The isl1:GFP, Tg(vsx1:GFP) ( Kimura et al., 2008), and moerw306 zebrafish are available from the National BioResource Project of Japan (http://www.shigen.nig.ac.jp/zebra/index_en.html) selleck ( Okamoto

and Ishioka, 2010). The procedures used for time-lapse imaging were those described previously (Ohata et al., 2009a and Tanaka et al., 2007). Labeling of WT cells with rhodamine-dextran (Invitrogen) and transplantation were performed according to standard protocols (Westerfield, 2007). The pGa981-6 and pEF-BOSneo-mDelta1-T7 plasmids were kind gifts from Dr. T. Honjo (Kyoto University) and the pEF-Fc plasmid was received from Dr. S. Nagata (Kyoto University). Plasmid construction, mutagenesis of plasmids, RT-PCR, generation of sense-capped mRNA, and analyses of amino acid sequence similarities were performed essentially as described previously (Hirate and Okamoto, 2006, Ohata et al., 2009a and Wada et al., 2006). The Moe amino acid sequences used for the sequence comparisons and the sequences of MOs (Gene Tools) are listed in Supplemental Experimental Procedures. Injections of mRNA- and MO-containing solutions were performed Bosutinib mw as previously described (Ohata et al., 2009a). Fixation of embryos, in situ hybridization, whole-mount staining, cryosection staining, retrograde labeling of the reticulospinal neurons, the cell-surface binding assay, and the luciferase assay were performed essentially as described previously (Eiraku et al., 2005, Ohata et al., 2009a,

Wada et al., 2005 and Westerfield, 2007). The rat monoclonal anti-Moe antibody used for anti-Moe blotting until (Figure 1Ch) was derived with the first 13 residues of the zebrafish Moe protein (MLSFFRRTLGRRS, Invitrogen) as an antigen. The other primary antibodies used in the present study are listed in Supplemental Experimental Procedures. F-actin was visualized with rhodamine-phalloidin (Invitrogen). Cell culturing, immunoprecipitation, immunoblotting, and the GST pull-down assay were performed essentially as described previously (Hodkinson et al., 2007, Ohata et al., 2009a and Ohata et al., 2009b). Transfection

was performed with the HilyMax transfection reagent (Dojindo) according to the manufacturer’s instructions. All results are expressed as mean ± standard error of the mean (SEM), and analyses were performed with the ImageJ, Excel, and Graphpad Prism programs. Two experimental groups were compared with the Student’s t test, and comparisons of more than three groups were analyzed with one-way factorial ANOVA and Tukey tests. Differences were considered significant for p < 0.05. The authors thank Drs. J. Aoki, M. Guo, S. Higashijima, T. Honjo, A.M. Jensen, T. M. Jessell, B. Margolis, N. Miyasaka, R. T. Moon, S. Nagata, Y. Yoshihara, and the Zebrafish International Resource Center for reagents and the transgenic zebrafish; Dr. A. B. Chitnis for the personal communication, Drs. M. Eiraku, M. Isoda, M. Itoh, M. Kengaku, A. Takashima, H. Takeda, and S. Tsuda for technical advice; Dr. A.

, 2009, Clopath et al., 2008, Frey, 2001, Frey and Morris, 1997 and Okada et al., 2009). This, in turn, Kinase Inhibitor Library high throughput would lead to a memory engram being formed, at the single-cell level, at synapses dispersed throughout the dendritic arbor. However, an alternative model combining STC with the phenomenon of local activity-induced protein synthesis (Martin and Kosik, 2002 and Steward and Schuman, 2001), namely the Clustered Plasticity Hypothesis (CPH) (Govindarajan et al., 2006), predicts that STC is biased toward

occurring between spines that are close together. This would result in memory engrams being preferentially formed at synapses clustered within dendritic compartments, such as a branch (Govindarajan et al., 2006). Competition

among synapses for limiting PrPs would further restrict the engram to such a dendritic compartment because spines close to the site of translation would use up limiting PrPs and reduce their concentration at more distant spines (Govindarajan et al., 2006). The advantages of the CPH include increased efficiency of long-term memory formation and retrieval, as well as a greater capacity for memory storage for an individual neuron (Govindarajan et al., 2006). A study of the link between the level of E-LTP at a given spine and the strength of its synaptic tag, the spatial limits over which STC can occur, and DAPT research buy the temporal dynamics of the STC at individual stimulated competing spines require already a

method that permits stimulations and response monitoring of single spines. However, the field stimulation and field recording methods that have been used in the past to study STC measure the average response of a population of unidentified stimulated synapses. Thus, we developed a method using two-photon glutamate uncaging at single spines on proximal apical dendritic branches of CA1 pyramidal neurons to examine the relationship between spines that participate in STC. The expression of L-LTP was assayed by examining spine volume using two-photon imaging of the fluorescent protein Dendra (Gurskaya et al., 2006), along with perforated patch-clamp electrophysiology in some experiments to measure the change in the postsynaptic response to the uncaging of glutamate. We found that STC is temporally asymmetric, is spatially localized, and is biased toward occurring between stimulated spines that reside on the same dendritic branch. In addition, while strongly stimulated spines facilitate induction of L-LTP at neighboring weakly stimulated spines, the stimulated spines then compete for expression of L-LTP. Lastly, we demonstrated that there is a bias toward L-LTP being induced at a single dendritic branch, as opposed to across branches.

, 2008). Subsequent histological analyses demonstrated that the Aβp3-42 peptide was well distributed among the majority of the plaques, thus presenting an effective target for opsonization, FcR engagement, and microglial phagocytosis. In addition to the roles of

epitope abundance and antibody affinity in triggering effector function, antibody isotype has also been shown to be critically important (Nimmerjahn and Ravetch, 2005). Utilizing Aβp3-x antibodies with isotypes of varying effector potency, we demonstrated that robust clearance of existing plaque was in agreement with reported ability to engage activating Fc receptors. Additionally, the plaque-lowering PD0332991 manufacturer ability of the Aβp3-x antibody was shown to be highly repeatable in a dose-response study. Differences were observed in the efficacy of plaque lowering between hippocampus and cortex for the anti-Aβp3-x antibodies that may be a result of the lower net levels of deposited Aβ or possibly the delay in deposition in this tissue relative to hippocampus (and thus less modified Aβ species). Interestingly, in contrast check details to 3D6 and other N-terminal antibodies,

the Aβp3-x antibody failed to show significant plaque lowering when used as a preventative measure. We attribute this observation to the lack of modified Aβ target in the young PDAPP mice during the course of treatment prior to amyloid formation and during initial deposition.

Additionally, the lack of efficacy in the prevention paradigm for the anti-Aβp3-x antibody suggests that Aβp3-x is not the major nucleating species for initial plaque deposition. Since the Aβp3-42 peptide appears to be solely located in deposits (Bibl et al., 2012), the only mechanism of action through which the Aβp3-x antibodies could lead to plaque lowering is through phagocytosis of existing plaque. Consistent with Endonuclease this mechanism, we observed that treatment with Aβp3-42 antibodies led to increased microglial colocalization with amyloid deposits in vivo. In regard to Aβ deposition, one critical parameter that is different between AD patients and PDAPP mice is the overall amount of Aβ deposited per unit time. Imaging studies with amyloid PET ligands have demonstrated that plaque accrual in AD patients is minimal after diagnosis (Ossenkoppele et al., 2012; Villemagne et al., 2011), whereas PDAPP mice have robust deposition even during the plateau phase (i.e., the time frame after the logarithmic phase of deposition), in which the Aβ levels can increase by more than 30% in as little as 3 months. Thus, plaque lowering in PDAPP mice probably represents a very high hurdle since the final “net” plaque lowering will be a function of clearance of pre-existing plaque in addition to the newly formed plaque during the course of the study.

We have shown that Cxcr7 is required in migrating interneurons to sustain normal levels of Cxcr4 receptors in response to Cxcl12. This defect does not seem to involve a transcriptional link between Cxcr4 and Cxcr7, as proposed in other systems ( Dambly-Chaudiere et al., 2007 and Wang et al., 2008), because Cxcr4 mRNA levels are normal in the absence of Cxcr7. Most notably, Cxcr4 receptors are found in the plasma membrane of Cxcr7 mutant interneurons

in the absence of Cxcl12, which indicates that Cxcr7 is not required for the normal trafficking of Cxcr4 to the membrane. This suggests that the defects observed in vivo might be linked to the regulation of receptor endocytosis. PLX-4720 For GPCRs, endocytosis serves as a mechanism to regulate cell-surface receptor levels, thereby modulating chemokine responsiveness. Upon internalization, learn more GPCRs can be recycled back to the plasma membrane or sorted to the lysosome for degradation, and the fraction of receptors that recycle back to the membrane depends on several factors ( Marchese et al., 2003). In the case of Cxcr4 receptors, nearly 70% of the activated receptors are targeted for degradation following prolonged Cxcl12 stimulation ( Kolodziej

et al., 2008, Marchese and Benovic, 2001 and Tarasova et al., 1998). This indicates that saturating signals of Cxcl12 render cells insensitive to the chemokine in a very short time. In this context, given its high affinity for Cxcl12 ( Balabanian et al., 2005a), expression of Cxcr7 in

migrating neurons may contribute to the regulation of Cxcl12 levels in the microenvironment of each cell to prevent the rapid desensitization of Cxcr4 receptors. In the absence of Cxcr7, synthesis of Cxcr4 to in migrating neurons might not be enough to cope with the degradation rate imposed by the surplus of Cxcl12 that the cells encounter as they enter the cortex. In sum, our experiments suggest that Cxcr7 fine-tunes the response of Cxcr4 to changing concentrations of Cxcl12, thereby enabling directional migration. It is conceivable that Cxcr7 might also be necessary for the proper internalization of Cxcr4 receptors, or that Cxcr7 may somehow protect a fraction of Cxcr4 receptors from degradation. This would imply strictly cell-autonomous functions of Cxcr7 in migrating interneurons, which we have not been able to test. However, our transplantation experiments strongly suggest that Cxcr4 expression can be rescued in Cxcr7 mutant interneurons when they migrate in a wild-type environment, which indicates that if Cxcr7 plays a strictly cell-autonomous role in vivo, it may only have a relative impact in the migratory behavior of this population of cells.

These prediction errors are then passed up the hierarchy in the reverse direction, to update conditional expectations. This ensures Selleck DAPT an accurate prediction of sensory input and all its intermediate representations. This hierarchal message passing can be expressed mathematically as a gradient descent on the (sum of squared) prediction errors ξ(i)=Π(i)ε˜(i), where the prediction errors are weighted by their

precision (inverse variance): equation(1) μ˜˙v(i)=Dμ˜v(i)−∂v˜ε˜(i)⋅ξ(i)−ξv(i+1)μ˜˙x(i)=Dμ˜x(i)−∂x˜ε˜(i)⋅ξ(i)ξv(i)=Πv(i)ε˜v(i)=Πv(i)(μ˜v(i−1)−g(i)(μ˜x(i),μ˜v(i)))ξx(i)=Πx(i)ε˜x(i)=Πx(i)(Dμ˜x(i)−f(i)(μ˜x(i),μ˜v(i))). The first pair of equalities just says that conditional expectations about hidden causes and states (μ˜v(i),μ˜x(i)) are updated based upon the way we would predict them to change—the first term—and subsequent terms that minimize prediction error. The second pair of equations simply expresses prediction error (ξv(i),ξx(i)) as the difference between conditional expectations about hidden causes and (the changes in) hidden states and their predicted values, weighed by their precisions (Πv(i),Πx(i)). These predictions are nonlinear functions of conditional expectations (g(i),f(i))(g(i),f(i))

at each level of the hierarchy and the level above. It is difficult to overstate the generality and importance of Equation (1)—it grandfathers nearly every known statistical estimation scheme, under parametric assumptions about Tryptophan synthase additive noise. Vorinostat in vivo These range from ordinary least

squares to advanced Bayesian filtering schemes (see Friston, 2008). In this general setting, Equation (1) minimizes variational free energy and corresponds to generalized predictive coding. Under linear models, it reduces to linear predictive coding, also known as Kalman-Bucy filtering (see Friston, 2010 for details). In neuronal network terms, Equation (1) says that prediction error units receive messages from the same level and the level above. This is because the hierarchical form of the model only requires conditional expectations from neighboring levels to form prediction errors, as can be seen schematically in Figure 4. Conversely, expectations are driven by prediction error from the same level and the level below—updating expectations about hidden states and causes respectively. These constitute the bottom-up and lateral messages that drive conditional expectations to provide better predictions—or representations—that suppress prediction error. This updating corresponds to an accumulation of prediction errors, in that the rate of change of conditional expectations is proportional to prediction error. Electrophysiologically, this means that one would expect to see a transient prediction error response to bottom-up afferents (in neuronal populations encoding prediction error) that is suppressed to baseline firing rates by sustained responses (in neuronal populations encoding predictions).

, 1969). These results suggest that the dLGN both maintains and sharpens retinal direction tuning in a subset of neurons and contains a preferred direction-biased superficial region.

Intriguingly, the DS neurons in this region overwhelmingly encode opposite directions along a single axis of motion. This surprising functional organization of opposing direction tuning prompted us to next investigate whether the dLGN integrates across opposing directions of motion to form axis-of-motion-selective neurons within the same region, in contrast to the role of the dLGN as a simple relay of segregated functional channels. In support of this hypothesis, 15 of the visually responsive neurons were highly selective for a particular axis of motion, at a single orientation of the grating (Figures 2E and 3B, ASI > 0.5). The proportion GSK1120212 order of axis-selective lateral geniculate neurons (ASLGNs) observed is also significantly different from chance (shuffled trials, p < 10−6, see Supplemental Experimental Procedures). The preferred axis of motion of these neurons was also overwhelmingly biased toward a single axis (axial Rayleigh test, p < 0.05, unimodal Rayleigh test, n.s.), corresponding to horizontal motion (Figure 3C). The axial Rayleigh test CH5424802 in vitro is significant (p < 0.05) for all ASI thresholds less than 0.5 for neurons that show a consistent axial bias or “sensitivity” (Hotelling T2 test, p < 0.05), suggesting

others that like direction selectivity, axis selectivity in the population lies on a continuum (Figure S2B). The preferred motion axis for axis-selective neurons was not significantly different than the axis for DS neurons

(Watson-Williams test; fitted distribution < 20° from horizontal axis). Furthermore, ASLGNs, pDSLGNs, and aDSLGNs were intermingled in depth within the superficial 75 μm of the dLGN (Figure 3D; one-way ANOVA, n.s.). ASLGNs, like DSLGNs, were more sharply tuned than DSRGCs (mean width at half-maximum = 61° ± 2° [SE] for ASLGNs compared to 115° reported for DSRGCs; Elstrott et al., 2008; t test, p < 0.05). Three of these neurons could be defined as On-Off cells. Cell 1 in Figure 2E shows On-Off responses in one such neuron. The similarity in response characteristics of ASLGNs and DSLGNs suggests that they may receive common, retinal input. This is further supported by parameters of the retinogeniculate circuit, as discussed below. DSLGNs and ASLGNs in the superficial region both have strong and statistically significant preferences for the same horizontal axis of motion. This suggests that anterior and posterior but generally not upward or downward DS inputs are likely to synapse in the superficial dLGN and that ASLGNs may arise from the integration of opposing DS inputs as a result of either specific connectivity mechanisms or random sampling from local axon terminals (random wiring).

Cattle were allowed to graze freely on natural pastures, characterized by annual grass species, and

supplemented with mineral salt, receiving water ad libitum. All animals were treated with levamisole (600 mg/100 kg body weight) three times (days 22, 43 and 64) to avoid endoparasite infestations along the vaccine trial, and managed under identical conditions in the same paddock during the whole trial. Cattle were managed in accordance with local institutional guidelines and all procedures were in accordance with international guidelines [36]. Vaccinated and control groups were formed by 18 and 20 animals, respectively. Antigens were administered subcutaneously. Each dose consisted of a mixture of recombinant proteins rBYC, rGST-Hl and rVTDCE (200 μg each, 0.5 mL) mixed with 0.5 mL of adjuvant (Montanide 888 and Marcol 52), emulsified according to the vortex Selleck Dorsomorphin method [37]. The control group received an emulsion of PBS (0.5 mL) plus adjuvant (0.5 mL). Both groups received three booster injections at 21-day intervals (days 22, 43, and 64). Blood samples (10 mL) were collected via caudal vein from pre-immunized and post-immunized cattle (days 1, 78 and 127), and used for sera recovery. Blood samples were centrifuged at 5000 × g for 10 min and sera

were stored at −20 °C. At days 1 and 127, all bovines were weighted. SDS-PAGE and Western blot analysis were performed as previously described [31]. Purified recombinant proteins (1 μg protein/lane) were applied to SDS-PAGE (14% gel). For Western Blot, the nitrocellulose membranes were incubated with cattle sera (diluted 1:100) collected on days 1 and 78. Levels of antigen-specific antibodies GSK2118436 ic50 in the serum samples were assessed by dot-blot. Nitrocellulose membrane circles of 0.5 cm of diameter were coated with 1 μg of each antigen in PBS. The membranes were dried and incubated for 1 h at 37 °C with blotto [38], followed by a second incubation with cattle

sera diluted in blotto (1:100) for 16 h at 37 °C. Washing times with blotto for 10 min ensued, and the peroxidase enough conjugated antibody diluted in blotto (1:5000) was added and incubated for 1 h at 37 °C. After three washes with PBS for 10 min, the membranes were incubated with 2.5 mg 3,3′-diaminobenzidine tetrahydrochloride, 10 μL H2O2, and 150 μL CoCl2 in 5 mL of PBS. The recognition levels were quantified by gel scanning, and were analyzed using the software Image J [39]. Along the vaccination trial, bovines were continuously exposed to tick infestation (since the beginning of the immunization process) because they were under natural conditions in a tick-infested pasture. Attached adult female ticks (sized between 4.5 mm and 8.0 mm) were counted on the left side of vaccinated and control groups, to follow the tick infestation rate [40]. Animals were immobilized and ticks were counted by the same investigator. All examinations were carried out at the same period of the day (morning/afternoon).

, 2009). Unexpectedly, CB2Rs were recently shown to mediate an activity-induced self-inhibition in medial Nintedanib order prefrontal cortical pyramidal neurons (den Boon et al., 2012). CB2Rs were localized to intracellular compartments and coupled to calcium-activated chloride channels to

decrease neuronal firing. The generalizability of autocrine eCB signaling to other brain regions should be examined. Growing evidence indicates that glia participate in eCB signaling (Stella, 2010). The synthetic machinery for eCB production was observed in oligodendrocytes (Gomez et al., 2010), astrocytes, and microglial cells (Hegyi et al., 2012). Likewise, cultured astrocytes and microglial cells can produce 2-AG or AEA (Stella, 2009). It is Lapatinib solubility dmso not yet clear whether eCBs produced by glial cells modulate synaptic transmission. On the other hand, several recent findings support a role for eCBs signaling to astrocytes and their ability to indirectly mediate synaptic function. At Schaffer collateral excitatory

synapses onto hippocampal CA1 pyramidal neurons, postsynaptic activity-dependent release of eCBs was shown to target not only presynaptic CB1Rs but also astrocytic CB1Rs (Figure 4A). Astrocytic CB1Rs unexpectedly coupled to PLC via Gq/11, which increased intracellular Ca2+ and triggered glutamate release (Navarrete and Araque, 2008). In support of these functional observations, CB1Rs in hippocampal astrocytes have recently been observed using immunoelectron microscopy (Han et al., 2012). Glutamate activated NMDA receptors (NMDARs) on CA1 pyramidal neurons and, at some synapses, triggered short-term facilitation of transmitter release, presumably by stimulating presynaptic mGluR1s (Navarrete and Araque, 2008, 2010). Interestingly, this short-term facilitation was not spatially restricted, being

observed over 70 μm away from the active pyramidal cell. Thus, eCBs could concomitantly STK38 suppress synaptic transmitter release by triggering DSE and indirectly potentiate synaptic transmission through astrocytes, both in a CB1R-dependent manner. While the functional significance of such plasticity is not yet clear, astrocytes may have long-distance neuromodulatory effects that are mediated by eCB signaling. eCB-mediated neuron-astrocyte communication has also been implicated in long-term plasticity. Spike timing-dependent LTD (tLTD) between neocortical pyramidal neurons is known to require activation of presynaptic NMDARs and CB1Rs (Bender et al., 2006; Nevian and Sakmann, 2006; Sjöström et al., 2003). Surprisingly, a recent study found that astrocytic CB1Rs were necessary and sufficient to mediate tLTD (Min and Nevian, 2012). eCBs originating from layer 2/3 pyramidal neurons activated astrocytic CB1Rs, which increased intracellular Ca2+, thereby releasing glutamate and stimulating presynaptic NMDARs (Figure 4B). Given the anatomical and functional evidence for presynaptic CB1Rs in neocortex (Domenici et al., 2006; Hill et al.

These behavioral data suggest that the cognitive impairment by repeated stress may be due to the Nedd4-1 and www.selleckchem.com/products/dorsomorphin-2hcl.html Fbx2-dependent loss of glutamate receptors in PFC. To understand the

potential mechanism underlying the region specificity of the effects of repeated stress on glutamate receptor expression and function, we examined the level of Nedd4-1 and Fbx2 in PFC, striatum, and hippocampus from control versus stressed young male rats. As shown in Figure 8E, the level of Nedd4-1 was significantly higher in PFC or striatum than in hippocampus from control animals (p < 0.01, n = 8). After repeated stress, Nedd4-1 was significantly elevated in PFC (∼70% increase, p < 0.01, n = 6 pairs) but was significantly reduced in striatum (∼35% decrease, p < 0.01, n = 7 pairs) and unchanged in hippocampus (p > 0.05, n = 8 pairs). Moreover, the level of Fbx2 was significantly

higher in PFC than in striatum or hippocampus from control or stressed animals (Figure 8F, p < 0.01, n = 7 pairs). These results provide a potential reason for the higher sensitivity of PFC to repeated stress than other brain regions, like GSK2118436 order the striatum and hippocampus. In the present study, we have identified glutamate receptors as an important molecular substrate of repeated stress. Given the significance of glutamatergic signaling in PFC-mediated cognitive processes (Goldman-Rakic, 1995 and Lisman et al., 1998), it is not surprising that repeated stress impairs the object recognition memory, which is reminiscent of the memory deficits following bilateral infusion of glutamate receptor antagonists directly into PFC. The loss of PFC glutamatergic responses could also underlie the stress-induced other behavioral impairments found earlier (Liston

et al., 2006, Cerqueira et al., 2005 and Cerqueira et al., 2007). Mounting evidence has suggested that stress induces divergent changes in different brain regions (de Kloet et al., 2005 and McEwen, 2007). Chronic stress causes atrophy of dendrites in the CA3 region, suppresses neurogenesis of dentate gyrus granule neurons, and impairs hippocampal-dependent cognitive functions (McEwen, 1999 and Joëls enough et al., 2007). High levels of corticosterone or chronic stress also impair long-term potentiation (LTP) and facilitate long-term depression (LTD) induced by electrical stimulation in hippocampus (Kim and Diamond, 2002 and Alfarez et al., 2003). On the other hand, chronic stress has been shown to enhance amygdala-dependent fear conditioning (Conrad et al., 1999) and anxiety-like behavior (Mitra et al., 2005), which may be correlated to the stress-induced dendritic growth and spinogenesis in this region (Vyas et al., 2002 and Mitra et al., 2005). In this study, we have demonstrated that glutamatergic transmission in PFC pyramidal neurons is significantly suppressed in young male rats exposed to repeated stress, without the apparent loss of synapses.

Outcomes were measured at baseline, 13, and 65 weeks at physiotherapy practices not involved in the trial by three trained research assistants

who were blinded to group allocation. Blinding was maintained by instructing participants not to talk about their intervention to the research assistants. Patients were included if they had osteoarthritis of the hip or knee according to the clinical BI 6727 price criteria of the American College of Rheumatology (Altman et al 1986, Altman et al 1991) and were between 50 and 80 years of age. They were excluded if they had other pathology explaining the complaints; complaints in less than 10 out of 30 days; intervention for these complaints with exercise in the preceding six months; indication for hip or knee replacement within one year; contraindication for exercise; inability to understand the Dutch language; and a high level of physical functioning defined as < 2 on the walking ability and physical function sections of the Algofunctional

index (Faucher et al 2003, Lequesne et al 1987). They were recruited directly by the participating physiotherapists or in response to press releases in local newspapers (Veenhof et al 2005). Age, gender, height, weight, location of complaints, duration of complaints, and the presence of other chronic disorders were collected. X-rays of the hip and/or knee were scored by a rheumatologist according to the Kellgren Selleckchem Torin 1 unless and Lawrence scale; it consists of five levels where 0 = no osteoarthritis, 1 = doubtful osteoarthritis, 2 = minimal osteoarthritis, 3 = moderate osteoarthritis, and

4 = severe osteoarthritis (Kellgren and Lawrence 1957, Ravaud and Dougados 1997). Pain and physical functioning were measured with the WOMAC (Bellamy et al 1988). Physiotherapists working in primary care in the Utrecht region were included in the study. They were recruited using the NIVEL National Database of Primary Care Physiotherapists. A random sample of six hundred physiotherapists from Utrecht region was invited to participate. One hundred physiotherapists responded, of whom 87 (working in 72 practices) were willing and able to participate. The experimental group received a behavioural exercise program (see Appendix 1 on the eAddenda for details). The intervention was directed at a time-effective increase in the level of activities, with the goal of integrating these activities into daily living. The intervention also included individually-tailored exercises aimed at reducing any impairment limiting the performance of these activities. The complete protocol included written materials such as education messages, activity diaries, performance charts. The intervention consisted of a maximum of 18 sessions over a 12-week period, followed by five booster sessions in Week 18, 25, 34, 42, and 55. In Week 18 and 25, participants were allowed to receive 2 sessions.