Sensory System

The ambon damselfish Pomacentrus amboinensis and lemon damsel P. moluccensis are common site attached species of damselfish (family Pomacentridae) found throughout the Indo-Pacific on shallow reef habitats at the interface between the live coral and rubble reef edge (Fig. S1). Both species have a similar larval duration after a demersal egg phase and settle at similar sizes (P. amboinensis 17.8 d, 11.2 mm SL; P. moluccensis 19.4 d, 10.7 mm SL; [17] ). Metamorphosis is concomitant with settlement and in these species involves a major change in pigmentation (transparent to coloured) that occurs within hours, but involves little obvious change in shape [36] . However, settlement does involve major changes in physiology [37] and it is likely that marked changes also occur in the sensory systems [38] . A laboratory-based habitat selection experiment has previously shown that both species preferentially settle to healthy live coral [39] . Both species settle naturally to patches of mixed live and dead coral. Both are also planktivores as juveniles and eat a similar array of prey items (Text S1). A tagging study of 295 newly settled P. amboinensis on the continuous reef edge found that fish moved little over the first 3 months after settlement (mean = 0.63 m [40] ). It is likely that P. moluccensis has a similar degree of site attachment (pers. obs.).
Research on newly settled P. amboinensis has shown that fish enter the reef with high variability in their behavioural traits (e.g. boldness, aggression) and these traits are displayed in a manner that is consistent on small time scales of hours to days ([41] (link), [42] (link), Mero, Meekan and McCormick unpublished data]. Establishment of dominance hierarchies occurs within minutes of settlement within the species, which can rapidly lead to the eviction of subordinates from small habitat patches [31] (link). Because of the rapid establishment of territories and the high juvenile mortality, it was decided that 60 min was an ecologically relevant time to use for the establishment of residents in the priority experiments for the present study.
The present datasets were collected at Lizard Island (14° 40′S 145° 28′ E) on the northern Great Barrier Reef, Australia, between October 2007 and March 2010. Both newly metamorphosed juveniles and recently settled juveniles from the reef were used for field experiments. Light traps (see [43] for design; small trap) were used to collect both fish species at the end of their larval phase prior to their settlement to the reef. These newly metamorphosed fish were separated by species and placed into 60 L aquaria with aerated flowing seawater. Fish were kept for 24 h and fed newly hatched Artemia sp. twice per day ad libitum to allow recovery from (or acclimation to) the stress of capture, prior to use in experiments. Juveniles were collected from a shallow fringing reef at the back of Lizard Island using a solution of dilute clove oil and hand nets. All fishes used in the experiments were placed into a small clip-seal bag with a small amount of aerated seawater and measured with calipers (±0.1 mm) and then transferred into individually labeled 1 L clip-seal bags for transport. To reduce transport and handling stress, fish in bags were transported to the field site in a 30 L bin of seawater (to reduce temperature fluctuations) under subdued light conditions.

Vibrotactile noise was applied using two C-3 tactors (Engineering Acoustics, Inc., Casselberry, FL, USA) attached to the volar and dorsal wrist of the paretic arm using adhesive tapes (Figure 1). White noise signals low-pass filtered at 500 Hz drove the tactors, as in the previous study (Enders et al., 2013 (link)). The tactors were attached on the wrist to minimize interruption with manual tasks, while still affecting finger tactile sensation as shown in the previous study (Enders et al., 2013 (link)). The intensity of the vibrotactile noise was set to 60% of the sensory threshold found at the beginning of testing. That intensity was used as it is approximately the optimal noise level to affect the sensory system per the literature (Wells et al., 2005 (link)) and our previous study with that noise intensity to the wrist has shown to improve fingertip tactile sensation in chronic stroke survivors (Enders et al., 2013 (link)). The vibrotactile noise generators were attached for the duration of the testing and were turned off or on at the beginning of each block depending on the noise condition. Subjects were blinded to the noise, as they could not feel the noise.

For HDTVi, vectors for HDTVi were prepared using the EndoFree-Maxi Kit (Qiagen) and resuspended in a sterile 0.9% NaCl solution/plasmid mix containing 10 μg of pX330-p53 (Addgene 59910) or pT3-N90-beta-catenin (Addgene 31785), and 10 μg of CMV-SB13 Transposase. CRISPR-Cas9 vector system carrying sgRNAs targeting Trp53 together with the Sleeping Beauty Transposon system overexpressing CTNNB1-N90 vector in sterile saline constituted a total volume of 10% of the mouse body weight were injected into the lateral tail vein of 6-week-old C57BL/6 J mice in 6–8 s^{15 (link),16 (link)}. HDTVi-induced tumors were harvested 3 weeks after HDTVi. For staining and lineage tracing assays, 3–6 mice (male and female) were used per group. 6-week-old mice male and female mice were purchased from The Jackson Laboratory, including C57BL/6J mice (JAX stock, #000664), Acta2-iCre-PolyA (Gem Pharmatech, #T036743), Cd36em1(flox) (Shanghai Model Organisms Center, #NM-CKO-200086), Mifem1(flox) (Shanghai Model Organisms Center, #NM-CKO-2110274), Lratem1(2A-Cre) (Shanghai Model Organisms Center, #NM-KI-190097), R26-CAG-LSL-tdTomato (Shanghai Model Organisms Center, # NM-KI-225042). All animals were housed in a pathogen-free facility with 24-h access to food and water. Animal experiments in this study were approved by and performed in accordance with the institutional animal care and use committee at the Zhongshan hospital, Fudan University. Mice were euthanized by cervical dislocation under anesthesia.

Primary human cortical astrocytes were prepared from fetal tissue provided by Advanced Bioscience Resources (Alameda, CA) as described previously.⁴² Astrocytes were initially cultured in DMEM supplemented with 10% fetal bovine serum, non‐essential amino acids, pen/strep, sodium pyruvate and glutamine. Astrocytes were serum starved for the experiments. The KMWT1 cell line is a mouse glioma established from the spontaneous glioma tumor. We have used CRE‐inducible oncogenic lentiviruses to generate it. These viruses expressing p53 shRNA and shRNA to NF1 induce tumors in Gfap‐Cre mice with histopathology and molecular signatures similar to human GBM. Tumors are induced using oncogenic lentiviruses in B6.Cg‐Tg(Gfap‐cre)77.6Mvs/2J driver mice that express the mouse Gfap promoter‐driven CRE in astrocytes and a subset of adult neural progenitor cells (inducing their differentiation to oligodendrocyte progenitor cells and their expansion). Spontaneous gliomas are generated within 6–9 weeks with 86% efficiency (n = 39) (Mockenhaupt et al, in revision). KMWT1 and U251‐MG cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose (Corning) supplemented with 10% tetracycline‐free fetal bovine serum (Biowest). The HeLa cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) with 1.0 g/L D‐glucose (Lonza) supplemented with 2 mM L‐glutamine (Sigma‐Aldrich) and 10% fetal bovine serum (Biowest). Cell lines modified with the Sleeping Beauty transposon system were additionally supplemented with 1 μg/mL puromycin (Invitrogen). Cell lines were maintained at 37°C in a humidified atmosphere with 5% CO₂. Cells were examined regularly for mycoplasma contamination using PCR.⁴³

Experimental heat pain was induced using a 3x3cm contact thermode (PATHWAY Pain & Sensory Evaluation System, Medoc ltd. Advanced Medical System, Israel) on the participants’ left thenar. During the experiment, the device was located in the control room and bridged to the scanner. Individual heat pain threshold and tolerance were determined using MEDOC Main Station 6.3. Ascending thermal stimulation cycles were presented and the mean value of the last three out of five consecutive threshold ratings was calculated. For pain threshold, subjects were asked to stop the stimulation via a mouse button when they started perceiving the stimulus as just painful, for pain tolerance they stopped at the maximum bearable heat. We calculated the specific stimulation intensity by building the mean between pain threshold and pain tolerance and adjusting the resulting intensity by running a Matlab routine. In a series of consecutive adjustment steps, a tonic heat stimulus of 27 s (ramp: 0.15 s per °C) was presented, followed by a VAS (see section ‘Rating procedure’) for intensity and unpleasantness ratings. Heat levels of the subsequent trials were adapted based on the respective intensity ratings until two consecutive ratings were in a range between 60 and 70. The adjusted stimulation intensity was used for both, the self-controlled and the computer controlled condition.