of maximum to minimum connection weight within-digit  is ~2x, whereas the ratio of maximum to minimum connection weight across digits is >100x (in favor of D1). As a result of syndactyly there is a significant shift such that there is a large, nearly symmetric continuous pattern with contributions from excitatory cells whose receptive fields are “double-digit” from both D1 and D2, and where the ratio of maximum to minimum connection strength is ~2.25x. The resulting distribution is indistinguishable from that of a normal baseline within-representational zone profile. Following syndactyly release the baseline refined map spatial pattern is reacquired.The spatial pattern of inhibitory to excitatory cell synaptic weights changes shares many, but not all of the features of those described for sensory afferent to excitatory, and excitatory to excitatory. The notable difference is that as a consequence of baseline refinement the excitatory cells receive inputs from inhibitory cells on both sides of the representational border. The source of inhibitory inputs from D2 is restricted to the two rows wide band immediately adjacent to the D1-D2 representational boundary. The spatial pattern of connection strengths is highly oriented across-digits, but nearly uniform within-digit representation. The results are consistent with and reveal at the level of individual synaptic weight patterns the basis for the observed double-digit inhibitory receptive fields. Following syndactyly release refinement, the baseline refined map spatial pattern is reacquired.The progression of changes across experimental conditions of the spatial pattern of excitatory to inhibitory cell synaptic weights is unique compared to those discussed above. The result of baseline refinement is a multi-peaked distribution of weights whose sources are from excitatory cells on both sides of the representational border. There is a concentration from excitatory cells whose own receptive fields lie on the opposite side of the representational border (on D2) and with a slight distal offset. A broader, less concentrated contribution arises from excitatory cells whose own receptive fields lie on the same side (D1) of the representational border. These are shown in Figure The above analysis was repeated on all applicable cortical column pairs straddling a representational border with similar results. These results establish a profile of characteristic spatial patterns of incoming excitatory and inhibitory synaptic weights that contribute to cortical representational discontinuities.Experimental Track II yields confirmatory control data (not shown) wherein the network undergoes substantially similar or nearly identical counts of stimulation but absent the syndactyly procedure. Throughout all phases, cycles and trial, the network maintains expected features of topographic organization and receptive field characteristics. Visual observation of topographic order and convergence of the network were confirmed with numerical measures of topographic order (not shown)  from the results reported above. The study was repeated with additional parameter variations. These included: (a) a range of input patch sizes from 5 × 5 to 15 × 15; (b) additional cycles up to 20 to confirm convergence; (c) varying input stimulus duration from 50 to 250 ms; (d) input stimulus magnitude ; (e) varying the size of the local extent of connections from 3 × 3 to 4 × 4, and 5 × 5; (f) repeated trials (N = 5) with 45 × 45 networks with independent random number generator seeds; and (g) repeated trials (N = 3) with 75 × 75 networks with independent random number generator seeds. The results reported above were confirmed across all of these parameter variations and network trials.Multiple control experiments were conducted to test the hypothesis that the results obtained in this study are merely an artifact due the input stimulation pattern. In particular, during Baseline Refinement, there is a descending gradient in stimulation count moving away from midline toward the digit border or network edge. See Figure A final set of control experiments sought to characterize the width of the “band” of double-digit inhibitory cells straddling the digit representational discontinuities. In a network of size 30 × 30 the width of the band of double-digit inhibitory cells was confined to a single row on either side of the discontinuity for a total of two rows. In the present 45 × 45 case there are two rows of such cells on both sides of the discontinuity for a total of four rows. Across repeated trials with 45 × 45 and 75 × 75 networks, the first row immediately to either side of the discontinuity inhibitory cells uniformly took on the characteristic of double-digit, bi-modal, large receptive field extent. In the next unit distance row away from the discontinuity and on both sides, inhibitory cells clearly maintained a double-digit characteristic, but for cells on the D1 (D2) side the receptive field extent and response magnitude favored D1 (D2) sites. In all cases the divergent characteristic was restricted at most to the first two rows on either side of a representational discontinuity.all of the network connection weights. These inhibitory cells' input spatial patterns of synaptic weights, and receptive field extent, orientation and overlap on the input layer differ profoundly from those observed in: (a) the paired excitatory cell in the same cortical column, though with characteristic overlap; and (b) excitatory and inhibitory cells' properties in cortical columns located at a distance from the representational discontinuity. The stabilization of and reversible reorganization of the properties of these inhibitory cells contributes to the plasticity of simulated cortical representational discontinuity observed before, during and following release from simulated digital syndactyly between two of the digits in a simplified three-digit model of the hand. These simulations suggest that: (a) local and long-range receptive field structure and its plasticity may reflect fundamentally emergent properties of cortical neural networks of the present type; (b) where computational network connectivity is assigned a priori, results may mask these emergent properties; and (c) the magnitude of excitatory and inhibitory receptive field structure plasticity in and around representational discontinuities must be of greater magnitude—and thus may be more readily accessible to measurement by the neurophysiologist perhaps through the analysis of laminar differences—compared to within-representational zones.Simulation studies with a computational model of area 3b demonstrate novel emergent properties of inhibitory cells located exclusively in zones immediately adjacent to and on either side of cortical representational discontinuities. The model consists of a lattice of cortical columns wherein a simple covariance synaptic plasticity rule is paired with competitive normalization to tune Limitations and available extensions of the present study are in relation to variation of system parameters, certain as yet unexplored configuration which themselves may prove fruitful, and network stability conditions.N = 15 to N = 45 in steps of 5; and N = 75), input patch size (1 × 1–15 × 15), synaptic plasticity adaptation rate, number of cycles per run, input patch stimulation duration and trial length, among others. Variables not systematically varied are those relating to the anatomical spread of input node cortical layer connections, and the extent of local connectivity amongst connection types intrinsic to the cortical layer. One reason to not prioritize such analysis is that even with the present value of 7 × 7 local connectivity grids, the excitatory-excitatory cell connections values, for example, are observed to concentrate in sub-regions with rapid fall off to the edges. A systematic analysis could vary the local neighborhood connectivity from less than 7 × 7 to an all-to-all connection motif. Other limitations in the present study that could potentially be material include the use of only a single fixed input patch size in a given experiment and the presentation of only a single stimulus per trial. More complex characterizations of area 3b receptive field structure have been obtained experimentally  this study does not contribute directly to the question of inverse cortical magnification.Network stability is a consideration and by itself a potentially fruitful area for further exploration using networks of the present type  In other cases, whether temporal pathologies are observed, or not, the topographic map becomes “degenerate”: three to four representational zones emerge with exceptionally high cortical magnification of a few restricted zones on the input layer, and at times including double-digit receptive fields (even where the input stimulation patterns were single-digit only).Finally, as with much of cortical neural network modeling, correspondence is most easily drawn with a salient feature of cortical neuroanatomy—the cortical column. While it is possible to observe complex and meaningful spatial-temporal dynamics in these lumped cortical column models, the present results do not explicitly take into account feed-forward and feed-back mechanisms involving sub-cortical contributions Jones, .The contribution of the present study is both confirmatory and distinctly novel with respect to neurophysiological and computational studies of cortical columns and cortical neural networks.Allard et al.  demonstrin vitro adult rat neocortical functional borders have described diverse excitatory and inhibitory outcomes following tetanic stimulation  in adult monkey area 3b and rat SI cortex. The experimental protocol might proceed to implement a standard receptive field mapping protocol to analyze adjacent cortical representations that each have sufficiently high cortical magnification and sufficiently small layer IV excitatory receptive field sizes. Such conditions may be met in the cortical representation of the glabrous skin surface of the phalanges of adjacent fingers of the hand in the monkey, or at the face-forearm representation in the rat. By sufficiently high cortical magnification is meant a representation that is large enough to support identification of a “within-representational” zone distant from all representational discontinuities. By sufficiently extensive representational borders is meant a region of discontinuity that permits multiple penetrations along and on both sides of the discontinuity. And by sufficiently small within-representational layer IV excitatory receptive field sizes is meant that increases in excitatory (inhibitory) receptive field size within- and between recording penetrations should be readily and obviously detectable using standard receptive field mapping procedures.The present model predicts that with the above protocol the data should, in their simplest form, yield two characteristic, statistically significantly different, laminar difference profiles. One profile will correspond to the “within-representation” type  and the other will be the “boundary-adjacent” type . Furthermore, subject to experimental manipulation of the type described in this study, the experimenter will be able to cause reversible change from type BA to type WR, and from type WR to type BA. A variation on the above hypothesis is to induce plasticity in a representational discontinuity contemporaneously with single-unit or multi-unit recordings of both excitatory and inhibitory cells and taking into account laminar position.w), the weight adaptation step size (βw), the weight adaptation step size relaxation time constant (αβ), and normalization frequency . Anecdotally, the formation of topographic maps with representational discontinuities was most sensitive to the weight adaptation step size (βw). Excitatory receptive field organization was more sensitive than inhibitory receptive field organization. For example, with much larger values than those listed in Table The present computational model shares features in common with the literature of distributed self-organizing neural networks  so that its neural mechanism can be compared and contrasted with controls at the level of the evolution of spatial patterns by type of individual synaptic connections strengths. Finally, the present study suggests that perhaps there are additional features of neural networks that may be understood as fundamentally emergent.The present computational model differs from and extends previous results in several ways. First, afferent and lateral network connectivity details are not assigned This author is aware of few studies, until quite recently, comparable with the present study in which there is synaptic plasticity on all connections and, in particular, inhibitory synaptic plasticity  C++ simulation code and R analysis scripts used to generate the Figures in this paper have been placed in a The author confirms being the sole contributor of this work and approved it for publication.This work was entirely self-funded.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heat shock proteins (HSPs) are ATP-dependent molecular chaperones which aid folding of nascent polypeptides, maintain proteins in unstable conformations and prevent protein denaturation. These functions are essential in many biological contexts, including assembly and disassembly of macromolecular complexes, trafficking of proteins and regulation of enzyme activity . MitoticIn a recent study, we discovered that Hsp72, an inducible cytoplasmic isoform of the Hsp70 family, is essential to build a mitotic spindle capable of efficient chromosome congression and segregation .Secondly, abrogation of Hsp72 function led to reduced interpolar distances, reduced astral microtubules and misoriented spindles. Astral microtubules attach the spindle to the cell cortex to maintain its shape and position. These data indicate that Hsp72 has additional functions in astral microtubule organization or cortical attachment. This is likely to be independent of the ch-TOG-TACC3 complex as this is not thought to be required for astral microtubule function. Importantly, whilst Hsp72 depletion and chemical inhibition of Hsp70 give similar phenotypes, there are some clear differences. This can be explained by that fact that chemical inhibition blocks the activity of all Hsp70 isoforms and it is possible that other Hsp70 isoforms have roles in mitosis. On the other hand, chemical inhibition does not remove the protein and so the different phenotypes may result from Hsp72 having mitotic functions as a scaffold that are independent of its catalytic activity.Hsp72 is phosphorylated in mitosis by Nek6, a protein kinase that is also required for robust spindle assembly , 5. Nek6Interestingly, a phosphospecific antibody revealed that Hsp72 phosphorylated on T66 was not only localised to the spindle apparatus, but also enriched on spindle poles and kinetochores. This begs the question of whether phosphorylated Hsp72 has additional, perhaps distinct, substrates at the kinetochore. The absence of total Hsp72 may lead to loss of function of these proteins as well, exacerbating the K-fibre assembly and chromosome congression defects. Similarly, problems in cortical attachment of microtubules could contribute to loss of astral microtubules and spindle orientation defects. It will thus be important to ascertain whether (phosphorylated) Hsp72 may stabilize the plus ends of microtubules that contact kinetochores and the cell cortex.Besides their homeostatic function, HSPs have an important role in protecting cells from the proteotoxic stress that can arise in different pathological states . These i


Despite the evidence showing the promise of HIV treatment as prevention (TasP) in reducing HIV incidence, a variety of ethical questions surrounding the implementation and “scaling up” of TasP have been articulated by a variety of stakeholders including scientists, community activists and government officials. Given the high profile and potential promise of TasP in combatting the global HIV epidemic, an explicit and transparent research priority-setting process is critical to inform ongoing ethical discussions pertaining to TasP.We drew on the Arksey and O’Malley framework for conducting scoping review studies as well as systematic approaches to identifying empirical and theoretical gaps within ethical discussions pertaining to population-level intervention implementation and scale up. We searched the health science database PubMed to identify relevant peer-reviewed articles on ethical and implementation issues pertaining to TasP. We included English language articles that were published after 2009  by using search terms related to TasP. Given the tendency for much of the criticism and support of TasP to occur outside the peer-reviewed literature, we also included grey literature in order to provide a more exhaustive representation of how the ethical discussions pertaining to TasP have and are currently taking place. To identify the grey literature, we systematically searched a set of search engines, databases, and related webpages for keywords pertaining to TasP.Three dominant themes emerged in our analysis with respect to the ethical questions pertaining to TasP implementation and scale-up: (a) balancing individual- and population-level interests; (b) power relations within clinical practice and competing resource demands within health care systems; (c) effectiveness considerations and socio-structural contexts of HIV treatment experiences within broader implementation contexts.Ongoing research and normative deliberation is required in order to successfully and ethically scale-up TasP within the continuum of HIV care models. Based on the results of this scoping review, we identify several ethical and implementation dimensions that hold promise for informing the process of scaling up TasP and that could benefit from new research. The role of antiretroviral therapy (ART) in preventing the onward spread of HIV is now widely accepted as a crucial component within comprehensive HIV prevention efforts  based ona. This raises questions about the kinds of efforts that will be required within these large-scale programs to identify and engage HIV positive individuals into long-term care and treatment. As a result, questions related to an individual’s autonomy and the potential for coercive seeking and testing strategies during implementation warrant examination [Given the high profile and potential promise of TasP in combatting the global HIV epidemic, an explicit and transparent research priority-setting process is critical to meaningfully inform ongoing ethical discussions pertaining to TasP. This strategy requires a comprehensive and systematic synthesis of the key ethical issues and concerns related to the implementation and scaling up TasP as expressed by both scientists and community stakeholders. For example, within the TasP approach, clinical treatment guidelines within many settings now recommend ART for all individuals living with HIV, regardless of their individual CD4 count or based on an expanded set of parametersmination . Moreovemination . Despitemination . Nonethemination .In this paper, we draw on concepts and approaches from the field of population and public health ethics to critically interrogate TasP approaches in order to more fully consider and negotiate a balance of individual and collective interests and health outcomes . Our aimHIV, HIV infections, treatment as prevention, TasP, HIV prevention, and ethics. We included review articles, and used citation tracking to identify other relevant sources. We included only articles that included a discussion of the ethical issues pertaining to the implementation and/or scaling up of TasP. Articles that only discussed issues pertaining to TasP research  were excluded, as well as articles that focused exclusively on other HIV prophylaxis regimes . Empirical studies that did not engage with implementation/scale up or ethical issues were excluded.We drew on the Arksey and O’Malley framework for conducting scoping review studies ,14 as weSearches yielded 374 results in PubMed. We reviewed all abstracts for content, removed duplicates, and included those articles related to the ethical and implementation issues related to TasP, resulting in a total of 19 articles. We also hand searched the references of relevant articles and special journal issues within this field, and co-authors searched their own library archives, resulting in additional articles, for a total of 8 peer-reviewed articles. Finally, one article was added via citation chasing and one from the library of a reviewer, resulting in a total of 29 articles.The Centre for Reviews and Dissemination[Given the tendency for the exclusion of grey literature within systematic reviews , as wellemination. This inCo-authors BP and RK screened the search results for eligibility. Data containing ethical discussions of TasP advanced within the peer-reviewed and grey literature were extracted and managed in Microsoft Excel. Tables Using these questions as a rubric, we were guided by both deductive and inductive approaches as we employed techniques from thematic analysis to identify patterns and themes within the literature . The co-b. Three dominant themes emerged in our analysis with respect to the ethical questions pertaining to TasP implementation and scale-up: (a) balancing individual- and population-level interests; (b) power relations within clinical practice and competing resource demands within health care systems; (c) effec tiveness considerations and socio-structural contexts of HIV-related experiences within broader implementation contexts.Our search resulted in 29 peer-reviewed articles and 19 grey literature sources that were included in data extraction and literature mapping. Figure The ethical implications of initiating individuals on ART regardless of their CD4 count emerged as the most salient and contentious question related to the ethical implementation of TasP . This deIndividualistic  frameworks tend to prioritize individual autonomy against a set of communitarian frameworks that emphasize the interests of the broader population . Applicanot to seek early treatment was also described as an important ethical consideration regarding the successful scaling up of TasP [Questions about the ethics of early initiation of ART have also been closely embedded in matters of agency. If the individual freely chooses to initiate TasP , then TasP meets the ethical criteria set out within applied medical ethics – regardless of the potential for individual or population-level harms/benefits ,21. Howe of TasP ,25.Communitarian and utilitarian framings, on the other hand, offer a view that more strongly features the potential for TasP to prevent future cases as a primary consideration, counter-positioning previous HIV prevention approaches as ‘status quo’ and insufficiently equipped to decrease the spread of HIV . These ‘The second dominant theme that emerged in the analysis related to power relations within patient-physician interactions and health care systems more broadly . For exaversus prevention [Questions also have been raised in the literature around the prioritization and distribution of limited resources. Indeed, in the context of scarce resources, issues related to TasP have led some to question the extent to which efforts should be focused on treatment evention . For exaevention  – particevention ,35. Stilevention . Moreoveevention . Ultimatevention ,33.Issues related to the scarcity of HIV prevention and health promotion resources have also led to a set of arguments around targeting efforts at specific population sub-groups considered most at-risk for transmitting HIV for cost-specific reasons , though Others have suggested that explicit criteria be used in order to determine who should be prioritized to receive treatment. In general, children and monogamous sex partners (those least likely to transmit the virus) are characterized as low priority groups for TasP, whereas those who are the most likely to spread HIV should be high priority  . These aTasP is most widely referred to as one strategy within a set of HIV prevention programs, often referred to as a continuum, cascade or combination prevention strategy ,36,40,41The third emergent theme centered on questions related to the external validity of randomized controlled trials supporting the role of TasP, leading to concerns about whether existing trial data is sufficient to justify the widespread implementation of TasP ,37,43. WIt also has been postulated that effectiveness studies could reveal TasP as having unintentional inequity-enhancing consequences. For example, effectiveness trials might demonstrate the potential for a skewing of beneficial impacts among various population sub-groups  ,44. UninThe issue of adherence in ‘real world’ settings was widely discussed in the literature, with many suggesting that the levels of adherence attained in clinical trials cannot be attained within most settings globally – particularly within resource-poor settings that present a set of geographical and sociocultural challenges ,44,47,48Given these concerns, the social determinants of health were often cited as a key priority within the literature. For example, organizations such as UNAIDS and the World Health Organization recommend comprehensive approaches to HIV treatment and prevention that also acknowledge and respond to the underlying and upstream causes of HIV infection, including gender inequality and access to health care, employment and housing ,52. In sSome of the most impassioned arguments in the literature related to the socio-structural contexts of HIV-related experiences, with some authors characterizing TasP’s commitments to addressing the distal causes of HIV as rhetoric . For theIn the current scoping review, a variety of normative ethical theories were either implicitly or explicitly used to ground the arguments advanced in the literature regarding the implementation of TasP. Specifically, we explored three key themes that have important ethical and implementation implications: (1) Effectiveness considerations and socio-structural contexts of HIV-related experiences; (2) Power relations within clinical practice and competing resource demands within health care systems; and 3) Balancing individual- and population-level interests. While conflicting interests associated with various ethical frameworks may not be fully resolvable , ongoing discussion and deliberation in this area will help provide progress towards a reflective equilibrium in which a degree of ethical coherence and acceptability is acquired. Below we outline where priorities for empirical research will advance these ethical discussions. Based on the results of this scoping review, we identify several areas that hold promise for informing the ethical implementation and scale up of TasP and that could benefit from new research  as well as on population subgroups  . OngoingThere also is an urgent need to support new empirical research that characterizes how interactions between TasP and its various implementation contexts affect the health and social well being of individuals as well as populations. Limited data exist on issues pertaining to risk compensation behavior and the capacity for non-adherence or early initiation to lead to drug resistance  . While wIn addition, effectiveness studies offer opportunities to embrace heterogeneous experiences associated with TasP implementation as well as the social and structural factors that shape HIV-related experiences – particularly among vulnerable and socially disadvantaged population sub-groups. While clinical trials have provided information related to heterosexual sero-discordant couples, additional information related to men who have sex with men and other vulnerable population sub-groups  is urgently required in order to advance these discussions. Highly-controlled clinical trial settings with primarily homogenous samples do not have sufficient capacity to detect the diversity of pathways and mechanisms through which TasP might be implemented across contexts; thus, relying exclusively on information regarding implementation and ethical considerations gleaned from controlled trials may point us in unrealistically uniform future directions. Furthermore, homogenous samples effectively limit (by default) forms of variability that are important considerations in ethical implementation, including behavioural characteristics, cultural and ethnic traditions and gender norms. Building capacity in effectiveness studies also could enhance the likelihood of undertaking international comparative studies to further determine the extent to which context-based heterogeneity  shape or respond to the ethical and implementation dimensions of TasP.Many authors argued that if an individual is fully informed and has the capacity to consent  to the early initiation of TasP, public health practices associated with TasP are ethically justifiable. However, many questions arose as to whether all individuals exposed to TasP will have the capacity to be fully autonomous given the power relations manifest in many clinical interactions – particularly among vulnerable or disadvantaged population sub-groups. In addition, while the literature reviewed here does not surface concerns that clinicians might make a set of idiosyncratic ‘choices’  about who should or should not be offered TasP, these concerns have been identified in another body of literature  – that pdispersion of benefits. TasP represents an important area in which more research, evidence and philosophical deliberation may provide some resolution pertaining to the ethical implications of targeted public health practices. In order to provide moral guidance in this area, ongoing empirical and philosophical work should aim to characterize the extent to which a burden negatively impacts individuals and population sub-groups targeted for TasP.Within the realm of public health, there is concern that features of various targeted public health interventions (whereby a particular subgroup of population is targeted for an intervention or enhanced intervention focus) may exacerbate health inequity ,60, raisPerhaps one of the most salient and complex moral dilemmas that arises relates to issues around resource distribution within the context of scarcity and various global austerity measures. Based on our analysis of the literature included in the current scoping review, we suggest it would be productive to move beyond the ongoing ‘treatment versus prevention’ debate and focus energies on developing new empirical estimations regarding the costs (including opportunity costs) of scaling up TasP. To make informed decisions globally and locally about ethical scaling up of TasP, it will be important to understand the degree to which investments in TasP efforts may imply opportunity costs (or have a multiplier effect) within a broader comprehensive spectrum of care and more upstream prevention.Within these efforts, interdisciplinary and collaborative approaches are required to engage in normative assessments of various sets of evidence. Indeed, based on these finding, techniques associated with implementation and social science  and moral frameworks from the field of population and public health ethics  will play an important role in leveraging the successful and ethical implementation of TasP. Finally, in order to mitigate some of the conflicting arguments in this area, we suggest the source origins of both the methodological and philosophical frameworks should be addressed to the fullest extent possible, along with clear, transparent rationale with respect to how the empirical and normative interacted to arrive at a moral conclusion. Indeed, the evaluation of data in this area should continue to engage in critical approaches, given the inherent difficulty of transforming complex research findings  into moral conclusions .This scoping study was limited in its capacity to reveal all ethical issues that will arise within this area . In addition, articles in languages other than English were not included. Moreover, this study did not appraise the quality of ethical arguments  nor the quality of the evidence base on which various arguments rest. However, by drawing on both peer-reviewed and grey literature, our review does provide illustrations of some dominant ethical issues that demand new empirical work that could inform the ethical implementation and scaling up of TasP.New research and normative deliberation is required in order to successfully and ethically treat and prevent HIV through the inclusion of TasP within HIV continuum of care models. From an ethical and implementation perspective, the process of scaling up TasP should not be conceptualized or practiced as linear, but rather as a constant feedback loop in which new evidence constantly informs morally robust practices within and during its implementation.aBefore the use of ART for TasP, the CD4 count (a measure of white blood cells in units of cells/mm3) at which individuals were recommended to begin ART was generally accepted to be at approximately 350 cells/mm3 or less [3[3). In other settings , ART may be altogether unavailable for prevention purposes [ or less . Since t or less  with resr less [3 and somepurposes -69.bWhile there are similarities between the methods associated with systematic reviews and the methods and techniques of scoping reviews with respect to ‘mapping’ the extent, range and nature of various substantive areas , there are also important differences . As a result, we have modified the PRISMA flow chart to reflect our inclusion/exclusion processes.The authors declare that they have no competing interests.RK and WS developed the study design and protocol. BP carried out the literature search. BP and RK screened the literature collected for inclusion/exclusion criteria. KT provided analytic contributions to the (re) analysis of data and revisions to the manuscript upon important feedback from reviewers. All authors contributed to the thematic analysis and drafting of the findings. All authors reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6939/15/54/prepub


Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits  were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against  B. bovis and B. bigemina have a considerable impact on cattle health and productivity , S/N ratio = [μ(+)-μ(-)]/σ(-), % CVmax = 100x[σ(+)/μ(+)], and % CVmin = 100x[σ(-)/μ(-)], respectively, where μ(+) and σ(+) are the mean and standard deviation of pRBCs (positive control), respectively, and μ(-) and σ(-) are the mean and standard deviation of the uninfected bovine and equine RBCs (negative control), respectively.Statistical analyses to determine assay quality (or Z’) were performed according to . BrieflyB. bovis and T. equi pRBCs at 1% parasitemia were cultivated in 96-well plates at different HCT values  in M199 medium alone or with the indicated concentrations of diminazene aceturate  in 100 μl final volume. Non-parasitized RBCs were loaded into each well in triplicate and used as a blank control. RBC cultures were maintained for 4 days without replacement of the medium. On the fourth day, thin blood smears from each well were prepared and stained with Giemsa stain to be observed under the microscope to calculate parasitemia. Next, 100 μl of a lysis buffer containing 2× SGI nucleic acid stain was directly added to each well by gentle mixing. The plates were then incubated for 24 hours in a dark place at room temperature, and the fluorescence values were determined using a fluorescence plate reader as previously mentioned. Gain values were set to 100. The mean fluorescence values (after background subtraction of uninfected bovine and equine RBCs) were then plotted against the logarithm of drug concentrations. The 50% inhibitory concentration (IC50) values were calculated on the fourth day of culture using both the fluorescence-based assay and microscopic examination. In a separate experiment, two 96-well plates were used for the cultivation of B. bovis and T. equi pRBCs at 1% parasitemia at 2.5% and 5% HCTs, respectively. The pRBCs were cultivated for 4 days with replacement of the medium in triplicate wells for each concentration of the drug. On the fourth day, IC50 values of diminazene aceturate were determined as reported above. Each experiment was repeated three times.B. bovis, B. bigemina, T. equi, and B. caballi pRBCs were cultivated at 1% parasitemia in 96-well plates using 2.5% and 5% HCTs for bovine and equine parasites, respectively, and M199 or RPMI 1640 media alone or with the indicated concentrations of drugs, including diminazene aceturate, luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin. The pRBCs were cultivated for 4 days without replacement of the medium in triplicate wells for each concentration of the drug. Four plates were used in quadruplicate for each drug experiment in bovine Babesia parasites and in diminazene aceturate only for equine Babesia and Theileria parasites, and growth inhibition assays [Babesia and Theileria parasites, only one plate was used in each drug experiment for 50% inhibitory concentration (IC50) value calculation on the fourth day of culture after adding lysis buffer containing 2× SGI. The IC50 values were calculated on the fourth day of culture using both the fluorescence-based assay and microscopic examination. Each experiment was repeated three times.n assays  between 50 values were calculated using GraphPad Prism ver. 5. The mean IC50 values by fluorescence assay and microscopic examination for each tested drug were analyzed using an unpaired t-test with the GraphPad software available online . P < 0.05 was considered to be statistically significant.Data analysis was performed using GraphPad Prism ver. 5  using the one-way ANOVA. The ICBabesia parasites showed significant linear relationships with R2 values of 0.9945, 0.9328, and 0.9930 for B. bovis and R2 values of 0.9842, 0.9637, and 0.9387 for B. bigemina with HCTs of 2.5%, 5%, and 10%, respectively  were obtained at 2.5% and 10% HCTs for B. bovis, 2.5% HCT for B. bigemina, 5% and 10% HCTs for T. equi, and 5% HCT for B. caballi.The correlation between the fluorescence values and microscopy values was assessed in this assay using three different HCTs . Correlating relative fluorescence units (RFUs) with parasitemias for bovine vely Fig  and 1B. alli Fig  and 1D. max, and % CVmin were calculated for each of the three HCTs . For bovine Babesia parasites, the Z’ factors were similar for all HCTs within a range of 0.60 to 0.97  in three separate experiments. The results obtained by the two assays at 2.5% and 5% HCTs, for B. bovis and T. equi, respectively were not significantly different  in all tested drugs for the four parasites  by 10, 1000, 1000, 500 and 50000 nM luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin, respectively  in comparison with diminazene aceturate treated group in vitro cultures of four parasites  [50 values of nimbolide for B. bigemina and B. caballi parasites were, respectively, similar to and lower than that calculated for P. falciparum [Toxoplasma [Trypanosoma [Leishmania [50 values of luteolin against the growth of 3D7 and 7G8 P. falciparum have been reported to be 11000 ± 1000 nM and 12000 ± 1000 nM, respectively [Babesia and equine Theileria parasites determined by the current assay, which might indicate the susceptibility of Babesia parasites to this drug. Pyronaridine tetraphosphate is a Chinese drug that has a strong inhibitory effect against chloroquine-sensitive and-resistant strains of P. falciparum [50 value of pyronaridine has been reported to be 2 nM against the growth of KT1 and KT3 P. falciparum [Babesia and equine Theileria parasites in the current study. Enoxacin is one of the fluoroquinolones, a potent DNA gyrase inhibitor that had the lowest IC50 values against FCC1 and VNS strains of P. falciparum [50 values of enoxacin for Babesia and Theileria parasites were significantly higher than the IC50 values for P. falciparum, which ranged from 2300 to 4400 nM [50 values of diminazene aceturate for B. caballi were lower than and nearly similar to those previously calculated by microscopy [The drugs luteolin, pyronaridine tetraphosphate, and enoxacin had not yet been tested against lciparum , 27, 28.B. bovis , but itsgram/ml) . In contlciparum . Luteolilciparum . In addixoplasma , Trypanopanosoma , Leishmaishmania , and malishmania . The IC5ectively . These vlciparum , 35. Thelciparum . The IC5 4400 nM . Finallycroscopy , 20, resBabesia and Theileria parasites, respectively, offers a new approach for accurate, simple, and rapid detection of Babesia and Theileria parasites and large-scale screening of antibabesial drugs in an in vitro culture. In addition, luteolin and pyronaridine tetraphosphate drugs exhibited the best growth inhibition of bovine and equine hemoparasites next to diminazene aceturate, fellowed by nimbolide, gedunin and enoxacin. These drugs might be more effective if used as part of a combination therapy rather than a single therapy. Further studies are required for analyzing the synergistic or an antagonistic effect of these drugs when used in combination with each other, and to determine the best effective composition ratio for the growth inhibition of bovine and equine hemoparasites for clinical application.In summary, our optimized fluorescence-based assay with 2.5% and 5% HCTs without daily replacement of the medium for bovine and equine S1 FigB. bovis. (B) Fluorescence-based monitoring of pyronaridine tetraphosphate-induced growth inhibition of B. bovis. (C) Fluorescence-based monitoring of nimbolide-induced growth inhibition of B. bovis. (D) Fluorescence-based monitoring of gedunin-induced growth inhibition of B. bovis. (E) Fluorescence-based monitoring of enoxacin-induced growth inhibition of B. bovis. Statistically significant differences are indicated by asterisks (*P <0.05) between the drug-treated cultures and the control cultures. Each value represents the mean of triplicate wells after subtraction of the background fluorescence for non-parasitized RBCs.(A) Fluorescence-based monitoring of luteolin-induced growth inhibition of (PDF)Click here for additional data file.S2 FigB. bigemina. (B) Fluorescence-based monitoring of pyronaridine tetraphosphate-induced growth inhibition of B. bigemina. (C) Fluorescence-based monitoring of nimbolide-induced growth inhibition of B. bigemina. (D) Fluorescence-based monitoring of gedunin-induced growth inhibition of B. bigemina. (E) Fluorescence-based monitoring of enoxacin-induced growth inhibition of B. bigemina. Statistically significant differences are indicated by asterisks (*P <0.05) between the drug-treated cultures and the control cultures. Each value represents the mean of triplicate wells after subtraction of the background fluorescence for non-parasitized RBCs.(A) Fluorescence-based monitoring of luteolin-induced growth inhibition of (PDF)Click here for additional data file.S3 FigT. equi. (B) Correlation between gedunin and enoxacin concentrations and RFUs on B. caballi. Each value represents a mean of triplicate wells after subtraction of the background fluorescence for non-parasitized RBCs. Statistically significant differences are indicated by asterisks (*P <0.05) between the drug-treated cultures and the control cultures.(A) Correlation between gedunin and enoxacin concentrations and RFUs on (PDF)Click here for additional data file.S4 FigB. bovis (x-axis) and log concentration of diminazene aceturate (nM) (y-axis) by the fluorescence-based method without daily replacement of the medium (black) and with daily replacement of the medium (gray). (B) Growth inhibition of B. bovis by diminazene aceturate on the fourth day estimated by the fluorescence-based method. (C) Growth inhibition of B. bovis by diminazene aceturate on the fourth day estimated by the microscope-based method. Each value is presented as the mean of three triplicate wells ± SD after subtraction of the background fluorescence for non-parasitized RBCs.(A) Growth inhibition of (PDF)Click here for additional data file.S1 Tablemax = coefficient of variation at the maximum signal and % CVmin = coefficient of variation at the minimum signal.S/N ratio = Signal to noise, % CV(DOCX)Click here for additional data file.


In animal models, neonatal exposure to general anesthetics significantly increased neuronal apoptosis with subsequent behavioral deficits in adulthood. Although the underlying mechanism is largely unknown, involvement of extracellular signal-regulated kinases (ERKs) is speculated since ERK phosphorylation is decreased by neonatal anesthetic exposure. Importance of ERK phosphorylation for neuronal development is underscored by our recent finding that transient suppression of ERK phosphorylation during the neonatal period significantly increased neuronal apoptosis and induced behavioral deficits. However, it is still unknown as to what extent decreased ERK phosphorylation contributes to the mechanism underlying anesthetic-induced toxicity. Here we investigated the causal relationship of decreased ERK phosphorylation and anesthetic-induced toxicity in the developing brain. At postnatal day 6 (P6), mice were exposed to sevoflurane (2%) or the blood-brain barrier-penetrating MEK inhibitor, α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327) (50 mg/kg). Transient suppression of ERK phosphorylation by an intraperitoneal injection of SL327 at P6 significantly increased apoptosis similar to sevoflurane-induced apoptosis. Conversely, SL327 administration at P14 or P21 did not induce apoptosis, even though ERK phosphorylation was inhibited. Restoring ERK phosphorylation by administration of molecular hydrogen ameliorated sevoflurane-induced apoptosis. Together, our results strongly suggests that suppressed ERK phosphorylation is critically involved in the mechanism underlying anesthetic-induced toxicity in the developing brain. There is accumulating evidence indicating that several classes of anesthetics induce widespread apoptosis at clinically relevant concentration in the developing brain of a wide range of animals, including rodents12345678101234568As such, we hypothesized that reduced ERK phosphorylation may be critically involved in the mechanism underlying the apoptosis caused by neonatal exposure to general anesthetics. Thus, we examined how inhibition of ERK phosphorylation is associated with anesthetic-induced toxicity in the developing brain using a mouse model.To evaluate the effect of sevoflurane on ERK phosphorylation, we performed western blot analysis on forebrain tissue after 6 h-of anesthesia with 2% sevoflurane at P6 .P < 0.0001). Sevoflurane exposure also significantly reduced the phosphorylation levels of both ERK isoforms . Total ERK expression levels were similar for both isoforms (P > 0.05).Sevoflurane-treated forebrain tissue had significantly increased levels of cleaved poly-(adenosine diphosphate-ribose) polymerase (PARP), compared to controls, indicating increased levels of apoptosis (P = 0.0005), which is similar to our previous reportP < 0.0001; pERK2, t-test, t = 30.01, P < 0.0001) without decreasing total ERK expression levels for both isoforms (P > 0.05).Next, we examined the causal relationship of inhibition of ERK phosphorylation and apoptosis in the P6 mouse brain. We used SL327 to achieve transient suppression of ERK phosphorylation as our previous report14P = 0.035), this increase was relatively small compared to the increase seen at P6 , although this reduction was relatively small compared to the reduction at P6 , similar to the reduction at P6, apoptosis was not significantly altered by SL327 administration (P > 0.05), although ERK phosphorylation levels were attenuated . Similarly, mice administered SL327 at P21 did not exhibit significantly altered cleaved PARP levels compared to vehicle controls (P > 0.05), even though ERK phosphorylation was mostly (~94%) abolished in the SL327 group . Thus, at P14 or P21, the reduction in ERK phosphorylation by SL327 did not significantly increase level of apoptosis. Taken together, we can conclude that apoptosis caused by SL327 is age-dependent, similar to the sevoflurane-induced apoptosis. In addition, the reduction of ERK phosphorylation by sevoflurane at P6 appears to be larger than the reduction at P14 and P21, suggesting that basal ERK phosphorylation may be more susceptible to sevoflurane exposure at P6 compared to P14 and P21.The result prompted us to speculate that increase in apoptosis may be in proportion to the reduction in ERK phosphorylation. To test this hypothesis, we evaluated the relationship between apoptosis and ERK phosphorylation in mice administered SL327 (50 mg/kg) at P14 . Althougstration . These dstration . Mice ad+ (AC3+) significantly increased in mice treated with SL327 at P6 compared to vehicle controls  caspase-3controls , which icontrols .+ cells were further examined. Double labeling for AC3 and the neuron-specific marker, neuronal nuclear antigen (NeuN), revealed that apoptosis was induced in neurons in the sevoflurane- , or partial pressure of arterial carbon dioxide (PaCO2) between the various groups , a significant main effect of sevoflurane treatment , and a significant main effect of SL327 treatment  (P > 0.05). Thus, the increased apoptosis caused by SL327 was occluded in mice exposed to sevoflurane. This support the idea that the decreased ERK phosphorylation is critically involved in the mechanism underlying anesthetic-induced toxicity in the developing brain.To further investigate the relationship between the ERK pathway and anesthetic-induced apoptosis, we evaluated the effect of SL327 administration on apoptosis in the brain during sevoflurane anesthesia. Mice at P6 were randomly assigned into one of the following groups: (1) SL327 (−), sevoflurane (−); (2) SL327 (+), sevoflurane (−); (3) SL327 (−), sevoflurane (+); or (4) SL327 (+), sevoflurane (+). Western blot analysis demonstrated that SL327 and/or sevoflurane administration induced a significant increase in apoptosis . A two-w 0.0001) . HoweverP > 0.05). In contrast, over an additional 7 days, the mice in the sevoflurane group travelled significantly longer distances compared to the mice in the control group (P = 0.027). These data indicate that sevoflurane-treated mice are hyperactive in a familiar environment. Similarly, the total distance travelled by the mice in the SL327 group was not significantly different from that of the mice in the vehicle group (P > 0.05) over 3 consecutive days after placement in a new cage. However, SL327-treated mice travelled significantly longer distances over an additional 7 days, compared to the mice in the vehicle group (P = 0.044). Therefore, SL327-treated mice also displayed a hyperactive phenotype in a familiar environment, similar to sevoflurane-treated mice.We previously reported that SL327 administration at P6 induced learning impairments and social deficits later in adulthoodP = 0.028), indicating increased depression-like behavior. Similarly, mice administered SL327 exhibited a significant increase in immobility time compared to vehicle controls (P = 0.018). To further confirm the increased depression-like behavior in these mice, mice were also evaluated using the tail suspension test, another assay for assessing depression-like behaviorP = 0.028), indicating increased depression-like behavior. Similarly, mice administered SL327 exhibited significantly increased immobility time compared to vehicle controls (P = 0.049).Next, mice underwent the forced swim test, a widely used assay for assessing depressant-like behaviorTogether with our previous reports, these results indicate that SL327 administration at P6 induced pervasive behavioral deficitsAlthough our data suggest that neonatal exposure to sevoflurane induced brain apoptosis through a decrease in ERK phosphorylation, the mechanism underlying the reduced ERK phosphorylation level by sevoflurane exposure remains unknown.2) gas, an effective antioxidant, suppressed anesthesia-induced ROS production in the developing mouse brain18P < 0.0001; pERK2, one-way ANOVA, F = 22.86, P < 0.0001), concomitant with an attenuation of apoptosis (P < 0.0001)). Conversely, hydrogen did not alleviate SL327-induced apoptosis (Accumulating evidence indicates that oxidative stress may contribute to anesthetic-induced neurotoxicity in the developing brain. It was reported that a synthetic ROS (reactive oxygen species) scavenger, EUK-134, effectively inhibited anesthetic-induced ROS production in the developing rat brainpoptosis . Hydrogepoptosis . Thus, tet al. (2009) reported that anesthetic-induced neuro-apoptosis in the developing brain was attenuated by lithium’s alleviation of ERK suppressionet al. also reported that N-stearoyl-L-tyrosine attenuated anesthetic-induced neuro-apoptosis via upregulation of the ERK pathway in the developing brainIn the current study, we observed that ERK phosphorylation was decreased in mice brain exposed to sevoflurane at P6, as reported previously12et al. 09 reportTaken together, our results strongly suggest that ERK suppression is critically involved in the mechanism underlying anesthetic-induced toxicity in the developing brain, although we could not exclude the possibility of existence of ERK-independent mechanism. Consistent with this notion, the effects of SL327 at P6 was much similar to those of sevoflurane. Anatomical pattern of apoptosis by SL327 and the cell type undergoing apoptosis were similar in mice administered SL327 and sevoflurane. Behavioral deficits properties caused by SL327 were also similar to those by sevoflurane. SL327 administration at P6 caused profound and long-lasting deleterious effects on behaviors. In the current study, we observed that mice exposed to SL327 exhibited hyperactivity in a familiar environment compared to control mice, similar to mice exposed to sevoflurane at P6. Thus, altered ERK phosphorylation level in the developing brain may result in attention deficit hyperactivity disorder, one of the most prevalent childhood-onset psychiatric disorders, as adult. In addition, mice exposed to SL327 showed depression-like behavior in the forced swim and tail suspension tests, similar to mice exposed to sevoflurane at P6. We previously reported that administration of SL327 at P6 also caused other behavioral deficits, including learning impairments and social deficitsThe reduced levels of ERK phosphorylation were not proportional to the levels of cleaved PARP in our results. The level of ERK phosphorylation after sevoflurane administration at P6 (~30% of control level) was higher than the levels after SL327 administration (~5% of control level), although the apoptosis levels induced by these compounds were similar . One posOur results indicated that sevoflurane decreased ERK phosphorylation through the production of ROS, although how ROS accumulation results in decreased ERK phosphorylation in the developing brain is still unknown. It is also unclear how decreased ERK phosphorylation leads in turn to apoptosis. It might be possible that decreased ERK phosphorylation results in actin cytoskeleton disorganization and impaired dendritic branching since ERK plays important roles in actin cytoskeleton organizationOne might doubt that anesthetic-induced apoptosis might be due to poor respiratory care during anesthesia in small animals. In this connection, it should be noted that SL327 does not have anesthetic effects such as hypnosis and sedation. In addition, SL327 did not significantly alter pH, arterial partial oxygen, or carbon dioxide pressure , indicatThe deleterious effects of SL327 were age-dependent, similar to anesthetic-induced apoptosis in the developing brain1324Although the underlying mechanism is unclear, there seems to exist a distinct time-window, during which ERK activation plays important roles in the normal development of neural functions. This time-window might closely correlate with the “critical period”, during which animals display an elevated sensitivity to certain environmental stimuliIn conclusion, our results strongly suggest that suppression of ERK phosphorylation through oxidative stress would be critically involved in the mechanism underlying anesthetic-induced toxicity in the developing brain. It may be important to keep ERK phosphorylation during anesthesia to prevent any deleterious effects in the developing brain. Furthermore, our result suggest that various neurodevelopmental disorders may be generally linked to defects in ERK signaling in the developing brain.All experiments were conducted according to the institutional ethical guidelines for animal experiments of the National Defense Medical College  and Tokyo Metropolitan Hospital and Institute of Gerontology . All experimental protocols were approved by the Ethics Committee for Animal Experimentation at the National Defense Medical College or the Ethics Committee for Animal Experimentation at the Tokyo Metropolitan Hospital and Institute of Gerontology. Adult C57BL/6 mice were housed three or four per standard shoebox breeding cage  with autoclaved wood-chip bedding materials. Mice were maintained on a 12-h light-dark cycle (lights on from 07:00 to 19:00) with room temperature at 22 ± 1 °C with 46 ± 2% humidity. Mice had ad libitum access to water and food.Sevoflurane anesthesia was performed as previously describedERK isoforms, ERK1 and ERK2, are activated through the phosphorylation of tyrosine and threonine residues by the upstream kinase MEK. ERK phosphorylation is suppressed by the MEK inhibitor, SL327 , which penetrates the blood-brain barrier2 + H2), which was produced by the manufacturer . Total gas flow was 2 L/min.Hydrogen gas (1.3%) was supplied as described previouslyWestern blot analysis of forebrain protein lysate was performed as previously describedImmunohistochemistry was performed as previously describedFor chromogenic staining, immunoreactivity was detected using peroxidase-conjugated secondary antibody  and 3,3-diaminobenzine-tetrachloride . The sections were counter-stained with hematoxylin. For fluorescent staining, immunoreactivity was detected using Alexa-Fluor 488-conjugated goat anti-mouse IgG secondary antibody  for primary antibodies derived from mouse. For primary antibody derived from rabbit, Alexa-Fluor 546-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes) was used. Sections were examined using a Nikon fluorescence microscope system  with an electron-multiplying (EM) CCD digital camera . Immunostaining analysis was performed by an investigator blinded to the treatment conditions. Samples from at least 5 mice per experimental condition were examined.Behavioral studies were performed at 10 weeks of age, as described previouslyThe home-cage activity test assessed spontaneous locomotor activity, and was performed as described previouslyThe forced swim test, also known as the behavioral despair test, evaluates depression-like behavior in rodents, and was performed as described previouslyThe tail suspension test, another assay of depression-like behavior, was performed as described previouslyArterial blood sampling from the left cardiac ventricle was performed as previously describedP values of less than 0.05 were considered statistically significant. Data are presented as mean ± SEM.Statistical analysis was performed using GraphPad Prism 6.0 . Comparisons of the group means were performed using a Student’s t-test, a one-way analysis of variance (ANOVA), or a two-way ANOVA followed by the Bonferroni post hoc test. How to cite this article: Yufune, S. et al. Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain. Sci. Rep.6, 21859; doi: 10.1038/srep21859 (2016).


Riparian zones support some of the most dynamic and species-rich plant communities in cold regions. A common conception among plant ecologists is that flooding during the season when plants are dormant generally has little effect on the survival and production of riparian vegetation. We show that winter floods may also be of fundamental importance for the composition of riverine vegetation. We investigated the effects of ice formation on riparian and in-stream vegetation in northern Sweden using a combination of experiments and observations in 25 reaches, spanning a gradient from ice-free to ice-rich reaches. The ice-rich reaches were characterized by high production of frazil and anchor ice. In a couple of experiments, we exposed riparian vegetation to experimentally induced winter flooding, which reduced the dominant dwarf-shrub cover and led to colonization of a species-rich forb-dominated vegetation. In another experiment, natural winter floods caused by anchor-ice formation removed plant mimics both in the in-stream and in the riparian zone, further supporting the result that anchor ice maintains dynamic plant communities. With a warmer winter climate, ice-induced winter floods may first increase in frequency because of more frequent shifts between freezing and thawing during winter, but further warming and shortening of the winter might make them less common than today. If ice-induced winter floods become reduced in number because of a warming climate, an important disturbance agent for riparian and in-stream vegetation will be removed, leading to reduced species richness in streams and rivers in cold regions. Given that such regions are expected to have more plant species in the future because of immigration from the south, the distribution of species richness among habitats can be expected to show novel patterns. Riparian zones are dynamic, diverse, and fundamentally important landscape components in most parts of the world  was mapped and photographed six times between November and April during each of two consecutive years 2011–2013). Specific ice formations  were also indicated on the maps. To identify temporal changes in early and late winter, ice formation was surveyed using Wingscapes Time Lapse Plant Cameras, which were permanently placed at each reach. Cameras were set to take three pictures per day from October to the end of April. The cameras were, however, not reliable at temperatures below −15°C. Based on the winter survey, the reaches were classified into three different groups based on the maximum spatial extent of anchor-ice cover of the entire wetted areas; one anchor-ice-rich group, with a high spatial extent of anchor-ice formation in the study reach (>30% anchor-ice cover), one with low spatial extent of anchor ice (5–30%), and another that lacked an anchor-ice cover . The obtained data included winter temperature (°C), winter precipitation (mm), number of days during winter with temperatures both below and above zero, that is, Carex juncella + C.nigra, Galium spp., Hieracium spp., Salix myrsinifolia + S. phylicifolia, Sparganium spp., and Taraxacum spp. Bryophytes included mosses and liverworts. The vegetation cover was summarized as cover of grasses, forbs, and woody plants, respectively. To evaluate differences in cover of these functional groups in relation to presence of ice, altitude in meter above sea level (MSL), elevation above the stream channel, and substrate , we used generalized linear models (GLM) with quasi-Poisson distribution to correct for overdispersion. All analyses were performed using R version 2.15.2  and four that represent a future scenario of anchor-ice-rich reaches  and four anchor-ice-free reaches . A describes within-group homogeneity compared to the random exceptions: A < 0 with more heterogeneity within groups than expected by chance; A = 0 when heterogeneity within groups equals expectation by chance; A = 1 when all items are identical within groups.To evaluate changes in species composition before and after the multiple flooding experiments, we used a nonparametric multi-response permutation procedure (MRPP), to test effects of treatment between groups. The test was run for three different sets of groups. We defined the sets of groups as (1) plots along anchor-ice-rich and anchor-ice-free reaches before versus after the watering treatment, to separate the treatment effect from the natural disturbance; (2) plots from anchor-ice-rich versus anchor-ice-free reaches after watering treatment; and (3) untreated control plots from 2011 versus 2012 to account for in-between year differences. The MRPP provides the test statistic Analysis of species richness and multivariate analyses were performed in PC-Ord version 6.0. Differences in species richness were analyzed using analysis of variance (ANOVA). Analyses were also applied on the control plot data to exclude interannual differences. To evaluate the effects on riparian bryophytes exposed to the flooding experiment, we used linear mixed-effects models (LME) with two random factors that nested the plots within elevation and reach. The same type of analysis was used to compare the cover of different functional groups before and after the flooding experiments and in the control plots, with stream as a random factor. The R package nlme was used in the mixed-effects models analysis , snow-free days in early winter. The experiment was made in four of the reaches that were included in the multiple flooding experiment . The moss mimics were thereafter attached to boulders in Mattjokkbäcken N and Beukabäcken S  with Poisson distribution as the data were not normally distributed. Position on boulder (upstream or downstream) and current velocity at each boulder were also included as factors in the analysis. All analyses were performed using R version 2.15.2  show an increase in precipitation, temperature, days with shifts between freezing and thawing, and length of growing season. Scenarios are projected for the Ume/Vindel River drainage area, and the increase in climate variables has a steeper incline in the RCP 8.5 scenario than in the RCP 4.5 scenario , whereas a different pattern was shown for the anchor-ice-free streams , whereas for grasses, woody plants, and liverworts the altitude (MSL) of the stream reach was more important for their cover than the presence of ice (P < 0.05). Substrate and elevation above the stream channel at summer low flow did not show any relationship with vegetation cover and were not included in the final model. Anchor-ice-rich streams had a higher cover of the forb Filipendula ulmaria (4.5% vs. 1.4%), whereas the cover of the woody shrub Vaccinium myrtillus was higher along anchor-ice-free streams (8.2% vs. 1.05%).The climate change scenarios cover an altitudinal gradient (mountain to coast), which represents substantial differences in ice regimes, ranging from streams with a stable ice cover to ice-free streams. As an illustration of extreme cases, we let four streams represent an anchor-ice-rich, and four streams an anchor-ice-free situation. An inventory of the streams showed that these differences also played a role in terms of plant community composition. The anchor-ice-rich streams had a higher cover of forbs in the riparian zone (Estimate = Est.) = 1.2, V. myrtillus, Vaccinium vitis-idaea, and Luzula pilosa, which turned black and died , but the plant community composition was not affected by the experiment . Neither were there any differences in the plant community composition in the control plots in-between years  and woody plants had lower cover , whereas grasses were unaffected by the multiple floods. In the unwatered control plots, there were no significant changes in cover in any of the functional groups between the 2 years  increased by 12.3%, and V. myrtillus decreased by 17.3%, while there was no difference for grasses such as Deschampsia flexuosa. Thirteen new species had colonized the plots after the experiment, eight of which were forbs, two woody plants, and three grasses. However, four species disappeared: a forb (Pedicularis sceptrum-carolinum), a woody plant (Picea abies), and two grasses (Milium effusum and Elymus caninus).The multiple floods experiment caused visible damage to stems and leaves of ied Fig. A and B. ied Fig. . The comVaccinium vitis-idaea and V. myrtillus showed visible damage after the flooding experiment. Leaves and stems of Vaccinium had turned black and died , neither was there a difference in the plant community composition in the control plots between the 2 years , but plant community composition differed between anchor-ice-rich and anchor-ice-free reaches . A look at some of the dominant species showed a 7.5% decrease of V. myrtillus, a 26.3% increase of F. ulmaria, and an 11.7% increase of Equisetum spp. , but there was no difference for grasses such as D. flexuosa. There were no differences in cover of different functional groups  before and after the experiment (P > 0.05). However, eight new species had colonized the watered plots after the experiment, six of which were forbs and two woody plants.ied Fig. A. In somts Table . OverallP = 0.047). The reduction of moss transplants was larger in Mattjokkbäcken .There was a significant loss of moss transplants in both Mattjokkbäcken (anchor-ice rich) and Beukabäcken . The number of missing sticks was also higher in plots at lower elevation, closer to the stream channel . There was no difference in the amount of broken sticks between anchor-ice-rich and anchor-ice-free reaches . Neither was there any difference in the amount of broken sticks in relation to the elevation above the stream channel .There were a higher number of sticks missing along ice-rich streams . Available evidence suggests that an increase in anchor-ice production will lead to more species in the riparian zone, whereas a decrease and eventual disappearance of ice will reduce species richness  whereas forbs increased in cover (such as F. ulmaria). This suggests that the differences in plant communities along anchor-ice-rich reaches are species specific, with some being favored while others are disfavored. The watering experiments do not include all types of physical disturbance that ice and winter floods might induce on riparian vegetation. For example, ice breakup and ice scouring are not mimicked and could potentially have caused a strong direct effect on plant community composition. Along reaches where plants annually and repeatedly experience flooding or ice events, the effects on community composition are evident, showing the difference between the chronic disturbances versus short-term experiments (Table Vaccinium spp. were reduced in cover as they are evergreen and sensitive to freezing and may suffer from frost burn if they start photosynthesizing while their basal parts are still being frozen into ice (Bokhorst et al. V. myrtillus shoots were damaged and reduced in cover by both flooding experiments, opening up patches for forbs to colonize (Fig. F. ulmaria. Plants close to the stream channel may also be affected by ice movement and ice jamming during ice breakup and by ice sheets detaching from the riparian zone (Lind et al. The community composition did not change in any of the experiments even though the species richness increased. Furthermore, there was a decrease in the percent cover of dwarf-shrubs (such as ts Table . The reaize Fig. A. This mFontinalis, making it more sensitive to disturbance. The fact that the Fontinalis transplants were dried, and probably less elastic, could also have played a role. According to Muotka and Virtanen (Fontinalis species are rather indifferent in their choice of habitat although they form their densest stands at the most stable sites, in this case the anchor-ice-free sites.The reason we did not observe any difference in riparian bryophyte cover after the flooding experiments probably reflects the fact that bryophytes are more resistant to freezing than are vascular plants (Minami et al. Virtanen , FontinaWe conclude that a dynamic flooding regime during winter, often associated with anchor-ice formation, may be a key component for favoring riparian plant diversity along boreal streams and rivers. The climate change scenarios for this boreal area indicate that ice-induced winter floods will be less likely by the end of the present century, suggesting that the species-rich riparian vegetation in streams and rivers in cold regions will change slowly from forb to dwarf-shrub dominance and that the species richness will decrease accordingly. A common view among researchers is that predicted climate changes will lead many species to shift their distribution limits northward and upward and result in generally higher species richness in currently cold regions (Pauli et al.